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$A $ 1#(#< $(& ,(,%,$%# %4# +")3#AA )E "#-#$A,(1 %4# (#?-/ A/(%4#A,W#& LH0 E")* %4# %"$(A3",+%,)( *$34,(#"/F M#"*,($%)"A $"# E)B(& &)?(A%"#$* )E %4# 1#(# %) .# %"$(A3",.#&D $(& %/+,3$--/ )33B" &,"#3%-/ $E%#" $(/ :l "#1B-$%)"/ #-#*#(%AD AB34 $A %4# +)-/$&#(/-$%,)( )" +)-/90< A,1($-F S4,-# *$(/ A%B&,#A E)3BA )( +")*)%#" A%"#(1%4 $A $ &#%#"*,($(% )E 1#(# #Z+"#AA,)( -#N#-AD %4# %#"*,($%)" $-A) +-$/A $( ,*+)"%$(% ")-# ,( LH0 +")3#AA,(1 $(& 3)(%",.B%#A %) N$",$.,-,%/ ,( LH0 4$-EX-,E#D $(& B-%,*$%#-/ 1#(#X#Z+"#AA,)(F >)-/$&#(/-$%,)(D $A %4# ($*# ,*+-,#AD ,A %4# +)A%X%"$(A3",+%,)($- $&&,%,)($- )E *B-%,+-# $&#(,(# 90< (B3-#)%,&#A %) %4# %$,- )E $ *#AA#(1#" LH0 %"$(A3",+%F M4# +B"+)A# $(& *#34$(,A* )E +)-/$&#(/-$%,)( N$"/ $*)(1 3#-%/+#AD .B% +)-/$&#(/-$%,)( 1#(#"$--/ A#"N#A %) +")*)%# %"$(A3",+% -)(1#N,%/ ,( #BC$"/)%#A $(& +")*)%# %"$(A3",+%
"$&$%,)( ,( +")C$"/)%#AF

!51/#5X10&6 A.5%&:#0&1: >")C$"/)%,3 %#"*,($%,)( *#34$(,A*A E$-- B(&#" %?) 1#(#"$- 3$%#1)",#AK "4)X&#+#(&#(% $(& "4)X ,(&#+#(&#(%F L4) E$3%)" ,A $ 4#-,3$A# ?4,34 $AA,A%A LH0 +)-/*#"$A# ,( %4# %#"*,($%,)( )E %4# %"$(A3",+%F L4)X&#+#(&#(% %#"*,($%)"A $"# ()% BAB$--/ #*+-)/#& ,( +-$A*,&X.$A#& #Z+"#AA,)( A/A%#*AD A) %4#A# ?,-- ()% .# &#%$,-#& 4#"#D .B% $&&,%,)($- "#E#"#(3#A $"# +")N,&#& $% %4# #(&F H#$"-/ $-- 3)**)( .$3%#",$- #Z+"#AA,)( +-$A*,&A BA# L4)X,(&#+#(&#(% %#"*,($%)"AD ?4,34 ,(3-B&# ($%B"$--/ )33B"",(1 %#"*,($%)"AD AB34 $A M8 $(& ""(JD $A ?#-as engineered high-efciency terminators such as T0.

L4)X,(&#+#(&#(% %#"*,($%,)( ,A $-A) C()?( $A ,(%",(A,3 %#"*,($%,)(D $(& "#-,#A )( %4# E)"*$%,)( )E $ U!X",34 4$,"+,( ,( %4# LH0 %"$(A3",+% E)--)?#& ./ $ ?#$C-/ bound poly-uracil tract as shown in the gure to the

",14%F M4# %#"%,$"/ A%"B3%B"# )E %4# 4$,"+,(XGH0 3)*+-#Z ,A %4)B14% %) &#A%$.,-,W# %4# %"$(A3",+%,)( 3)*+-#ZD ,(,%,$%,(1 3-#$N$1# )E %4# %"$(A3",+%F No terminator is 100% efcient at halting transcription of

%4# %#*+-$%# $(& ,(,%,$%,(1 %4# &#A,"#& 3-#$N$1# #N#(%D $-%4)B14 A)*# #(1,(##"#& %#"*,($%)"A 3)*# 3-)A# 9wgcx
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A=3G?@,AB3F ,@- !BE\, F?[@,EF ;DB@AW-> terminator will sufce. Many commercial expression vectors use double terminators to reduce unwanted %"$(A-$%,)( )E &)?(A%"#$* #-#*#(%AF 0 high afnity terminator  *$/ .# &#A,"#& E)" *B-%,X3,A%")(,3 3)(A%"B3%A where high termination efciency is necessary to minimize transcriptional read-through. Chris Voigt’s lab has

34$"$3%#",W#& $ A#% )E +")C$"/)%,3 %#"*,($%)"A $(& &#+)A,%#& A#N#"$- ?,%4 0&&1#(# 97
!51/#5X10&6 !1"X#'.:X"#0&1:  Although mostly thought of as a eukaryotic-specic process, prokaryotes also add poly(A) tails to certain RNAs.

b(-,C# %4# #BC$"/)%,3 *#34$(,A* ?4,34 "#[B,"#A $ 3)(A#(ABA A#[B#(3# E)" %4# $&&,%,)( )E $ +)-/90< %$,-D %4# addition of a poly(A) tail on a prokaryotic transcript is non-specic and can be added to any accessible 3’ end.

M4# +"#A#(3# )E %4# +)-/90< %$,- %$"1#%A %4# LH0 %) %4#
"$&)A)*#D ?4,34 3)(%$,(A #(W/*#A %4$% 3B% LH0 ()% protected by secondary structure. Because it lacks specicity, it is thought that poly(A)s are used to control the

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A#3)(&$"/ A%"B3%B"# %) ,(&B3# %"$(A3",+% 3-#$N$1#D A,*,-$" %) %4# L4)X,(&#+#(&#(% %#"*,($%,)( E)B(& ,( +")C$"/)%#AF M4,A ,A &,EE#"(#(% %4$( >)-/*#"$A#A O $(& OOD ?4,34 .)%4 "#[B,"# .,(&,(1 )E %#"*,($%,)( E$3%)"AF  0-%4)B14 .)%4 $"# %#"*,($%,)( E$3%)" &#+#(&#(%D >)-/*#"$A#A O $(& OO #*+-)/ &,EE#"#(% *#34$(,A*A %) %#"*,($%# %"$(A3",+%,)(F >)-/*#"$A# O BA#A $ +")3#AA A,*,-$" %) %4# +")C$"/)%,3 L4)X&#+#(&#(% *#34$(,A*D ?4#"#$A >)-/*#"$A# OO %#"*,($%,)( ,A *)"# 3)*+-#Z $(& ,(N)-N#A %?) LH0 +)-/*#"$A#X$AA)3,$%#& +")%#,(AD !>V` $(& !A%`D ?4,34 $"# "#A+)(A,.-# E)" "#3"B,%,(1 %4# 3-#$N$1# $(& +)-/$&#(/-$%,)( #(W/*#AD ,( $ +")3#AA %4$% A##*A %) 3)B+-# %#"*,($%,)( ?,%4 +)-/$&#(-/$%,)(F 2$**$,-$( #Z+"#AA,)( +-$A*,&A $"# +",*$",-/ BA#& %) 3"#$%# *LH0 $(& %4# 3)**)(-/ BA#& *$**$-,$( %#"*,($%)"A 9V]\6D 4URD JURD $(& ".U-).< ,(3-B&# %4# A#[B#(3# *)%,E 00b000 ?4,34 +")*)%#A .)%4 +)-/$&#(/-$%,)( $(& %#"*,($%,)(F IB% )E %4)A# -,A%#&D %4# V]\6 -$%# +)-/0 $(& ".U-). +)-/0 $"# %4)B14% %) .# more efcient in terminating transcription due to the presence of additional helper sequences (2-3).

 0A $--B&#& %) $.)N#D %#"*,($%,)( $(& +)-/$&#(/-$%,)( )E >)-/*#"$A# OO %"$(A3",+%A 9$(& %4#"#E)"# *LH0A< $"#

$%& 0;*C&/O&( &F9+/";780 B;@"+(&*"@+78;* C8)*+@ (8/&07C 0@&+O+)& +7 7%& 0@&+O+)& C8)*+@ +*( +((878;* ;G + B;@"T' 7+8@ 7; 7%& ,DW' 7/+*C0/8B7? !" arunreginald at en.wikipedia, CC BY-SA 3.0. https://en.wikipedia.org/wiki/Post-transcriptional_modication

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E)" +)-/$&#(/-$%,)(F M4# $&&,%,)( )E %4# +)-/90< %$,- ,A ,*+)"%$(% E)" A%$.,-,%/ )E %4# *LH0D +")%#3%,)( E")*
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Further Reading 1. Peters JM, Vangeloff AD, Landick R. Bacterial Transcription Terminators: The RNA 3′-End Chronicles. h)B"($- )E *)-#3B-$" .,)-)1/F 5677F >B.2#& >2OGK 57\:g5g8F >B.2#& !#(%"$- >2!OGK >2!:e55576F 2. Schek N, Cooke C, Alwine JC. Denition of the upstream efciency element of the simian virus 40 late +)-/$&#(/-$%,)( A,1($- ./ BA,(1 ,( N,%") $($-/A#AF 2)-#3B-$" $(& !#--B-$" J,)-)1/F 7gg5F >B.2#& >2OGK 7:::6\5F >B.2#& !#(%"$- >2!OGK >2!:e6\8eF 3. U,- 0D >")B&E))% HhF >)A,%,)(X&#+#(&#(% A#[B#(3# #-#*#(%A &)?(A%"#$* )E 00b000 $"# "#[B,"#& E)" efcient rabbit beta-globin mRNA 3’ end formation. Cell. 1987. PubMed >2OGK :cef7:7F 4. R$1#" VD `"$*# `2D !)--,(A 0MD JB"(A h;D 2$,%-$(& HhF 0( O(%#"($- >)-/$&#(/-$%,)( V,1($- VB.A%$(%,$--/ O(3"#$A#A ;Z+"#AA,)( Y#N#-A )E Y#(%,N,"BAXG#-,N#"#& M"$(A1#(#A .B% R$A %4# >)%#(%,$- %) L#&B3# ],"$- M,%#" ,( $ >")*)%#"XG#+#(&#(% 2$((#"F RB* U#(# M4#"F 566fF >B.2#& >2OGK 7fe585\8F 5. yBEE#"#/ LD G)(#--) h;D M")() GD R)+# MhF S))&34B3C R#+$%,%,A ],"BA >)A%%"$(A3",+%,)($- L#1B-$%)"/ ;-#*#(% ;(4$(3#A ;Z+"#AA,)( )E M"$(A1#(#A G#-,N#"#& ./ L#%")N,"$- ]#3%)"AF h)B"($- )E ],")-)1/F 7gggF >B.2#& >2OGK 7668\7:eF >B.2#& !#(%"$- >2!OGK >2!76\6\eF 6. Wodrich H, Schambach A, Kräusslich H-G. Multiple copies of the Mason–Pzer monkey virus constitutive LH0 %"$(A+)"% #-#*#(% -#$& %) #(4$(3#& RO]X7 U$1 #Z+"#AA,)( ,( $ 3)(%#Z%X&#+#(&#(% *$((#"F HB3-#,3 03,&A L#A#$"34F 5666F >B.2#& >2OGK76e\f8f7F >B.2#& !#(%"$- >2!OGK >2!765cf5F

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"$&#& ./ "#A%",3%,)( #(&)(B3-#$A#AF 0 1,N#( +")C$"/)%# %/+,3$--/ 4$A 1#(#A encoding one or a few restriction modication systems containing methyltransferases that add methyl groups to specic DNA sequences and companion endonucleases that recognize and cleave the same DNA sequence if

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AB34 $A !-$O )" t.$OD )"D 3)(N#"A#-/D $3%,N$%,(1 #(W/*#A AB34 $A G+(OF `)" #Z$*+-#D 3-#$N$1# ./ t.$O *$/ .# .-)3C#& &B# %) *#%4/-$%,)( ,E %4# #(W/*#lA "#3)1(,%,)( A,%# 9M!M0U0< ,A preceded by GA or followed by TC. As shown in the gure below, a Dam methylase recognition site (underlined

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J-)3C#& ./ )N#"-$++,(1 *#%4/-$%,)( J-)3C#& ./ )N#"-$++,(1 *#%4/-$%,)( H)% $EE#3%#&

 5g = >$1#

DN#20.5 )+ ON#0 &$ # !"#$%&'P

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

G=A`\E,A?B@ ,@- 3=FA3?DA?B@ =@d\G=F ;DB@AW->

U,+)& 0/&+7&( FC8*) P*+Bg&*&RDS

D1:051""&:9 G.0NX"#0&1: `,($--/D /)B 3$( 3)(%")- *#%4/-$%,)( ./ $-%#",(1 /)B" 34),3# )E .$3%#",$F `)" #Z$*+-#D ,E /)B *BA% BA# $ "#A%",3%,)( site that will be blocked by Dam or Dcm methylation, you can ensure this site remains unblocked by rst cloning /)B" GH0 ,(%) $ &$*_Q&3*_ A%"$,( )E >? 0;@8  AB34 $A h2776 $(& "#X+B",E/,(1 ,%F M4#A# A+#3,$-,W#& >? 0;@8  A%"$,(A have been specically engineered to be Dam and Dcm methylase-decient, and, as such, produce DNA that is

B(*#%4/-$%#& $% %4)A# A,%#AF >-#$A# C##+ ,( *,(& %4$% &$*XQ&3*X A%"$,(A *$/ 4$N# $( ,(3"#$A#& "$%# )E *B%$%,)( (as these would also be decient in mis-match repair functions of Dam), so these strains should not be used for

-)(1 %#"* A%)"$1#F

Further Reading r r r r

L#N,#? )E Restriction Modication Systems H;JlA L#A)B"3# G#A3",.,(1 G$* $(& G3* 2#%4/-$A#A >$+#" )( %4# G,A3)N#"/ )E G+(O L#N,#? &#A3",.,(1 &$*XQ&3*X >? 0;@8 

 :6 = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

JEC=_O`?A= FD3==@?@[ !" H&CC80+ [&@0%1 '(()&*& - HF* I1 234= 

H)? %4$% ?# 4$N# 3)N#"#& $(%,.,)%,3 A#-#3%,)( , we can talk about an even more specic method of screening /)B" 3-)(,(1 "#$3%,)(F J#,(1 $.-# %) A#-#3% E)" 3)-)(,#A %4$% 3)(%$,( /)B" +-$A*,& ,A $ 1"#$% A%$"% ?4#( 3-)(,(1D .B% 4)? $.)B% .#,(1 $.-# %) 34))A# %4)A# %4$% 3)(%$,( $ +-$A*,& ?,%4 $( ,(A#"%T J-B#X?4,%# A#-#3%,)( ,A $ ?,&#-/ BA#& *#%4)& %) &) @BA% %4$%p Y#%lA .#1,( $% %4# .#1,((,(1F M4# ?#--X34$"$3%#",W#& .$3%#",$- @+0  )+#")( 3)(%$,(A $ 1#(# 3$--#& @+0h  %4$% #(3)&#A E)" %4# #(W/*#  βX1$-$3%)A,&$A#F ;Z+"#AA,)( )E %4# @+0  )+#")( ,A ,(&B3#& ./ -$3%)A#D $(& $-A) ./ $ -$3%)A# $($-)1B#D O>MU 9,A)+")+/-  βXGX7X%4,)1$-$3%)+/"$()A,&#MU .,(&A $(& ,($3%,N$%#A %4# @+0   )+#")( "#+"#AA)"D %4#"#./ $--)?,(1 @+0  #Z+"#AA,)(F S4#( #Z+"#AA#&D %4#  βX1$-$3%)A,&$A# #(W/*# 3$( ."#$C &)?( $ &/#X-,(C#& AB.A%"$%# 3$--#& ZX1$- 9cX.")*)X\X 34-)")X:X,(&)-/-X βXGX1$-$3%)X+/"$()A,&#< ,(%) 1$-$3%)A# $(& $( ,(A)-B.-# .-B# +,1*#(% 9\X34-)")X:X.")*X,(&,1)
J"4._ON&0. F65..:&:9 &: 0N. E#H V3,#(%,A%A &,A3)N#"#& %4$% &#-#%,(1 $ A#3%,)( E")* %4# @+0h  gene (a mutation called lacZΔM15) creates a nonfunctional β-galactosidase enzyme. Providing DNA encoding this section of amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell 8* 7/+*C 3)*+-#*#(%A %4# *B%$%,)( $--)?,(1 E)" $ EB(3%,)($- #(W/*#F M4,A process is called α-complementation.

Blue-White screening. A) A white cell in which cloning was successful and the β-galactosidase gene (lacZ) was disrupted (split wedge on black plasmid)  preventing the cell from turning blue. B) Blue cell in which cloning was unsuccessful and the β-galactosidase gene is retained in the plasmid (un-split E&()&S 7F/*8*) 7%& 0&@@ <@F&? $%& B8& 0%+/7C 8* &+0% 0&@@ C%;E E%&7%&/ ;/ *;7 7%& )&*;,80 ,F7+78;* 8* @+0h 8C 0;,B@&,&*7&( R!S 0/&+78*) + GF@@ B8&1 ;/ *;7 R'S @&+O8*) + E&()& ;F7 ;G 7%& B8&?

M4# A/A%#* &#A3",.#& $.)N# ?$A +B% %) +"$3%,3$- BA# ,( %4# E)--)?,(1 ?$/F Scientists engineered a multiple cloning site (MCS) into the α-peptide (represented as an orange wedge in the gure above) and inserted it into a plasmid, creating an α-complementation cloning vector. When a cloning

"#$3%,)( 1)#A %) +-$( $(& /)B" GH0 9"#&< ,A 3-)(#& ,(%) %4,A 2!VD %4# α-peptide gets interrupted as shown in (A), and thus will not complement the a β-galactosidase mutation in the host cell. An unsuccessful cloning reaction leaves the α-peptide intact, and therefore the cell will have a functional β-galactosidase enzyme through α-complementation (cell B).

 :7 = >$1#

DN#20.5 )+ ON#0 &$ # !"#$%&'P

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

JEC=_O`?A= FD3==@?@[ ;DB@AW->  0A A4)?( )( %4# "#+"#A#(%$%,N# +-$%# )( %4# +"#N,)BA +$1#D 3)-)(,#A ?,%4 $( ,(A#"%X3)(%$,(,(1 +-$A*,& 4$N# $ non-functional β-galactosidase and remain the lovely whitish-cream color of standard >? 0;@8 F I( %4# )%4#" 4$(&D intact β-galactosidase produces pigment from x-gal (included in the transformation plate medium), turning the .$3%#",$- 3)-)(/ .-B#F S# A4)B-& ()%# %4$% O>MU ,A $-A) ,(3-B&#& ,( %4# *#&,B* %) #(AB"# %"$(A3",+%,)( )E %4# @+0

)+#")(F

A&2$ I15 J"4._ON&0. F65..:&:9 g

C$. # 911' 61:051"K M"$(AE)"* %4# .$3C.)(# +-$A*,& ?,%4)B% ,(A#"%F 0-- 3)-)(,#A )( %4,A +-$%# A4)B-&

.# .-B#D ,(&,3$%,(1 %4$% /)B" O>MU $(& ZX1$- $"# ?)"C,(1 $A %4#/ A4)B-& .#F g

-1:W0 54$N 0N. 2516.$$: It is important to give your plates enough time for any intact β-galactosidase

%) .# #Z+"#AA#& $(& +")3#AA ZX1$- ,(%) .-B# +,1*#(% 97eX56 4)B"A
3.I5&9.5#0. X145 2"#0.$K >-$3,(1 +-$%#A $% \s ! E)" $ E#? 4)B"A $E%#" %4# ,(,%,$- )N#"(,14% ,(3B.$%,)(

,(3"#$A#A +,1*#(% +"#3,+,%$%,)(D #(4$(3#A %4# .-B# 3)-)" )E (#1$%,N# 3)-)(,#AD $(& $--)?A E)" .#%%#" &,EE#"#(%,$%,)( .#%?##( .-B# $(& ?4,%# 3)-)(,#AF g

A#/. 6#5. &: %#/&:9 X145 2"#0.$K tX1$- ,A -,14% $(& %#*+#"$%B"# A#(A,%,N# $(& (##&A %) .# $&&#& %)

*#&,$ $E%#" $B%)3-$N,(1F OE A+"#$& )( %)+ )E +"#X*$&# +-$%#AD *$C# AB"# ,% ,A #N#(-/ &,A%",.B%#& $(& allow sufcient drying time before use. g

J.7#5. 1I I#"$. 21$&0&].$K J-B#X?4,%# A3"##(,(1 )(-/ ,(&,3$%#A %4# +"#A#(3# )E 0H ,(A#"%D ()% necessarily YOUR insert. Any cloning artifact that disrupts the α-peptide DNA will also lead to a white

3)-)(/F g

,:' #"$1 I#"$. :.9#0&].$K M4#A# $"# "$"#D .B% ,E $ A*$-- E"$1*#(% ,A ,(A#"%#& ,(XE"$*#D "#$&X%4")B14 can lead to a functional β-galactosidase enzyme and a blue colony. Blue-white screening is a good way to narrow down candidates for more specic analysis, like PCR or restriction digest.

g

G#/. $45. X14 4$. # 2512.5 D@ -).& *"',&7 (i.e. contains the lacZΔM15 mutation): XL1-Blue, DH5α,

GR76JD h276gD VMJY\D h2776D $(& M)+76 $"# $ E#? #Z$*+-#AF g

G#/. $45. X14 4$. # 2512.5 2"#$%&' (i.e. contains the α-complementation cloning MCS): pGEM-T,

+b!7f $(& +b!7gD $(& +J-B#A3",+% $"# $ E#? 3)**)( N#3%)"AF J-B#X?4,%# A3"##(,(1 ,A @BA% %4$% _ $ A3"##(,(1 +")3#AAF O% &)#A ()% A#-#3% )(-/ %4)A# 3#--A %4$% 4$N# %$C#( B+ $ +-$A*,& $(& %4BA A4)B-& .# BA#& ,( 3)(@B(3%,)( ?,%4 A#-#3%,)( *#%4)&AF !)*.,(,(1 A#-#3%,)( $(& A3"##(,(1 #(AB"#A %4$% %4# ?4,%# 3)-)(,#A /)B A## $"# ?4,%# &B# %) AB33#AAEB- 3-)(,(1 $(& ()% .#3$BA# %4# 3#-- E$,-#& %) take up the α-complementation plasmid. This way you can quickly and easily identify colonies that not only have your plasmid (antibiotic resistance), but also conrm those plasmids have your insert (blue-white screening).

 :5 = >$1#

DN#20.5 )+ ON#0 &$ # !"#$%&'P !@F&TE%87& P0/&&*8*) 

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

JEC=_O`?A= FD3==@?@[ ;DB@AW-> B0N.5 AX2.$ 1I F65..:&:9 G.0N1'$  0-%4)B14 .-B#X?4,%# A3"##(,(1 ,A +").$.-/ %4# *)A% ?,&#A+"#$& ?$/ %) A#-#3% E)" +-$A*,&A 3)(%$,(,(1 $( ,(A#"%D %4#"# $"# )%4#" *#%4)&AF >)A,%,N# A#-#3%,)( N#3%)"A #(3)&# $ 1#(# ?4,34D ?4#( #Z+"#AA#&D ,A -#%4$- %) %4# 3#--F !-)(,(1 E"$1*#(%A $"# ,(A#"%#& ,(%) $( 2!V ,( % 4# 3#(%#" )E %4,A 1#(#D &,A"B+%,(1 %4# -#%4$-,%/F M4,A ,A A,*,-$" to α-peptide DNA disruption in the blue-white screen. Antibiotic selection is also used in conjunction with this

*#%4)& %) #(AB"# %4$% +)A,%,N# 3)-)(,#A &) 4$N# %4# +-$A*,& 3)(%$,(,(1 %4# -#%4$- 1#(#F 0( #Z$*+-# ,A %4# 33&J 1#(# A/A%#*F M4#"# $"# $-A) *#%4)&A %) A#-#3% E)" +-$A*,&X3)(%$,(,(1 3#--A ?,%4)B% BA,(1 $(%,.,)%,3 "#A,A%$(3#F M4#A# "#-/ )( 3#-- -,(#A ABA3#+%,.-# %) )" &#+#(&#(% )( 3#"%$,( *#&,$ 3)*+)(#(%AD $(& $"# "#A3B#& ./ 1#(#A AB++-,#& )( %4# transformed plasmid. Such plasmids may contain genes that allow for t he use of a particular substrate in dened

*#&,B*D ?,%4)B% ?4,34 %4# 3#--A 3$(()% "#+")&B3# %) E)"* 3)-)(,#A 9BA#& %) 3)*+-#*#(% $BZ)%")+4,3 3#-- -,(#A
 :: = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

DBGGB@ E,J D@ EF=G  FA3,?@F !" .+77%&E L&/&*01 '(()&*& - W;O 51 234I

^)BlN# ?)"C#& 4$"& &#A,1(,(1 /)B" +-$A*,& _ /)B 3$"#EB--/ A#-#3%#& %4# )+%,*$- +")*)%#" E)" /)B" 1#(# )E ,(%#"#A%D /)B +$,(A%$C,(1-/ 3-)(#& ,(%) %4# +#"E#3% #*+%/ .$3C.)(#D /)B *$&# AB"# %) $&& %4# ",14% %$1A $(& $ nuclear localization signature (NLS) to your gene, you put a uorescent

+")%#,( &)?(A%"#$*D A#+$"$%#& ./ $( OL;V #-#*#(%F ^)B &,& $ -)% )E ?)"Cp JB% -#%lA %$C# $ *)*#(% %) "#3)1(,W# %4# +")C$"/)%,3 *,(,)(A %4$% 3$"",#& )B% %4# -$.)"X,(%#(A,N# +")3#AA )E "#+-,3$%,(1 /)B" (#? +-$A*,&K %4# >C0%&/80%8+ 0;@8 .$3%#",B*F O%lA 4$"& %) 3)B(% %4# (B*.#" )E &,EE#"#(% 3)**#"3,$- A%"$,(A )E >? 0;@8  3B""#(%-/ $N$,-$.-# _ $ [B,3C U))1-# A#$"34 AB11#A%A %4#"# $"# 4B(&"#&AF M4,A )(-/ ,(3-B&#A 1#(#"$- -$. A%"$,(A &#A,1(#& E)" AB.3-)(,(1 )" +")%#,( #Z+"#AA,)(F OE /)B ?#"# %) ,(3-B&# 3BA%)*,W#& A%"$,(AD %4# (B*.#" ,A +").$.-/ ,( %4# %4)BA$(&Ap M4# 1)$- )E %4,A A#3%,)( ,A %) +")N,&# #()B14 .$3C1")B(& E)" /)B %) &,A%,(1B,A4 %4# E#$%B"#A )E $(/ 3)**)( -$. A%"$,( $(& &#%#"*,(# ?4#%4#" ,% ,A $++")+",$%# E)" +")+)1$%,(1 /)B" +-$A*,& )" 3$""/,(1 )B% /)B" #Z+#",*#(%F

`&$015X 1I D@ -).&  F05#&:$ >? 0;@8  $"# 1"$*X(#1$%,N#D ")& A4$+#& .$3%#",$ %4$% ?#"# ($*#& $E%#" G"F M4#)&)" ;A34#",34D %4# A3,#(%,A% who rst described them in 1885. >? 0;@8 $"# *$,(-/ E)B(& ,( %4# ,(%#A%,($- %"$3% )E $(,*$-AF M4#"# $"# *$(/ &,EE#"#(% ($%B"$--/ )33B"",(1 A%"$,(A )E >? 0;@8 D A)*# )E ?4,34 $"# &#$&-/ %) 4B*$(AF M4# *$@)",%/ )E $-- 3)**)(D 3)**#"3,$- -$. A%"$,(A )E >? 0;@8  BA#& %)&$/ $"# &#A3#(&#& E")* %?) ,(&,N,&B$- ,A)-$%#AD %4# dX75 A%"$,( $(& %4#

J A%"$,(F dX75 ?$A ,A)-$%#& E")* $ +$%,#(% ,( 7g56 $(& #N#(%B$--/ -#& %) %4# 3)**)( -$. A%"$,( 2U7eccD ?4,34 -#& %) GRc$-+4$ $(& GR76. 9$-A) C()?( $A MI>76? 0;@8  J A%"$,(F

D1%%1: D@ -).&  F05#&:$ C$.' &: 0N. E#H Most of the commercial strains you nd today are marketed for a specic purpose: fast growth, high-throughput

3-)(,(1D ")B%,(# 3-)(,(1D 3-)(,(1 B(A%$.-# GH0D +"#+$",(1 B(*#%4/-$%#& GH0D $(& *)"#F 2$(/ *B%$%,)(A %4$% *$C# %4#A# E#$%B"#A +)AA,.-# $"# +"#A#(% ,( *)A% 3)**#"3,$- A%"$,(AD #A+#3,$--/ *B%$%,)(A %4$% *$C# *$@)" ,*+")N#*#(%A AB34 $A %4)A# %4$% ,(3"#$A# +-$A*,& /,#-& $(&Q)" GH0 [B$-,%/F M$.-# 7 .#-)? )B%-,(#A $ E#? )E %4# *)"# 3)**)( 1#(#%,3 34$(1#A E)B(& ,( >? 0;@8  A%"$,(A $(& M$.-# 5 )B%-,(#A 3)**)( -$. A%"$,(A )E >? 0;@8 F

 :\ = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

DBGGB@ E,J D@ EF=G  FA3,?@F ;DB@AW-> A#H". )+ D1%%1: [.:. G40#0&1:$ K14:' &: D@ -).&  F05#&:$ [.:.$

-.$65&20&1:

(+,

GH0 0&#(,(# *#%4/-$A# *B%$%,)( 9U0M!<

(0,

GH0 !/%)A,(# *#%4/-$A# *B%$%,)( 9!!SUU<

(*+H 

2B%$%,)( ,( $ 34$+#")(,( 1#(#

&*('1 &*('4

Endonuclease I (nonspecic cleavage of

L 

&AGH0 < *B%$%,)( R)A% &)#A 9`l< )" &)#A ()% 9`X< 3)(%$,( %4# E#"%,-,%/ +-$A*,&F

K4:60&1:#" D1:$.U4.:6.

>"#+$",(1 B(*#%4/-$%#& GH0D ,*+)"%$(% ?4#( %"/,(1 %) 3B% ?,%4 3#"%$,( "#A%",3%,)( #(W/*#A 9#ZK !-$O )" t.$O< >"#+$",(1 B(*#%4/-$%#& GH0D ,*+)"%$(% ?4#( %"/,(1 %) 3B% ?,%4 3#"%$,( "#A%",3%,)( #(W/*#A %4$% $"# *#%4/-$%,)( A#(A,%,N#F O(3"#$A#A %4# A%$.,-,%/ )E 3#"%$,( #Z+"#AA#& +")%#,(A O*+")N#A +-$A*,& /,#-&  0 -)? 3)+/X(B*.#" +-$A*,&D #(3)&#A +")%#,(A (##&#& E)" .$3%#",$- 3)(@B1$%,)(F U#(#A -,A%#& )( `z $"# ?,-&X%/+# B(-#AA ,(&,3$%#& )%4#"?,A# M7QMc >4$1# "#A,A%$(3#

)"/'1 )"/'`M 

E#"",3 4/&")Z$*$%# B+%$C#D ,")( B+%$C# "#3#+%)" *B%$%,)(F 2B%$%,)( ,( 1$-$3%)A# *#%$.)-,A* +$%4?$/ GH0 1/"$A# *B%$%,)(

%C(D.P

%C(DR/9T1 ,9iS

3#--A 3$(()% 1")? )( @BA% 1$-$3%)A# !)(E#"A "#A,A%$(3# %) ($-,&,Z,3 $3,& B(*#%4/-$%#& GH0 ()%
"$&#&D 3#-- A%,-- 3$( *#%4/-$%# GH0

%C(PR/9T1,9TS

B(*#%4/-$%#& GH0 ()%
"$&#&D 3#-- 3$(()% *#%4/-$%# GH0

G%F' RG;/,&/@" 7;*'S )+@ 

Y$3 I+#")( 2B%$%,)(A .-B#Q?4,%# A3"##(,(1 )E 3-)(#A

@+0  @+0UQ

-$3 "#+"#AA)" )N#"+")&B3#&D #Z+"#AA,)( E")* +Y$3 "#+"#AA#& *)"#

@+0h 

β-galactosidase activity abolished

@+0b 

Y$3%)A# +#"*#$A# ,($3%,N$%#&D -$3%)A# 3$(()% .# %$C#( B+ ./ 3#--

 :c = >$1#

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DN#20.5 )+ ON#0 &$ # !"#$%&'P

DBGGB@ E,J D@ EF=G  FA3,?@F ;DB@AW-> A#H". )+ D1%%1: [.:. G40#0&1:$ K14:' &: D@ -).&  F05#&:$ ;61:08> [.:.$ ,/0'1 ,0/!a  mrr, Δ(mcrC-mrr) /&0'1 /&0'41 /&0'4J

/&0!aV /&@' ;/ /&@'4  B7/0T00('

-.$65&20&1:

O($3%,N$%,)( )E +$%4?$/ %4$% 3-#$N#A  0--)?A E)" B+%$C# )E E)"#,1( *#%4/-$%#& 3/%)A,(# GH0 9*#%4-/$%#&< GH0 O($3%,N$%,)( )E +$%4?$/ %4$% 3-#$N#A  0--)?A E)" B+%$C# )E E)"#,1( *#%4/-$%#& $&#(,(# )" 3/%)A,(# GH0 9*#%4-/$%#&< GH0 2B%$%,)( ,( $ GH0X&#+#(&#(% 0M>$A# %4$% L#&B3#A +-$A*,& "#3)*.,($%,)(D ,A #AA#(%,$- E)" "#3)*.,($%,)( $(& 1#(#"$- ,(3"#$A#A +-$A*,& A%$.,-,%/ GH0 "#+$,"  ;Z)(B3-#$A# ] $3%,N,%/ $.)-,A4#& O(3"#$A#& +-$A*,& A%$.,-,%/D "#&B3#& "#3)*.,($%,)( L#-$Z#& +4#()%/+#D *B%$%,)( #-,*,($%,(1  0--)?A LH0 A/(%4#A,A ,( $.A#(3# A%",(1#(% E$3%)"  )E +")%#,( A/(%4#A,A >")+$1$%,)( )E 33&JX3)(%$,(,(1 +-$A*,&A

%7& (&;D 

K4:60&1:#" D1:$.U4.:6.

high transformation efciency

3)(A%,%B%,N# #Z+"#AA,)( )E 1#(#A E)" &#)Z/",.)A# A/(%4#A,A

 0--)?A B+%$C# )E -$"1# +-$A*,&A high electroporation efciency

%&&

VB++"#AA,)( )E %4# $*.#" 9b0U< A%)+ 3)&)( ./ ,(A#"%,(1 1-B%$*,(# VB++"#AA,)( )E %4# $*.#" 9b0U< A%)+ 3)&)( ./ ,(A#"%,(1 %/")A,(#

CFB>II R)@*jIIS CFBL R7"/$S  λ-thi-1 or thi1

2B%$%,)( ,( %4,$*,(# *#%$.)-,A*

+/+

&,A"B+%,)( )E $"$.,()A# *#%$.)-,A* +$%4?$/

@&F!

β-isopropyl malate dehydrogenase

 B/;'!

,($3%,N$%#& *B%$%,)( ,( +")-,(# .,)A/(%4#A,A +$%4?$/

/BC^

2B%$%,)( ,( AB.B(,% V75 )E :6V ",.)A)*#

"#[B,"#A #Z)1#()BA %4,$*,(# E)" 1")?%4 ,($.,-,%/ %) B%,-,W# $"$.,()A# $A $ 3$".)( A)B"3# "#[B,"#A #Z)1#()BA -#B3,(# A)B"3# E)" 1")?%4 "#[B,"#A #Z)1#()BA +")-,(# A)B"3# E)" 1")?%4 !)(E#"A "#A,A%$(3# %) A%"#+%)*/3,(

 :e = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

DBGGB@ E,J D@ EF=G  FA3,?@F ;DB@AW-> A#H". L+ E#H F05#&:$ 1I D@ -).&  F05#&:

 

@#045#" 3.$&$0#:6.

!5&%#5X C$.

[.:10X2.

33&J VB"N,N$- 5

HQ0

`)" +")+$1$%,(1 +-$A*,&A #Z+"#AA,(1 %4# 33&J 1#(# 9,*+)"%$(% ,( U$%#?$/ 3-)(,(1
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK /BC^ RP7/DS &*('4 *FBg G%F'XXUP2 

GJ:F7

V%"#+%)*/3,(

`)" +")+$1$%,(1 +-$A*,&A #Z+"#AA,(1 %4# 33&J 1#(# 9,*+)"%$(% ,( U$%#?$/ 3-)(,(1
F- gyrA462 endA1 glnV44 Δ(sr1/&0'S ,0/! ,// %C(P23R/ !T1 T ,! S +/+4I )+@K2 @+0b4 B/;'2 /  xyl5 Δleu mtl1 /BC^23RP, )

GR76J

V%"#+%)*/3,(

2!76e7 &#",N$%,N#F U#(#"$3-)(,(1 $(& A%)"$1#D .-B#Q?4,%# A3"##(,(1D -#B3,(# $BZ)%")+4F

L T &*('4 (&;D i /&0'4 )+@>4= galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139  Δ(ara,leu)7697 mcrA Δ(mrrhsdRMS-mcrBC) StrR λ T

DH5α

HQ0

U#(#"$- 3-)(,(1 $(& A%)"$1# )E 3)**)( +-$A*,&AD .-B#Q?4,%# A3"##(,(1F

L T &*('4 )@*jII 7%8T4 /&0'4 /&@'4 )"/'`M (&;D *FBg BF/!23 Φ80dlacZΔM15 Δ(lacZYA-argF) U169, hsdR17(r K T ,K i ), λ–

RJ767

V%"#+%)*/3,(

h276g

HQ0

R/.",& )E >? 0;@8 d75 $(& >? 0;@8 J L T ,0/! ,// %C(P23R/ !T ,! ST /&0'4J 9*)A%-/ d75D %4)B14
h2776

V%"#+%)*/3,(

2!76e7

V%"#+%)*/3,(

2U7ecc

HQ0

"#+#%,%,N# GH0F `)" A%)",(1 +-$A*,&A %4$% A4)B-& ()% .# *#%4/-$%#&D $--)?A E)" *#%4/-$%,)( A#(A,%,N# "#A%",3%,)( #(W/*#A %) 3B% %4# +-$A*,& $E%#" +"#+$"$%,)(F >$"#(% )E GR76JQMI>76 $(& &#",N#& A%"$,(AD 3)**)( -$. 3-)(,(1 $(& A%)"$1# A%"$,(F iS,-& %/+#j dX75 A%"$,(F

%C(D45R/ K T,K i S /BC^ 7%/ @&F 7%8 @+0b )+@K )+@$ +/+ tonA tsx dam dcm glnV44 Δ(lac proAB) e14- [F’ traD36 proABi @+0U Q lacZΔM15] hsdR17(r K T,K i S L T Δ(ara-leu)7697 [araD139] !c/   Δ(codB-lacI)3 galK16 galE15 λ T &4IT ,0/'3 /&@'4 /BC^4=3RC7/DS CB;$4 ,0/!4 %C(D2R/ T,i S L T λT 8@OgT /G
 :8 = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

DBGGB@ E,J D@ EF=G  FA3,?@F ;DB@AW-> A#H". L+ E#H F05#&:$ 1I D@ -).&  ;61:0W'> F05#&:

>,"7

 

@#045#" 3.$&$0#:6.

HQ0

!5&%#5X C$.

`)" 3-)(,(1 $(& *$,(%#($(3# )E $ plasmids with R6Kγ origin; contains

$ *B%$(% $--#-# )E %4# +," 1#(# %4$% *$,(%$,(A %4# +-$A*,&A $% P5c6 3)+,#A +#" 3#--F h276gX&#",N#&F `)" A%)"$1# )E +-$A*,&A %4$% 4$N# %4# +)%#(%,$- %) "#3)*.,(#F ;Z$*+-#D %4# YMLA ,( -#(%,X $(& "#%")XN,"$+-$A*,&AF G#",N#& E")* RJ767F `)" A%)"$1# )E +-$A*,&A %4$% 4$N# %4# +)%#(%,$- %) "#3)*.,(#F ;Z$*+-#D %4# YMLA ,( -#(%,X $(& "#%")XN,"$- +-$A*,&AD #(&0vD BA# 3$"# ,( +"#+$",(1 GH0 E")* %4,A A%"$,(F 2!76e7 &#",N$%,N#F U#(#"$- 3-)(,(1 $(& A%)"$1#D .-B#Q?4,%# A3"##(,(1F

[.:10X2. F- Δlac169 rpoS(Am) robA1 0/&a=43 %C(D=4I &*(' /&0'4 uidA(ΔMluI)::pir-116 

V%.-5

HQ0

V%.-:

V%"#+%)*/3,(

M)+76

V%"#+%)*/3,(

tY7 J-B#

M#%"$3/3-,(#

J-B#Q?4,%# A3"##(,(1 $(& ")B%,(# 3-)(,(1D ($-,&,Z,3 $3,& "#A,A%$(%F

tY76 U)-&

M#%"$3/3-,(# $(& !4-)"$*+4#(,3)-

!-)(,(1 $(& +")+$1$%,)( )E -$"1# &*('4 )@*jII /&0'4 7%8T4 +-$A*,&AD 4,14 3)*+#%#(3/D ($-,&,Z,3 $3,& gyrA96 relA1 lac Hte Δ(mcrA)183  Δ(mcrCB-hsdSMR-mrr)173 tet D "#A,A%$(%F

LT &*('4 )@*jII 7%8T4 /&0'4 gyrA96 relA1 Δ(lac-proAB) mcrA  Δ(mcrBC-hsdRMS-mrr) λT LT )@*jII /&0'4J ,0/! ,// %C(P23R/!T1 ,!TS +/+T4I )+@K2 @+0b4 B/;'2 /BC^23 A"@T= @&F ,7@T4

F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 )+@>4= )+@K4M /BC^RP7/ D  ) endA1 λT &*('4 )"/'`MR*+@ D  S 7%8T4 /&0'4 relA1 lac glnV44 F’[ ::Tn10 proAB i @+0U Q Δ(lacZ)M15] hsdR17(r K T ,K i S

F’[proAB lacI QZΔM15 Tn10(Tet D  '," a,D  )] 

Note: Generally inactivating mutations in specic genes are signied with a minus sign (-) as is typically C7+*(+/(\ fFC7 %+O8*) 7%& )&*& @8C7&( 8*(80+7&C 87 8C *;*TGF*078;*+@? UG + )&*& 8C (&@&7&( 7%+7 8C *;/,+@@" *;7&( E87% a Greek delta (Δ).

 :f = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

=8 DBE? FA3,?@F KB3 !3BA=?@ =h!3=FF?B@ !" HF@8+* $+"@;/T6+/9&/1 '(()&*& - L&< 431 234= 

S# +"#N,)BA-/ "#N,#?#& %4# A$-,#(% E#$%B"#A )E A#N#"$- +)+B-$" A%"$,(A )E ;F 3)-, E)" GH0 +")+$1$%,)(F S4,-# 1"#$% E)" 3-)(,(1 +B"+)A#AD %4#A# >? 0;@8 A%"$,(A $"# ()% BAB$--/ ?#-- AB,%#& E)" "#3)*.,($(% +")%#,( #Z+"#AA,)(F 2$(/ 34$--#(1#A 3$( $",A# ?4#( )N#"X#Z+"#AA,(1 $ E)"#,1( +")%#,( ,( >? 0;@8 F S# ?,-- "#N,#? %4# +)%#(%,$- +,%E$--A )E "#3)*.,($(% +")%#,( #Z+"#AA,)( $(& A)*# )E %4# *)A% +)+B-$" 3)**#"3,$- A%"$,(A &#A,1(#& %) $N),& %4#*F

ONX -1 ? @..' #: =Y25.$$&1: F05#&:P >")%#,( #Z+"#AA,)( E")* 4,14X3)+/ (B*.#" +-$A*,&A $(& +)?#"EB- +")*)%#"A ?,-1"#$%-/ #Z3##& %4$% )E $(/ ($%,N# 4)A% +")%#,(D BA,(1 B+ N$-B$.-# "#A)B"3#A ,( %4# 3#-- %4BA -#$&,(1 %) A-)?#& 1")?%4F 0&&,%,)($--/D A)*# +")%#,( +")&B3%A *$/ .# %)Z,3 %) %4# 4)A% ?4#( #Z+"#AA#&D +$"%,3B-$"-/ %4)A# %4$% $"# ,(A)-B.-#D $3% )( GH0D )" $"# #(W/*$%,3$--/ $3%,N#F `)" %4,A "#$A)(D "#3)*.,($(% +")%#,(A $"# %/+,3$--/ #Z+"#AA#& ,( >? 0;@8 #(1,(##"#& %) $33)**)&$%# 4,14 +")%#,( -)$&A BA,(1 ,(&B3,.-# +")*)%#" A/A%#*A 9?4,34 ?,-- .# &,A3BAA#& -$%#"
;F 3)-,

g

 0 E#? *B%$%,)(A $"# 3)**)( %) $-- )" *)A% #Z+"#AA,)( A%"$,(A %) $33)**)&$%# 4,14 +")%#,( -#N#-A ,(3-B&,(1K

1%2A: Strains harboring this mutation are decient in outer membrane protease VII, which reduces

+")%#)-/A,A )E %4# #Z+"#AA#& "#3)*.,($(% +")%#,(AF g

"1: 2510.#$.: Strains where this is completely deleted (designated lon or Δlon) similarly reduce

+")%#)-/A,A )E %4# #Z+"#AA#& +")%#,(AF g

N$'FJ ;5 J_ %J_>K M4#A# A%"$,(A 4$N# $( ,($3%,N$%#& ($%,N# "#A%",3%,)(Q*#%4/-$%,)( A/A%#*F M4,A *#$(A %4#

A%"$,( 3$( (#,%4#" "#A%",3% ()" *#%4/-$%# GH0F

g

'6%K V,*,-$"-/D A%"$,(A ?,%4 %4,A *B%$%,)( $"# B($.-# %) *#%4/-$%# 3/%)A,(# ?,%4,( $ +$"%,3B-$" A#[B#(3#F

 :g = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

D@ EF=G FA3,?@F KB3 !3BA=?@ =h!3=FF?B@ ;DB@AW-> A#H". )+ D@ -).& =Y25.$$&1: F05#&:$ W;7&X '@@ C7/+8*C +/& (&/8O&( G/;, 7%& >?0;@8 ! C7/+8*1 &A0&B7 7%;C& ,+/9&( E87% NN E%80% +/& K42? N 8*(80+7&C 7%+7 C7/+8* 0;,B;*&*7 8C + B@+C,8(? F05#&:

3.$&$0#:6.

f.X K.#045.$

JY579G;:<

HQ0

JY579G;:< +Y/AVu

!4-)"$*+4#(,3)- +Y/AV #Z+"#AA#A M8 9+Y/AV< -/A)W/*# %) "#&B3# .$A$- #Z+"#AA,)( -#N#-Ao #Z+"#AA,)( N#3%)" 3$(()% 4$N# +7c0 )",1,( )E "#+-,3$%,)( !4-)"$*+4#(,3)- +Y/A; 4$A 4,14#" M8 9+Y/A;< -/A)W/*# #Z+"#AA,)( %4$( +Y/AVo #Z+"#AA,)( N#3%)" 3$(()% 4$N# +7c0 )",1,( )E "#+-,3$%,)( HQ0 Y$3CA EB(3%,)($- LH$A#; ?4,34 "#AB-%A ,( -)(1#" %"$(A3",+% 4$-EX-,E#

JY579G;:< +Y/A;u

JY57 A%$" 9G;:<

[.:10X2.

J$A,3 O>MUX,(&B3,.-# A%"$,( LT ;,B$ @;* %C(P!R/!T 3)(%$,(,(1 M8 LH0> 9G;:< ,!TS )+@ (0, RV>JS

C$.

U#(#"$- +")%#,( #Z+"#AA,)(

LT ;,B$ @;* %C(P!R/!T ,!TS )+@ (0,RV>JS  B^"CP Ra+,DS

;Z+"#AA,)( )E %)Z,3 +")%#,(A

LT ;,B$ @;* %C(P! R/!T ,!TS )+@ (0,RV>JS  B^"C> Ra+,DS

;Z+"#AA,)( )E %)Z,3 +")%#,(A

LT ;,B$ @;* %C(P!R/!T ,!TS )+@ (0, /*&4J4 RV>JS

U#(#"$#Z+"#AA,)(o ()% "#3)**#(&#& E)" %)Z,3 +")%#,(A U#(#"$- +")%#,( #Z+"#AA,)(

JY57X07

M#%"$3/3-,(#

0"$.,()A#X,(&B3,.-# #Z+"#AA,)( )E M8 LH0>o O>MU *$/ A%,-- .# "#[B,"#& E)" #Z+"#AA,)(

LT ;,B$ @;* %C(P!R/!T ,!TS )+@ (0, +/+!XX$5DW'6T7&7' [malB+]K-12(λS)

JYL 9G;:<

M#%"$3/3-,(#

RecA-decient; best for

LT ;,B$ @;* %C(P!R/!T ;Z+"#AA,)( )E mB-) gal dcm(DE3) Δ(srl- B(A%$.-# +")%#,(A /&0'SJ3MXX$*43 R$&7DS

+-$A*,&A ?,%4 "#+#%,%,N# A#[B#(3#AF R2V78\ 9G;:
L,E$*+,3,(

MB(#" 9G;:<

HQ0

RecA-decient; allows for

3-)(,(1 $(& #Z+"#AA,)( ,( A$*# A%"$,(

LT /&0'4 %C(DR/K42T ,K42iS RV>JS RD8GDS

LT ;,B$ @;* %C(P!R/!T !)(%$,(A *B%$%#& -$3 +#"*#$A# ?434 $--)?A E)" ,!TS )+@ (0, -,(#$" 3)(%")- )E #Z+"#AA,)( @+0b4RV>JS

;Z+"#AA,)( )E B(A%$.-# +")%#,(A ;Z+"#AA,)( )E %)Z,3 )" ,(A)-B.-# +")%#,(A

 \6 = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

D@ EF=G FA3,?@F KB3 !3BA=?@ =h!3=FF?B@ ;DB@AW-> A#H". )+ D@ -).&  =Y25.$$&1: F05#&:$ ;61:0W'> W;7&X '@@ C7/+8*C +/& (&/8O&( G/;, 7%& >?0;@8 ! C7/+8*1 &A0&B7 7%;C& ,+/9&( E87% NN E%80% +/& K42? N 8*(80+7&C 7%+7 C7/+8* 0;,B;*&*7 8C + B@+C,8(? F05#&:

L)A#%%$5 9G;:
Y#*)57 9G;:
M8 ;Z+"#AA

3.$&$0#:6.

f.X K.#045.$

[.:10X2.

!4-)"$*+4#(,3)- U))& E)" iB(,N#"A$-j %"$(A-$%,)(o `X )*+M 4A&VJ9"JX *JX< 9+L0L;< 3)(%$,(A 8 $&&,%,)($- %LH0A E)" 1$- &3* 9G;:< +L0L;5 "$"# 3)&)(A ()% ()"*$--/ BA#& ,( 9!$*L< >? 0;@8 F ;Z+"#AA,)( N#3%)" 3$(()% 4$N# +7c0 )",1,( )E "#+-,3$%,)( fhuA2 [lon] ompT gal !4-)"$*+4#(,3)- L4$*()A#X%B($.-# M8 LH0> (λ DE3) [dcm] ΔhsdS/ 9+Y#*)< #Z+"#AA,)( $--#N,$%#A ,(3-BA,)(  B^&,; Ra+,DS .)&/ E)"*$%,)(F ;Z+"#AA,)( N#3%)" 3$(()% 4$N# +7c0 )",1,( )E "#+-,3$%,)( O>MUX,(&B3,.-# #Z+"#AA,)( )E M8 G%F'2 @+0hXX$5 )&*&4 LH0> E")* %4# 1#()*#o &)#A ()% [lon] ompT gal sulA11 "#A%",3% *#%4/-$%#& GH0 DR,0/T5JXX,8*8$*43T

C$.

;Z+"#AA,)( )E #BC$"/)%,3 +")%#,(A ;Z+"#AA,)( )E %)Z,3D ,(A)-B.-#D )" *#*."$(# +")%#,(A U#(#"$+")%#,( #Z+"#AA,)(

-TetS)2 [dcm] R(zgb243XX$*43TT$&7PS &*('4  Δ(mcrC-mrr)114::IS10 

*7cuu +L;>\u

d$($*/3,( 9+L;>\<

I",1$*,5 9G;:
V%"#+%)*/3,( $(& M#%"$3/3-,(#

!,AX"#+"#AA,)( )E %4# >? 0;@8 Mc +")*)%#" 9E)B(& )( N#3%)"A AB34 $A +a; )" A,*,-$" MU 9-$3 "#+"#AA)" )( %4# +L;>\ +-$A*,&
F-, Φ80ΔlacM15, thi, @+0T1 ,7@T1 /&0'i1 K,D 

;Z+"#AA,)( )E %)Z,3 +")%#,(A

 Δ(ara-leu)7697 ΔlacX74  ΔphoA PvuII phoR +/+V4J` +%Ba )+@> )+@K rpsL F′[lac+ lacIq pro] RV>JS );/=22XX$*43 7/A! RP7/D1 $&7DS

;Z+"#AA,)( )E ,(A)-B.-# +")%#,(A

 \7 = >$1#

!"#$%&'$ )*)+ , -.$/012 3.$1456. ;< 5' ='&0&1:>

DN#20.5 )+ ON#0 &$ # !"#$%&'P

D@ EF=G FA3,?@F KB3 !3BA=?@ =h!3=FF?B@ ;DB@AW-> `17 -1.$ ?:'46&H". =Y25.$$&1: O15/P  0A *#(%,)(#& $.)N#D *$(/ #Z+"#AA,)( +-$A*,&A B%,-,W# ,(&B3,.-# +")*)%#"AD ?4,34 $"# k,($3%,N#l B(%,- $( ,(&B3#" AB34 $A O>MU ,A $&&#& %) %4# 1")?%4 *#&,B*F O(&B3%,)( %,*,(1 ,A ,*+)"%$(%D $A you typically want to make sure your cells have rst reached an

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c9+L;>\< BA# $ -$3 "#+"#AA)" %) $3% &,"#3%-/ )( %4# #Z+"#AA,)( +-$A*,& ,( )"&#" %) "#+"#AA %"$(A3",+%,)( E")* $ 4/.",& +")*)%#"F  0-%4)B14 %4# G;:QM8 LH0> A/A%#* ?)"CA ?#-- E)" *)A% #Z+#",*#(%AD %4# -$3 +")*)%#" 3$( i-#$CDj *#$(,(1 %4$% $ -)? -#N#- )E #Z+"#AA,)( #Z,A%A #N#( ?,%4)B% %4# $&&,%,)( )E O>MUF M4,A ,A *)A%-/ $ +").-#* E)" %)Z,3 +")%#,( +")&B3%AD ?4,34 3$( +"#N#(% %4# 3B-%B"# E")* "#$34,(1 %4# &#A,"#& &#(A,%/ ?,%4,( $ "#$A)($.-# %,*#X E"$*#F `)" %4#A# 3$A#AD A)*# A%"$,(A 3$""/ $( $&&,%,)($- *#$AB"# )E 3)(%")- AB34 $A %4# +Y/A +-$A*,&D ?4,34 AB++"#AA#A .$A$- M8 #Z+"#AA,)(F M4# +Y/A +-$A*,& 3)(%$,(A $ 34-)"$*+4#(,3)- "#A,A%$(3# 3$AA#%%# E)" +)A,%,N# A#-#3%,)( $(& $ +7c0 )",1,( )E "#+-,3$%,)(D *$C,(1 ,% ,(3)*+$%,.-# ?,%4 )%4#" +7c0 +-$A*,&AF +Y/A 3)*#A ,( %?) avors—pLysS and pLysE—the difference being that the latter provides tighter control of basal expression.

ON#0 &I ? -1:W0 F.. !510.&: B].5.Y25.$$&1:P The strains described above should generate sufcient expression levels for most purposes, but what do you do

?4#( /)BlN# %",#& $ 3)**)( A%"$,( $(& &)(l% 1#% %4# &#A,"#& -#N#- 9)" $(/< +")%#,( #Z+"#AA,)(T Y)? #Z+"#AA,)( outcomes can result from variety of sources, so fear not—there are a few simple troubleshooting measures that

3$( 4#-+ 1#% /)B .$3C )( %"$3CK g

D1%2#0&H&"&0XK G)B.-#X34#3C /)B" +-$A*,& .$3C.)(# $(& #Z+"#AA,)( A%"$,( %) *$C# AB"# %4#/ $"#

3)*+$%,.-#F 0( $"$.,()A#X,(&B3,.-# +-$A*,& ?,-- ()% #Z+"#AA ,( $( O>MU ,(&B3%,)( A%"$,( E)" #Z$*+-#D ()" ?,-- $ +7c +-$A*,& .# 3)*+$%,.-# ?,%4 $ +Y/A A%"$,(F ^)B" A%"$,( *$/ "#[B,"# $&&,%,)($- $(%,.,)%,3 A#-#3%,)( )" $ A+#3,$- 1")?%4 *#&,$D )" ,E /)B" +-$A*,& ,A -)?X3)+/D 3)(A,&#" "#&B3,(1 %4# $(%,.,)%,3 3)(3#(%"$%,)(F g

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[5170N G.'&#K !4$(1,(1 *#&,$ ,A %",3C/D .#3$BA# %4#"# 3$( .# $ %"$&#X)EE .#%?##( 1")?%4 "$%# $(&

+")%#,( [B$-,%/F `)" *$(/ +")%#,(AD $ ",34 *#&,$ AB34 $A MJ )" 5t^M ,A )+%,*$- .#3$BA# )E %4# 4,14 3#--X&#(A,%/ %4#/ AB++)"%o 4)?#N#"D *,(,*$- *#&,$ AB++-#*#(%#& ?,%4 2g A$-%A *$/ .# +"#E#"$.-# ,E %4# +")%#,( +")&B3% ,A A#3"#%#& %) %4# *#&,B* )" ,E A-)? #Z+"#AA,)( ,A "#[B,"#& &B# %) A)-B.,-,%/ 3)(3#"(AF g

?:$1"4H". #:' F.65.0.' !510.&:$: The most common purication protocols are designed for soluble,

3/A%)A)-,3 +")%#,( +")&B3%AD .B% %4,A ,A ()% $-?$/A $34,#N$.-#F >")%#,(A ?4,34 3)(%$,( 4/&")+4).,3 regions or multiple disulde bonds may aggregate and become insoluble. These insoluble globs of misfolded protein are known as inclusion bodies, and can be recovered and puried using a A+#3,$+")%)3)-F 0-%#"($%,N#-/D "#&B3,(1 %4# 3)(3#(%"$%,)( )E ,(&B3#" )" $&&,(1 $( afnity tag such as GST *$/

4#-+ ?,%4 A)-B.,-,%/ ,AAB#AF

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D`,!A=3 L+ DBGGB@ DEB@?@[ A=D`@?iC=F

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B].5]&.7 S4#( 3-)(,(1 ./ "#A%",3%,)( &,1#A% $(& -,1$%,)(D /)B BA# "#A%",3%,)( #(W/*#A %) 3B% )+#( $ +-$A*,& 9.$3C.)(#< $(& ,(A#"% $ -,(#$" E"$1*#(% )E GH0 9,(A#"%< %4$% 4$A .##( 3B% ./ 3)*+$%,.-# "#A%",3%,)( #(W/*#AF 0( #(W/*#D GH0 -,1$A#D %4#( 3)N$-#(%-/ .,(&A %4# +-$A*,& %) %4# (#? E"$1*#(% %4#"#./ 1#(#"$%,(1 $ 3)*+-#%#D 3,"3B-$" +-$A*,& %4$% 3$( .# #$A,-/ *$,(%$,(#& ,( $ N$",#%/ )E .,)-)1,3$- A/A%#*AF

:O&/O8&E ;G 7%& /&C7/8078;* 0@;*8*) B/;0&CC? !;7% 7%& B@+C,8( R<@F&1 <+09<;*&S +*( 7%& VW' C&QF&*0& ;G 8*7&/&C7 R)/&&*1 8*C&/7S +/& 0F7 E87% /&C7/807 8;* &*Y",&C 7; )&*&/+7& 0;,B+78<@& ;O&/%+*)C 7%+7 +@@;E 7%&, 7; <8*( 7;)&7%&/? ^8)+C& 8C FC&( 7; ,+9& <;*(C <&7E&&* 7%& 8*C&/7 +*( <+09<;*& 0;O+@&*7?

J#E)"# .#1,((,(1 %4# "#A%",3%,)( &,1#A% $(& -,1$%,)( +")3#AAD /)B A4)B-& 3$"#EB--/ 34))A# /)B" .$3C.)(# $(& ,(A#"% X %4#A# .)%4 *BA% 4$N# 3)*+$%,.-# 3B% A,%#A E)" "#A%",3%,)( #(W/*#A %4$% $--)? /)B" ,(A#"% %) .# +-$3#& ,(%) %4# .$3C.)(# ,( %4# +")+#" )",#(%$%,)(F `)" ,(A%$(3#D ,E /)B ?#"# 3-)(,(1 $ 1#(# ,(%) $( #Z+"#AA,)( N#3%)"D /)B ?)B-& ?$(% %4# A%$"% )E %4# 1#(# %) #(& B+ @BA% &)?(A%"#$* )E %4# +")*)%#" E)B(& ,( %4# .$3C.)(#F O&#$--/D %4# .$3C.)(# ?,-- 3)(%$,( $ N$",#%/ )E "#A%",3%,)( #(W/*# 3B% A,%#A 9"#A%",3%,)( A,%#A< &)?(A%"#$* )E %4# +")*)%#" $A +$"% )E $ *B-%,+-# 3-)(,(1 A,%# 92!VL;VMID /)B 4$N# $ EB-- +-$A*,& "#$&/ %) .# BA#& ,( /)B" #Z+#",*#(%AF

V&7+8@&( ;O&/O8&E ;G 7%& /&C7/8078;* 0@;*8*) B/;0&CC? P&& 7&A7 G;/ (&7+8@C?

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3=FA3?DA?B@ DEB@?@[ ;DB@AW->  Alternatively, this whole process can be completed using a single enzyme if your insert is anked on both sides

./ %4$% #(W/*#lA "#A%",3%,)( A,%#AD .B% %4# ,(A#"% 3$( %4#( $((#$- %) %4# .$3C.)(# ,( #,%4#" $ E)"?$"& )" "#N#"A# )",#(%$%,)( A) /)Bl-- (##& A)*# ?$/ %) N#",E/ %4$% %4# ,(A#"% #(&#& B+ ,( %4# &,"#3%,)( /)B ?$(% X BAB$--/ ./ V$(1#" A#[B#(3,(1 )" EB"%4#" "#A%",3%,)( &,1#A%AF Of course there’s much more detail and verication required for the process t o work well, so let’s go over the

&#%$,-A A%#+X./XA%#+F

)8 -&9.$0&1: V#% B+ "#A%",3%,)( &,1#A%A E)" /)B" ,(A#"% 9)" &)()" +-$A*,&< $(& +-$A*,& .$3C.)(#F J#3$BA# /)B -)A# A)*# DNA during the gel purication step, it is important to digest plenty of starting material. We recommend 1.5-2 μg of insert and 1 μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible

.# 3B% ?,%4 .)%4 #(W/*#AD $(& %4#"#E)"# ,% ,A ,*+)"%$(% %4$% %4# &,1#A% 1) B(%,- 3)*+-#%,)(F M4# %,*# "#[B,"#& E)" 3)*+-#%# &,1#A%,)( N$",#A E)" &,EE#"#(% #(W/*#AF 2$(/ 3)*+$(,#A ()? A#-- E$A% &,1#A% #(W/*#A %4$% 3$( &,1#A% -$"1# $*)B(%A )E GH0 ,( $A -,%%-# $A 76 *,(B%#AD .B% 34#3C ?,%4 /)B" #(W/*#lA *$(BE$3%B"#" %) #(AB"# %4$% /)Bl"# 3B%%,(1 E)" %4# +")+#" &B"$%,)( $(& BA,(1 %4# +")+#" 3)(&,%,)(AF

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3=FA3?DA?B@ DEB@?@[ ;DB@AW-> 2. Isolate Your Insert and Vector by Gel Purication H)? %4$% /)BlN# 3B% /)B" ,(A#"% $(& N#3%)"D B(E)"%B($%#-/ /)B 3$(l% @BA% %4")? %4# &,1#A%,)( *,Z%B"#A %)1#%4#"F ^)B (##& %) ,A)-$%# /)B" ,(A#"% $(& .$3C.)(# E")* %4# #(W/*#A BA#& %) &,1#A% %4#* $A ?#-- $A $(/ +,#3#A 3B% )B% )" )EE )E %4#*F 0( #$A/ ?$/ %) &) %4,A ,A 1#purication. In gel purication, you use a voltage

&,EE#"#(3# $3")AA $ 1#- *$%",Z 9BAB$--/ $1$")A#< %) +B-- /)B" (#1$%,N#-/ 34$"1#& GH0 %4")B14 %4# 1#-F 0A indicated in the gure on the left, your digested DNA

9$(& B(&,1#A%#& 3)(%")-A< $"# -)$&#& $% %4# %)+ )E %4# 1#,( ?#--A +)A,%,)(#& %)?$"& %4# $()&# 9X 34$"1#
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3=FA3?DA?B@ DEB@?@[ ;DB@AW-> M4#A# A%$,(A "#[B,"# /)B %) #,%4#" A%$,( /)B" 1#- $E%#" /)B "B( /)B" A$*+-#A )" $&& %4# A%$,( $A %4# 1#- ,A .#,(1 *$&# 9+)A% )" +"# "B( ,( %4# %$.-# $.)N#D "#A+#3%,N#-/
)B% %4# .$(&AF J#3$BA# )E %4,A ?# "#3)**#(& %4$% /)B BA# $ ?,&# 1#- 3)*.D "B( %4# 1#- )( %4# A-)?#" A,&#D $(& AC,+ -$(#A .#%?##( A$*+-#AF O( $&&,%,)( %) $ GH0 -$&&#" A%$(&$"&D ,% ,A $-A) $ 1))& ,&#$ %) "B( $( B(3B% A$*+-# )E #$34 +-$A*,& %) 4#-+ ?,%4 %")B.-#A4))%,(1 ,E /)B" &,1#A%A &)(l% -))C $A #Z+#3%#&F Once you have cut out and puried your insert and recipient plasmid backbone bands away from the gel via your E$N)",%# gel purication *#%4)&D ,% ,A ,*+)"%$(% %) &#%#"*,(# %4# 3)(3#(%"$%,)( )E "#3)N#"#& GH0 $A %4,A ?,-- .#

BA#EB- E)" %4# -,1$%,)( A%#+F

<8 E&9#0. \145 ?:$.50 &:01 \145 Z.6015  In the ligation step, you mix your puried, cut backbone and insert in a single tube allowing the compatible

)N#"4$(1A 1#(#"$%#& ./ "#A%",3%,)( &,1#A%,)( %) $((#$- %) )(# $()%4#" $(& E)"* $ 3)*+-#%#D 3,"3B-$" +-$A*,&F ^)B %4#( $&& GH0 -,1$A# %) 3)N$-#(%-/ -,(C %4# E"$1*#(%A %)1#%4#" $% %4# #Z+#(A# )E 0M> 9A## .#-)?
S# "#3)**#(& $")B(& 766(1 )E %)%$- GH0 ,( $ A%$(&$"& -,1$%,)( "#$3%,)(F ^)B ,&#$--/ ?$(% $ i"#3,+,#(% +-$A*,& : insert ratio” of approximately 1:3. Since the number of base pairs for each varies, it is difcult to calculate this

.$A#& )( GH0 3)(3#(%"$%,)( $-)(#F I(# *#%4)& ,A %) 3)(&B3% 5 -,1$%,)(A E)" #$34 +-$A*,& /)B $"# %"/,(1 %) 3"#$%#D ?,%4 N$"/,(1 "$%,)A )E "#3,+,#(% +-$A*,& %) ,(A#"%F O% ,A 3",%,3$- %) A#% B+ $ (#1$%,N# 3)(%")- -,1$%,)( "#$3%,)( ?,%4 () ,(A#"% GH0 $&&#&F M4,A ?,-- $--)? /)B %) &#%#"*,(# 4)? *$(/ 3)-)(,#A /)B A4)B-& #Z+#3% ,( %4# %"$(AE)"*$%,)( &B# %) .$3C1")B(& "#X3,"3B-$",W$%,)( $(& 3)(%$*,($%,)( E")* B(3B% +-$A*,&F

R8 A5#:$I15%#0&1: M"$(AE)"* /)B" -,1$%,)( "#$3%,)( ,(%) /)B" .$3%#",$- A%"$,( )E 34),3#F `)--)? %4# *$(BE$3%B"#"lA ,(A%"B3%,)(A E)" /)B" 3)*+#%#(% 3#--AF For most standard cloning, you can transform 1-2 μl of your ligation reaction into competent cells such as

GRc$-+4$ )" MI>76F OE BA,(1 *B34 -#AA %)%$- GH0 9{7 (1< )" ,E /)B $"# 4$N,(1 %")B.-# 1#%%,(1 3)-)(,#AD /)B *,14% want to use higher competency cells. Additionally, if your nal product is going to be very large (>10kb) you

*,14% ?$(% %) BA# #-#3%")X3)*+#%#(% 3#--A ,(A%#$& )E %4# *)"# 3)**)( 34#*,3$--/X3)*+#%#(% 3#--AF  \f = >$1#

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3=FA3?DA?B@ DEB@?@[ ;DB@AW-> ^)B A4)B-& +#"E)"*D $% *,(,*B*D %?) %"$(AE)"*$%,)(A $E%#" $ -,1$%,)(K )8 !)(%")- %"$(AE)"*$%,)( 3)(%$,(,(1 %4# -,1$%,)( *,Z%B"# ?,%4 .$3C.)(# $-)(#o L8 M"$(AE)"*$%,)( 3)(%$,(,(1 -,1$%,)( *,Z%B"# ?,%4 ,(A#"% $(& .$3C.)(#F

V$*+-# "#AB-%A ,(&,3$%,N# )E AB33#AAEB- $(& B(AB33#AAEB- -,1$%,)(A $"# ,(&,3$%#& .#-)?F 0 AB33#AAEB- -,1$%,)( ?,-- 4$N# E#? 3)-)(,#A )( %4# .$3C.)(# $-)(# +-$%# $(& *$(/ 3)-)(,#A )( %4# .$3C.)(# v ,(A#"% +-$%# 9)" $% -#$A% *)"# 3)-)(,#A %4$( %4# .$3C.)(# $-)(# +-$%#
B(AB33#AAEB- 5 $(& : $.)N#
/)B $&& -,1$A#F OE /)B &) ()% A## $(/ 3)-)(,#AD /)B A4)B-& 3)(&B3% $ +)A,%,N# 3)(%")- %) #(AB"# %4$% /)B" %"$(AE)"*$%,)( ?)"C#&F ^)B A4)B-& $-A) N#",E/ %4$% /)B $"# +-$%,(1 )( %4# $++")+",$%# $(%,.,)%,3 $(& %"/ N$"/,(1 %4# i"#3,+,#(% +-$A*,& K ,(A#"% "$%,)j ,( %4# -,1$%,)( "#$3%,)(F

S8 !45&IX 0N. K&:&$N.' !"#$%&'

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3=FA3?DA?B@ DEB@?@[ ;DB@AW-> I(3# ,% -))CA -,C# /)B" -,1$%,)( 4$A ?)"C#&D /)B ?,-- (##& %) +,3C ,(&,N,&B$- .$3%#",$- 3)-)(,#A $(& 34#3C %4#* E)" AB33#AAEB- -,1$%,)(F >,3C :X76 3)-)(,#A &#+#(&,(1 )( %4# (B*.#" )E .$3C1")B(& 3)-)(,#A )( /)B" 3)(%")- +-$%# (the more background, the more colonies you will need to pick) and grow overnight cultures for DNA purication. The simplest purication you can do is a miniprep, but if you need larger quantities of DNA, you’ll need to do a midiprep or a maxiprep. In these purications, you generally lyse the bacteria; add chemicals to precipitate out the high molecular weight genomic DNA; lter the remaining plasmid DNA through a column that binds the plasmid DNA and lets other materials pass through; and, nally, selectively elute the plasmid DNA from the column using a particular buffer or water. See column manufacturers for more detail. Columnless purication

+")%)3)-A 3$( .# E)B(& 4#"#F

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Once your complete plasmid has been veried, you’re ready to get experimenting!

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 0&&1#(#lA +-$A*,&A $"# BA#& ?,%4 $ ?,&# N$",#%/ )E "#A%",3%,)( #(W/*#X.$A#& 3-)(,(1 *#%4)&AF ;$34 *#%4)& 4$A ,%A )?( +-BA#A $(& *,(BA#AD .B% U)-&#( U$%# 3-)(,(1 4$A .##( #A+#3,$--/ BA#EB- ?,%4,( .)%4 %4# A/(%4#%,3 .,)-)1/ $(& 1#()*# #(1,(##",(1  elds. We’ll walk you through how to apply this precise and easy-to-use system %) /)B" 3-)(,(1 #EE)"%AF Golden Gate cloning technology relies on Type IIS restriction enzymes, rst discovered in 1996. Type IIS

"#A%",3%,)( #(W/*#A $"# B(,[B# E")* i%"$&,%,)($-j "#A%",3%,)( #(W/*#A ,( %4$% %4#/ 3-#$N# )B%A,&# )E %4#," recognition sequence, creating four base anking overhangs. Since these overhangs are not part of the

"#3)1(,%,)( A#[B#(3#D %4#/ 3$( .# 3BA%)*,W#& %) &,"#3% $AA#*.-/ )E GH0 E"$1*#(%AF S4#( &#A,1(#& 3)""#3%-/D the recognition sites do not appear in t he nal construct, allowing for precise, scarless cloning.

M4# 3-)(,(1 A34#*# ,A $A E)--)?AK %4# 1#(# )E ,(%#"#A% ,A &#A,1(#& ?,%4 M/+# OOV A,%#A 9AB34 $A JA$O )" J.AO< %4$% $"# -)3$%#& )( %4# )B%A,&# )E %4# 3-#$N$1# A,%#F 0A $ "#AB-%D %4#A# A,%#A $"# #-,*,($%#& ./ &,1#A%,)(Q-,1$%,)( and do not appear in the nal construct. The destination vector contains sites with complementary overhangs that direct assembly of the nal ligation product. As shown below, a fragment with 5’ overhang TGGA and 3’

)N#"4$(1 M!!U 3$( .# -,1$%#& ,(%) $ N#3%)" 3)(%$,(,(1 %4)A# )N#"4$(1AF ;(%"/ GH0 )N#"4$(1A *$/ .# +"#A#(% in the original plasmid (Option 1) or added using PCR-based amplication (Option 2).

 c7 = >$1#

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[BE-=@ [,A= DEB@?@[ ;DB@AW-> ,']#:0#9.$ 1I [1"'.: [#0. D"1:&:9 U)-&#( U$%# 3-)(,(1 ,A )(# )E %4# #$A,#A% 3-)(,(1 *#%4)&A ,( %#"*A )E 4$(&AX)( %,*#D $A &,1#A%,)( $(& -,1$%,)( 3$( .# &)(# ,( )(# :6X*,(B%# "#$3%,)(F M4# &#A%,($%,)( N#3%)" $(& #(%"/ N#3%)"9A< $"# +-$3#& ,( $ A,(1-# %B.# 3)(%$,(,(1 %4# M/+# OOV #(W/*# $(& -,1$A#F 0-%4)B14 %4# )",1,($- &#A%,($%,)( N#3%)" v ,(A#"% *$/ A+)(%$(#)BA-/ "#X-,1$%#D %4,A %"$(A,#(% 3)(A%"B3% "#%$,(A EB(3%,)($- M/+# OOV A,%#A $(& ?,-- .# "#X&,1#A%#&F O( 3)(%"$A%D E)"*$%,)( )E %4# &#A,"#& -,1$%,)( +")&B3% ,A ,""#N#"A,.-# .#3$BA# %4,A 3)(A%"B3% &)#A ()% "#%$,( %4# #(W/*# "#3)1(,%,)( sites. As a result, the ligation process is close to 100% efcient. Another strength of Golden Gate cloning is

,%A A3$-$.,-,%/F b(,[B# \ .$A# )N#"4$(1A 3$( .# BA#& %) $AA#*.-# *B-%,+-# E"$1*#(%A X B+ % ) 76 E"$1*#(%A $"# 3)**)(-/ $AA#*.-#& ,( $ A,(1-# "#$3%,)(p M4#A# )N#"4$(1A A+#3,E/ %4# &#A,"#& )"&#" )E E"$1*#(%AD $(& %4# loss of enzyme recognition sites after ligation favors formation of the construct of interest. Although efciency

*$/ "#$A# ?,%4 $( ,(3"#$A#& (B*.#" )E E"$1*#(%AD )" %4# -,1$%,)( )E N#"/ A*$--QN#"/ -$"1# E"$1*#(%AD %4#A# +").-#*A 3$( .# )N#"3)*# ./ A3"##(,(1 $ 4,14#" (B*.#" )E +)%#(%,$- 3-)(#AF U)-&#( U$%# $AA#*.-/ 4$A $ E#? $&N$(%$1#A )N#" )%4#" 3-)(,(1 *#%4)&AF ;Z)(B3-#$A#X.$A#& *#%4)&A -,C# U,.A)( $AA#*.-/ "#[B,"# 56X\6 .+ )E 4)*)-)1/ $% %4# #(&A )E GH0 E"$1*#(%A %) A+#3,E/ $AA#*.-/ )"&#"D A) E"$1*#(%A ?,%4 cl )" :l A#[B#(3# 4)*)-)1/ 3$(()% .# $AA#*.-#& BA,(1 %4,A *#%4)&D .B% 3$( .# $AA#*.-#& ?,%4 U)-&#( U$%#F M4# +)+B-$" U$%#?$/ 3-)(,(1 A/A%#* +")&B3#A 3)(A%"B3%A ?,%4 $( $%%J "#3)*.,($%,)( A3$" #(3)&,(1 #,14% $*,() $3,&AD .B% U)-&#( U$%# $AA#*.-/ 3$( .# &#A,1(#& %) .# A3$"-#AAF U)-&#( U$%# $AA#*.-/ ,A $-A) -#AA #Z+#(A,N# %4$( *$(/ 3)**#"3,$- 3-)(,(1 *#%4)&AF

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[BE-=@ [,A= DEB@?@[ ;DB@AW-> [1"'.: [#0. #:' FX:0N.0&6 J&1"19X V/(%4#%,3 .,)-)1,A%A 4$N# -#N#"$1#& %4# +)?#" )E U)-&#( U$%# 3-)(,(1 ,(%) $ *)&B-$" 3-)(,(1 A%"$%#1/F V)*#X times referred to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efciently

$AA#*.-# B+ %) A,Z GH0 E"$1*#(%A $% $ %,*#F 0A ?,%4 $-- U)-&#( U$%#X.$A#& *#%4)&AD %4,A A/A%#* #Z+-),%A %4# $.,-,%/ )E M/+# OOV #(W/*#A %) 3B% )B%A,&# %4#," "#3)1(,%,)( A,%# $(& +#"*,%A GH0 E"$1*#(%A ?,%4 3)*+$%,.-# )N#"X hangs to be efciently assembled. Scientists can engineer unique enzyme recognition sites that ank their DNA

E"$1*#(% ,( $( ,(N#"A# )",#(%$%,)(F M4,A $--)?A E)" *B-%,+-# GH0 3)*+)(#(%A 9+")*)%#"AD 1#(#AD %#"*,($%)"AD #%3F< %) .# $AA#*.-#& ,( %4# 3)""#3% )"&#" ,( $ A,(1-# "#$3%,)(F `)" &#%$,-#& U)-&#( U$%# +")%)3)-AD 3)*+-#%# ?,%4 4#-+EB- %,+A $(& %",3CAD A## M4# V$,(A.B"/ Y$. ?#.A,%# )" ;(1-#" q 2$",--)(#%F

[1"'.: [#0. #:' [.:1%. =:9&:..5&:9 O( #$"-/ 5677D %4# J)1&$()N# $(& ])/%$A 1")B+A &#A3",.#& $ (#? U)-&#( U$%#X.$A#& %#34()-)1/ E)" 1#()*# #&,%,(1 ?4,34 $--)?#& E)" %4# )"&#"#& $AA#*.-/ )E *B-%,+-# GH0 E"$1*#(%A %) 3"#$%# M0Y #EE#3%)" (B3-#$A#AF M4#A# +-$A*,&A ?#"# &#A,1(#& %) B%,-,W# %4# JA$O $(& JA*JO M/+# OOV A,%#A AB34 %4$% 3BA%)* M0Y $""$/A 3)B-& be constructed quickly and efciently in just a few steps. More recently, CRISPR technology has adapted

U)-&#( U$%# 3-)(,(1 E)" ,(A#"%,(1 %4# $++")+",$%# )-,1)(B3-#)%,&#A A+#3,E/,(1 $ 1LH0 %$"1#% A#[B#(3# ,(%) $ !$AgX3)(%$,(,(1 +-$A*,& AB34 $A +t::6F M4,A 3-)(,(1 A%"$%#1/ ()% )(-/ *$C#A ,% #$A/ %) 3"#$%# $ A,(1-# 1LH0X#Z+"#AA,(1 +-$A*,&D .B% ,% 3$( $-A) .# $&$+$%#& %) #Z+"#AA *B-%,+-# 1LH0AF 0&&1#(# 4$A %?) U)-&#( U$%#X.$A#& 1LH0 $AA#*.-/ *#%4)&A  available, which allow you to efciently clone up to 7 gRNAs into one &#A%,($%,)( N#3%)"D *$C,(1 *B-%,+-#Z,(1 #$A/F

-&$#']#:0#9.$ 1I [1"'.: [#0. D"1:&:9 U)-&#( U$%# 3-)(,(1 ,A ()% 766x A#[B#(3#X,(&#+#(&#(%K %) $N),& B(&#A,"#& &,1#A%,)(D %4# M/+# OOV A,%# BA#& *BA% ()% .# +"#A#(% ?,%4,( %4# E"$1*#(%A /)B A##C %) $AA#*.-#F I(# ?$/ %) ?)"C $")B(& %4,A ,A %) i&)*#A%,3$%#j your fragment: PCR-based amplication can be used to create silent point mutations at internal recognition sites

%4BA #-,*,($%,(1 %4#A# E")* /)B" 1#(# )E ,(%#"#A%F >!L +")&B3%A $"# %4#( &,1#A%#& ?,%4 %4# M/+# OOV #(W/*#D $(& %4# *,Z%B"# ,A -,1$%#& E)--)?,(1 $ 4#$% ,($3%,N$%,)( A%#+F OE /)B" 1#(#A )E ,(%#"#A% )" &#A%,($%,)( N#3%)" 3)(%$,( *B-%,+-# ,(%#"($- "#A%",3%,)( A,%#A %4$% *$/ ()% .# $*#($.-# %) i&)*#A%,3$%,)(jD /)B *,14% ?$(% %) 3)(A,&#" BA,(1 $( $-%#"($%,N# *#%4)& -,C# U$%#?$/ 3-)(,(1 )" U,.A)( $AA#*.-/F 0()%4#" ,*+)"%$(% 3)(A,&#"$%,)( ,A %4# &#A,1( of anking overhangs. Although there are theoretically 256 distinct anking sequences, sequences that differ by

)(-/ )(# .$A# *$/ "#AB-% ,( B(,(%#(&#& -,1$%,)( +")&B3%AF S4#%4#" /)B $"# BA,(1 %4# U)-&#( U$%# *#%4)& %) 3"#$%# !LOV>LQ!$Ag 3)(A%"B3%AD $AA#*.-# A%$(&$"& +-$A*,&A +$"%A ,( &,EE#"#(% 3)*.,($%,)(AD )" )%4#" (#? $(& #Z3,%,(1 $++-,3$%,)(AD %4,A A/A%#* ,A $( ,(3"#&,.-/ powerful tool for cloning complicated constructs in a single, high-efciency step.

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[BE-=@ [,A= DEB@?@[ ;DB@AW-> Further Reading 1. Lee, Jae H., et al. “Sequential amplication of cloned DNA as tandem multimers using class-IIS "#A%",3%,)( #(W/*#AFj U#(#%,3 $($-/A,AK .,)*)-#3B-$" #(1,(##",(1 7:Fe 97ggeB.2#& >2OGK g778ffg8 2. ;(1-#"D !$")-$D L)*/ d$(&W,$D $(& V/-N#A%"# 2$",--)((#%F i0 )(# +)%D )(# A%#+D +"#3,A,)( 3-)(,(1 *#%4)& ?,%4 4,14 %4")B14+B% 3$+$.,-,%/Fj >-)V )(# :F77 9566fB.2#& >2OGK 7fgfc7c\ 8 3. Cermak, Tomas, et al. “Efcient design and assembly of custom TALEN and other TAL effector-based 3)(A%"B3%A E)" GH0 %$"1#%,(1Fj HB3-#,3 $3,&A "#A#$"34 95677B.2#& >2OGK 57\g:ef8 8 4. ;(1-#"D !$")-$D $(& V/-N#A%"# 2$",--)((#%F iU)-&#( 1$%# 3-)(,(1Fj GH0 !-)(,(1 $(& 0AA#*.-/ 2#%4)&A 9567\B.2#& >2OGK 5\:gc:e78

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AB!B DEB@?@[ !" ^8+**+ PE+*C;* - :07;<&/ 251 234M

M)+)A,)*#"$A# .$A#& 3-)(,(1 9MI>I 3-)(,(1< ,A $ GH0 3-)(,(1 *#%4)& %4$% &)#A ()% BA# "#A%",3%,)( #(W/*#A )" -,1$A#D $(& "#[B,"#A () +)A%X>!L +")3#&B"#AF V)B(&A #$A/ ",14%T M4# %#34(,[B# "#-,#A )( %4# .$A,3 $.,-,%/ )E 3)*+-#*#(%$"/ .$A#+$,"A $&#(,(# 90< $(& %4/*,(# 9M< %) 4/.",&,W# $(& E)"* 4/&")1#( .)(&AF M4,A +)A% E)3BA#A )( iA%,3C/ #(&j MI>I 9$-A) 3$--#& MI>IXM0< 3-)(,(1o 4)?#N#"D %4# MI>I 3-)(,(1 %#34(,[B# 4$A $-A) .##( $&$+%#& E)" .-B(% #(& 3-)(,(1F

F1 `17 -1.$ AB!B D"1:&:9 O15/P  0A ,--BA%"$%#& ,( %4# `,1B"# .#-)?D %4# i0j )N#"4$(1 )( %4# .-B# >!L +")&B3% ,(A#"% 3)*#A E")* BA,(1 M$[ polymerase for the amplication step since Taq polymerase leaves a single deoxyadenosine (A) at the 3’ ends

)E >!L +")&B3%AF M4# 3)*+-,*#(%$"/ iMj ,( %4# +$," 3)*#A E")* $ %)+),A)*#"$A# OX-,(#$",W#& .$3C.)(#F GH0 %)+),A)*#"$A# O 9&#+,3%#& $A $ 1"##( 3-)B&< EB(3%,)(A .)%4 $A $ "#A%",3%,)( #()(B3-#$A# $(& $A $ -,1$A# ./ 3-#$N,(1 $(& "#@),(,(1 AB+#"3),-#& GH0 #(&A %) E$3,-,%$%# "#+-,3$%,)(F The TOPO technique specically uses Vaccinia virus-

,A)-$%#& %)+),A)*#"$A# O $A %4,A #(W/*# "#3)1(,W#A %4# GH0 A#[B#(3# czX9!QM
&,EE#"#(% GH0 A%"$(& 3)*#A $-)(1D ,% 3$( $%%$3C %4,A 3)N$-#(% .)(& %4BA @),(,(1 %4# %?) GH0 A%"$(&A $(& "#-#$A,(1 %)+),A)*#"$A# 95I C,%A +")N,&# N#3%)"A )" 3-)(,(1 $"*A ?,%4 )N#"4$(1,(1 :z &#)Z/%4/*,&,(# 9M< "#A,&B#A %4$% $"# 3)N$-#(%-/ -,(C#& %) %)+),A)*#"$A#F ]#3%)"A ,( %4#A# C,%A $-A) )E%#( 4$N# %4# %)+),A)*#"$A# A,%# ,(A#"%#& ,(%) $ .#%$X1$-$3%)A,&$A# 3$AA#%%# $--)?,(1 $ "#A#$"34#" %) +#"E)"* .-B#X?4,%# A3"##(,(1 $E%#" %"$(AE)"*$%,)( X A#-E @),(,(1 )E %4# N#3%)" #(&A "#AB-%A ,( %4# +")&B3%,)( )E .-B# 3)-)(,#A %4$% &) ()% (##& %) .# +,3C#& $(& A#[B#(3#& E)" +)%#(%,$- +)A,%,N# 3-)(#AF I(3# /)B ,(%")&B3# /)B" :lX#(& i0j )N#"4$(1 ,(A#"%D %4# *$1,3 )E MI>I 3-)(,(1 4$++#(AF

J#$&6 !516.'45. Y#%lA ."#$C &)?( %4# A%#+A (##&#& E)" MI>I 3-)(,(1K 7F D5.#0. \145 !D3 !51'460K G#A,1( A%$(&$"& +",*#"A 9() (##& %) $&& B(,[B# "#A%",3%,)( A,%#A )( %4# #(&A< $(& $*+-,E/ /)B" A#[B#(3# )E ,(%#"#A% ?,%4 M$[ +)-/*#"$A# BA,(1 /)B" E$N)",%# >!L +")%)3)-F 5F F.0 C2 0N. AB!B D"1:&:9 3.#60&1:K 2,Z %)1#%4#" %4# >!L +")&B3% $(& MI>I ]#3%)"F :F ?:64H#0. S G&:40.$ #0 311% A.%2.5#045.K ^)B 3$( +-$3# /)B" "#$3%,)( )( ,3# ,E /)B $"# +-$((,(1 %) %"$(AE)"* ",14% $?$/ IL /)B 3$( A%)"# %4# "#$3%,)( $% X56 |! )N#"(,14%F \F A5#:$I15% AB!B D"1:&:9 3.#60&1: &:01 D1%2.0.:0 D.""$K ^)B 3$( BA# /)B" A%$(&$"& -$. +")%)3)- E)" %4,Ao cc = >$1#

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cF F.".60 #:' ,:#"Xe. )* ON&0. 15 E&9N0 J"4. D1"1:&.$: You can conrm the presence of your insert by PCR, "#A%",3%,)( &,1#A%D )" A#[B#(3,(1F

!51 A&2c r G) ()% $&& cl +4)A+4$%#A %) /)B" >!L +",*#"Ao /)B (##& %4$% E"## 4/&")Z/1")B+p r ^)B *$/ ?$(% %) ,(3-B&# #Z%"$ #Z%#(A,)( %,*# $E%#" %4# -$A% 3/3-# )E >!L %) *$C# AB"# %4$% %4# i0j 1#%A $&&#& %) $-- >!L +")&B3%AF r d##+ ,( *,(& %4$% M$[ +)-/*#"$A# 4$A $( #"")" "$%# )E $.)B% 7 ,( :Dc66 .$A#AF M/+,3$--/ +)-/*#"$A#A ?,%4 +"))E"#$&,(1 EB(3%,)($-,%/ $"# BA#& ,( +-$3# )E M$[ %) "#&B3# #"")" "$%#Ao 4)?#N#"D +"))E"#$&,(1 +)-/*#"$A#A ?,-- $-A) "#*)N# $-- B(+$,"#& :l #(&A ,( /)B" >!L +")&B3%F OE /)B (##& %) "#$A# #"")" "$%#D +-#$A# BA# )(# )E %4#A# *#%4)&A %) #(AB"# /)B" ,(A#"% "#%$,(A %4# :l 0 )N#"4$(1K bA# $ *,Z%B"# )E +"))E"#$&,(1 #(W/*# $(& M$[D ?,%4 M$[ BA#& ,( $( #Z3#AA "$%,) )E 76K7F ○ U#- +B",E/ /)B" >!L +")&B3% $(& ,(3B.$%# ,% ?,%4 .BEE#"D M$[ $(& &0M>A $% 85 |! E)" 76X ○ 7c *,(F r S4#( *,Z,(1 %4# >!L +")&B3% ?,%4 %4# MI>I N#3%)"D /)B *$/ ?$(% %) $&& #Z%"$ A$-% %) /)B" "#$3%,)(K M)+),A)*#"$A# O ,A "#-#$A#& E")* %4# N#3%)" ?4#( %4# >!L +")&B3% $(& N#3%)" -,1$%#o 4)?#N#"D ,% 3$( +)%#(%,$--/ "#.,(& $(& (,3C %4# (#?-/ -,1$%#& GH0F V$-% 4#-+A +"#N#(% %)+),A)*#"$A# O E")* "#.,(&,(1D ?4,34 "#AB-%A ,( *)"# ,(%$3% *)-#3B-#AF 9H)%# %4$% %4# $*)B(% )E A$-% /)B $&& ?,-- &#+#(& )( ?4#%4#" /)B $"# +-$((,(1 )( %"$(AE)"*,(1 /)B" "#$3%,)( ,(%) 34#*,3$--/ )" #-#3%")X3)*+#%#(% >? 0;@8  X #Z3#AA A$-% 3$BA#A $"3,(1 &B",(1 #-#3%")+)"$%,)( ?4,34 ?)B-& 3$BA# %4# #-#3%")+)"$%,)( %) E$,-
S4#( ,(3B.$%,(1 $% "))* %#*+#"$%B"#D ,% ,A ()% "#3)**#(&#& %4$% /)B #Z3##& %4# c *,(%B%#

time limit (lower transformation efciencies have been reported with longer i ncubation); however,

/)B *$/ (##& %) ,(3B.$%# E)" 56X:6 *,(B%#A ,E /)B" >!L +")&B3% ,A $% $ -)? 3)(3#(%"$%,)( )" /)B $"# 3-)(,(1 $( #Z%"#*#-/ -$"1# ,(A#"%F r V,(3# %4# A%$(&$"& -,1$%,)( "#$3%,)( ,A E$,"-/ [B,3CD *$C# AB"# /)B A%$/ )"1$(,W#& $(& +"#+$"# #N#"/%4,(1 /)B (##& E)" %4# (#Z% A%#+ .#E)"# +")3##&,(1F r >"#X?$"*,(1 /)B" $(%,.,)%,3X3)(%$,(,(1 +-$%# +",)" %) +-$%,(1 /)B" %"$(AE)"*$%,)( *$/ $--)? /)B %) A## 3)-)(,#A ?,%4,( f 4)B"AF

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AB!B DEB@?@[ ;DB@AW-> Further Reading 1.

V4B*$( VF iL#3)*.,($%,)( *#&,$%#& ./ N$33,(,$ N,"BA GH0 %)+),A)*#"$A# O ,( ;A34#",34,$ 3)-, ,A sequence specic.” Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10104-8. PubMed >2OGK 7ecf8geF >B.2#& !#(%"$- >2!OGK >2!c5f8eF 2. H)N#- $++")$34 %) *)-#3B-$" 3-)(,(1 $(& +)-/(B3-#)%,&# A/(%4#A,A BA,(1 N$33,(,$ GH0 %)+),A)*#"$A#F V4B*$( VF h J,)- !4#*F 7gg\ G#3 5:o5eg9c7B.2#& >2OGK 88gf58cF

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F=iC=@D= ,@- E?[,A?B@_?@-=!=@-=@A DEB@?@[ ;FE?D> !" .+/" g&+/8*)1 '(()&*& - P&B7&,<&/1 234= 

OE 3-)(,(1 *#%4)&A 4$& +#"A)($-,%,#AD VYO! 9A#[B#(3#X $(& -,1$%,)(X,(&#+#(&#(% 3-)(,(1< ?)B-& .# $ %"B# "#.#-F Not only does this system not use site-specic recombination, it also doesn’t require a ligation step! Based on %4# ").BA% A/A%#* )E 4)*)-)1)BA "#3)*.,($%,)( E)B(& ,( >? 0;@8 D VYO! ,A $ 34#$+D A%$(&$"&,W#&D $(& "$+,& *B-%,X

+$"% GH0 $AA#*.-/ *#%4)& X "#$& )( %) -#$"( 4)? %) BA# ,% ,( /)B" "#A#$"34F

AN. K&5$0 F0.2+ E&9#0&1:_?:'.2.:'.:0 D"1:&:9 Y,1$%,)(X,(&#+#(&#(% 3-)(,(1 9YO!<  was rst developed in the 1990s. While traditional restriction enzyme cloning BA#& A4)"% A%,3C/ #(&AD YO! #*+-)/#& %4# #Z)(B3-#$A# $3%,N,%/ )E M\ GH0 +)-/*#"$A# %) 3"#$%# -)(1#"D i34#?#&X .$3Cj )N#"4$(1A )E $.)B% 76X75 .$A#AF I(-/ )(# % /+# )E &HM> ?)B-& .# +"#A#(% ,( %4# "#$3%,)( *,ZD -,*,%,(1 the exonuclease activity to the rst occurrence of that nucleotide. At that position, T4 would perform the favored

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

, FE?D G#/.1].5+ F.U4.:6. #:' E&9#0&1:_?:'.2.:'.:0 D"1:&:9

.&+*C ;G FC8*) P^Ua 

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F=iC=@D= ,@- E?[,A?B@_?@-=!=@-=@A DEB@?@[ ;FE?D> ;DB@AW-> O( 5668D YO! "#3#,N#& $( ,*+)"%$(% B+&$%#D 3)B"%#A/ )E 0&&1#(# &#+)A,%)" V%#+4#( ;--#&1#F R,A (#? *#%4)&D ($*#& A#[B#(3# $(& -,1$%,)(X,(&#+#(&#(% 3-)(,(1 9VYO!? 0;@8 D $ ").BA% 4)*)-)1)BA "#3)*.,($%,)( A/A%#* $--)?A E)" %4# "#+$," )E 1$+A $(& )N#"4$(1A .$A#& )( "#1,)(A )E A#[B#(3# 4)*)-)1/F M4,A +")3#AA 3$( )33B" %4")B14 )(# )E %?) +$%4?$/AK L#30X*#&,$%#& "#3)*.,($%,)( )" L#30X,(&#+#(&#(% A,(1-#XA%"$(&#& $((#$-,(1F ;--#&1# "#$-,W#& %4$% 4# 3)B-& ,(%")&B3# ,*+#"E#3% i"#3)*.,($%,)( ,(%#"*#&,$%#Aj 1#(#"$%#& %4")B14 >!L $(& ,*+"#3,A# M\ exonuclease activity, rather than the carefully designed DNA fragments used in LIC (see gure on the previous

+$1#? 0;@8  would be able to “repair” the plasmid, generating recombinant DNA. Adding puried RecA to the +"#X%"$(AE)"*$%,)( ,(3B.$%,)( #(4$(3#A %4# "#+$," +")3#AAD $--)?,(1 VYO! %) .# BA#& ?,%4 N#"/ A*$-- $*)B(%A )E GH0 9#F1F : (1
AN. !5#60&6#"&0&.$ 1I J.&:9 FE?D To start the SLIC cloning process (see gure

)( %4# +"#N,)BA +$1#
cl $(& :l 4)*)-)1/ "#1,)(AF M4,A E"$1*#(% $(& -,(#$",W#& +-$A*,& $"# %4#( +$"%,$--/ &,1#A%#& BA,(1 M\ +)-/*#"$A# ,( %4# $.A#(3# )E &HM>AF O( %4# A#3)(& A%#+D %4# $&&,%,)( )E $ A,(1-# &HM> A%)+A %4# #Z)(B3-#$A# "#$3%,)(F M4#A# +")&B3%A $"# %4#( 3)*.,(#&D $((#$-#&D $(& %"$(AE)"*#& ,(%) >? 0;@8 F M) *$C# *$%%#"A #N#( #$A,#"D VYO! ,A $-A) 3)*+$%,.-# ?,%4 ,(3)*+-#%#-/ A/(%4#A,W#& 9,>!L< E"$1*#(%AF OE >!L &)#A ()% ,(3-B&# a nal extension step, many of the products

?,-- 4$N# A,(1-#XA%"$(&#& )N#"4$(1A &B# %) ,(3)*+-#%# #Z%#(A,)(D $(& %4#A# E"$1*#(%A 3$( ,(&B3# "#3)*.,($%,)(F 2,Z#& >!L 3$( $-A) .# BA#& %) 3"#$%# $( ,(A#"%F M?) >!L +")&B3%A $"# BA#& %) 1#(#"$%# $ %$"1#% 1#(# ?,%4 $ cl )" :l )N#"4$(1F S4#( %4# +")&B3%A $"# *,Z#& $(& $((#$-#&D 5cx )E %4# "#AB-%,(1 GH0 ?,-- 4$N# %?) A,(1-#XA%"$(&#& )N#"4$(1A %4$% 3$( ").BA%-/ A%,*B-$%# "#3)*.,($%,)(F

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F=iC=@D= ,@- E?[,A?B@_?@-=!=@-=@A DEB@?@[ ;FE?D> ;DB@AW-> SLIC is ideal for multicomponent assembly (see gure on previous page), as overlapping sequence homology species the order of multiple fragments, and the assembly is scarless. With 40 bp homology regions, a ve piece assembly reaction is highly efcient (~80%). Ten-fragment assembly can also be successful, but at a lower efciency (~20%).

`17 -1.$ FE?D D1%2#5. 01 B0N.5 D"1:&:9 G.0N1'$P VYO!lA -,*,%$%,)(A $",A# E")* ,%A &#+#(&#(3# )( A,(1-#XA%"$(&#& )N#"4$(1AF M4#A# )N#"4$(1A *BA% .# $33#AA,.-# %) $--)? E)" 3)*+-#*#(%$"/ .$A# +$,",(1D A) VYO! 3$(l% .# BA#& ,E %4# )N#"4$(1A ?)B-& 4$N# A%$.-# AAGH0 A#3)(&$"/ A%"B3%B"#F I(# 3)**)( #Z$*+-# ,A %4# A%#*X-))+ A%"B3%B"# )E %"$(A3",+%,)($- %#"*,($%)"AF  0()%4#" +)%#(%,$- ,AAB# ,A A#[B#(3# A,*,-$",%/F OE E"$1*#(%A ,( $ *B-%,3)*+)(#(% $AA#*.-/ 4$N# cl )" :l A#[B#(3# 4)*)-)1/ %) #$34 )%4#"D %4#/ *$/ .# $AA#*.-#& ,(3)""#3%-/F M) )N#"3)*# %4,A -,*,%$%,)(D )(# )+%,)( ,A %) +#"E)"* $ 4,#"$"34,3$- $AA#*.-/D $AA#*.-,(1 E"$1*#(%A ,( *B-%,+-# A%#+A %) $N),& BA,(1 *B-%,+-# E"$1*#(%A %4$% A4$"# 4)*)-)1/ ,( %4# A$*# "#$3%,)(F O( %4#A# 3$A#AD %4# BA# )E $()%4#" 3-)(,(1 *#%4)&D AB34 $A U)-&#( U$%# assembly, may also be benecial.

VYO! ,A *)A% )E%#( 3)*+$"#& %) U,.A)( $AA#*.-/D $()%4#" 3-)(,(1 *#%4)& .$A#& )( 4)*)-)1)BA "#3)*.,($%,)(F O(A%#$& )E BA,(1 M\ GH0 +)-/*#"$A#D UO.A)( $AA#*.-/ "#[B,"#A Mc #Z)(B3-#$A# ,( 3)*.,($%,)( ?,%4 >4BA,)( +)-/*#"$A# $(& GH0 -,1$A#F M4,A "#$3%,)( %$C#A +-$3# ,( )(# A%#+ "$%4#" %4$( %?) A%#+A "#[B,"#& E)" VYO!D $(& ligase may improve the efciency of multipart assembly. The higher temperature at which Gibson assembly

%$C#A +-$3# *$/ $-A) -,*,% E)"*$%,)( )E A#3)(&$"/ A%"B3%B"#A $% %4# #(&A )E E"$1*#(%AF M4# *$@)" $&N$(%$1# )E VYO! )N#" U,.A)( $AA#*.-/ ,A 3)A%D $A M\ +)-/*#"$A# ,A *B34 -#AA #Z+#(A,N# %4$( %4# #(W/*#A "#[B,"#& E)" U,.A)( $AA#*.-/F VYO! ,A $ A%$(&$"&,W#& *#%4)& E)" *B-%,XE"$1*#(% GH0 $AA#*.-/D $(& ,%A -)? 3)A% *$C#A ,% ,&#$- E)" "#A#$"34#"A doing large amounts of cloning. Assembly is scarless, unlike Gateway cloning, and the method’s exibility allows ,% %) .# BA#& ?,%4 &,EE#"#(% %/+#A )E >!LX1#(#"$%#& ,(A#"%AF J/ 4$"(#AA,(1 %4# +)?#" )E GH0 "#+$," ,( >? 0;@8 D /)B can assemble multiple fragments without the need for specic restriction sites or DNA ligase!

Further Reading 1. Y,D 2$*,# yFD $(& V%#+4#( hF ;--#&1#F iR$"(#AA,(1 4)*)-)1)BA "#3)*.,($%,)( ,( N,%") %) 1#(#"$%# "#3)*.,($(% GH0 N,$ VYO!Fj H$%B"# *#%4)&A \F: 95668B.2#& >2OGK 785g:fef 8 2. Y,D 2$*,# yFD $(& V%#+4#( hF ;--#&1#F iVYO!K $ *#%4)& E)" A#[B#(3#X$(& -,1$%,)(X,(&#+#(&#(% 3-)(,(1Fj U#(# V/(%4#A,AK 2#%4)&A $(& >")%)3)-A 95675B.2#& >2OGK 55:5f\5cF

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DD-J_A`= ABh?D f=\ AB =KK?D?=@A DEB@?@[ !" .80%+&@ g? ^&,8&FA - V&0&,<&/ k1 234M

OE /)Bl"# ,(%) 3-)(,(1D /)Bl"# +").$.-/ $?$"# %4$% %4#"# $"# A#N#"$- *#%4)&)-)1,#A 3B""#(%-/ $N$,-$.-# E)" $++")$34,(1 ,%F M4#A# ,(3-B&# %4# %"$&,%,)($- "#A%",3%,)( #(W/*#Q-,1$A#X*#&,$%#& *#%4)&D %4# *)"# "#3#(%-/ &#N#-)+#& U,.A)( 0AA#*.-/ !-)(,(1 $(& U$%#?$/} 3-)(,(1 %#34()-)1,#AD $A ?#-- $A A#N#"$- )%4#"AF ;$34 method is unique and relies on specic components that are key to the cloning reaction. Understanding the specic components is essential for choosing the correct cloning method for your own experiments, and here we ?,-- E)3BA )( $ B(,[B# 1#(# %4$% *$C#A %4# +)+B-$" U$%#?$/ M2 *#%4)& +)AA,.-#K 00(!F JB% ?4$% ,A 00(!D ?4$% role does it play in modern cloning, and why should you learn more about it? Read on to nd out how00(! 3$(

*$C# /)B" 3-)(,(1 #Z+#",*#(%A $ -,%%-# #$A,#"F One of the most time-consuming aspects of traditional cloning is the identication of clones that actually contain /)B" ,(A#"% )E ,(%#"#A%F V,*+-/ A%$%#&D 00(! *$C#A 3-)(,(1 #$A,#" ./ A#-#3%,(1 $1$,(A% N#3%)"A %4$% &,& ()% %$C# B+ /)B" ,(A#"%F JB% #Z$3%-/ 4)? &)#A 00(! $33)*+-,A4 %4,AT Y#%lA A%$"% ?,%4 $ .",#E 4,A%)"/ )E %4# 1#(# $(& 4)?

*)-#3B-$" .,)-)1,A%A 4$N# 4$"(#AA#& ,% %) #N)-N# 3-)(,(1 %#34()-)1/F

, !10.:0 A1Y&:888 M4# 00(! 1#(#D -)3$%#& )( %4# ` A#Z E$3%)" +-$A*,& )E >? 0;@8 D ,A +$"% )E $ %)Z,(X$(%,%)Z,( A/A%#* #(3)&#& ./ %4# 33& )+#")(D ?4,34 ,A "#A+)(A,.-# E)" +-$A*,& *$,(%#($(3# &B",(1 3#-- &,N,A,)(F 00(! 3)&#A E)" %4# %)Z,3 +")%#,( 9!3&J< %4$% $3%A $A $ GH0 1/"$A# +),A)(D -)3C,(1 B+ GH0 1/"$A# ?,%4 .")C#( &)B.-# A%"$(&#& GH0 $(& B-%,*$%#-/ 3$BA,(1 3#-- &#$%4F 00('D $()%4#" 1#(# E)B(& ,( %4# 33& )+#")(D 3)&#A E)" %4# $(%,%)Z,( +")%#,( 9!3&0< %4$% +")%#3%A %4# 3#-- $1$,(A% %4# %)Z,3 !3&JF !#--A %4$% -)A# 00(' %4")B14 %4# -)AA )E %4# ` +-$A*,&D AB33B*. %) %4# %)Z,3,%/ )E !3&JF

J.61%.$ # !17.5I4" D"1:&:9 A11" Molecular biologists rst saw the potential of this system for enhancing cloning efciency about 20 years ago and

&#N#-)+#& 3-)(,(1 N#3%)"A %) 4$"(#AA ,%F M4#A# N#3%)"AD 3$--#& +dOY7f $(& +dOY7gD 3)(%$,(#& %4# 00(! 1#(# ,( U,+)& G/;, !&/*+/(1 6?1 &7 +@? C%;E8*) 7%& BKU^4kc4` O&07;/C +*( E"$*# ?,%4 $( 2!VF >? 0;@8  %4$% ?#"# %"$(AE)"*#& ?,%4 (&,;*C7/+78*) 7%& 0;*0&B7 ;G /&C7/8078;* &*Y",&T,&(8+7&( (8C/FB78;*C ;G %4# #*+%/ N#3%)"A #Z+"#AA#& %4# 00(! 1#(# $(& ?#"# ccdB, leading to positive identication of desired clones. %4#"#E)"# B($.-# %) +")+$1$%# .#3$BA# !3&0 ?$A(l% $N$,-$.-# %) 3)B(%#"$3% %4# %)Z,(F OED 4)?#N#"D $( ,(N#A%,1$%)" ?#"# %) AB33#AAEB--/ 3-)(# $( ,(A#"% ,(%) %4# N#3%)"D %4# 00(! "#$&,(1 E"$*# ?)B-& .# &,A"B+%#& $--)?,(1 3#--A #Z+"#AA,(1 %4# "#3)*.,($(% +-$A*,& %) +")+$1$%#F  0(/ 3#--A %4$% 3)(%$,(#& ()(X"#3)*.,($(% N#3%)"A 9"#X-,1$%#& #*+%/ N#3%)"AD E)" ,(A%$(3#< ?)B-& A%,-- #Z+"#AA 00(! $(& %4#"#E)"# ?)B-& &,#F M4,A +")3#&B"# &"$*$%,3$--/ "#&B3#A %4# (B*.#" )E 3-)(#A %4$% &) ()% 3)(%$,( %4# recombinant plasmid and therefore makes the cloning process much more efcient, as one does not have to

%4)")B14-/ A3"##( 3)-)(,#A E)" %4# ,(A#"%F U$%#?$/} %#34()-)1/ 9&#N#-)+#& ./ O(N,%")1#(M2< ,A #AA#(%,$--/ $ *)"# *)&#"( N#"A,)( )E %4,A )-&#" A/A%#*D ?,%4 $&&#& $&N$(%$1#A %4$% $"# &,A3BAA#& ,( %4# A#3%,)( )( U$%#?$/ 3-)(,(1F `)3BA,(1 )( %4# 33&J $A+#3%D e7 = >$1#

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DD-J_A`= ABh?D f=\ AB =KK?D?=@A DEB@?@[ ;DB@AW-> Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briey, %4# N#3%)" i.$3C.)(#j ,( %4,A A/A%#* 3)(%$,(A 00(!F 0 AB33#AAEB- ,(A#"%,)( ?,-- 3)*+-#%#-/ "#+-$3# 33&J ?,%4 %4# investigator’s insert of interest. Hence correct clones are identied much more efciently, as those that do not

3)(%$,( %4# &#A,"#& ,(A#"% A4)B-& ()% 1")?F

U,+)& G/;, %77BXcc7&+0%@8*&?@C?%Ff8?+0?8@c52Mk2c7F7;/8+@Cc)+7&E+"c8*7/;(F078;*?%7,@ (&,;*C7/+78*) 7%& B/8*08B@& <&%8*( 7%& _CE+BB8*) ;F7l ;G 00(! E87% 7%& investigator’s gene of interest.

D6'J 3.$&$0#:0 D@ -).&  F05#&:$ D1%2".0. 0N. FX$0.% JB% 00(! $-)(# ,A(l% %4# )(-/ C#/ %) %4# A/A%#* _ 4)? 3$( )(# ?)"C ?,%4 $ +-$A*,&Q1#(# %4$% C,--A %4# 3#--A #Z+"#AA,(1 ,%T M4# $(A?#" -,#A ,( A+#3,$- A%"$,(A )E >? 0;@8  %4$% %)-#"$%# %4# #Z+"#AA,)( )E %4# %)Z,( 1#(#F I(# AB34 A%"$,( ,A GJ:F7D ?4,34 3)(%$,(A $ *B%$(% N#"A,)( )E GH0 1/"$A# 91/"0\e5< %4$% ,A "#A,A%$(% %) %4# %)Z,3 #EE#3%A )E !3&JF 0()%4#" 3)**#"3,$--/ $N$,-$.-#D !3&JX"#A,A%$(% A%"$,( ,A 0 0(! VB"N,N$-~ E")* O(N,%")1#(M2F bA,(1 #,%4#" GJ:F7 )" 00(! VB"N,N$-M2, one can efciently propagate and prep plasmids containing the 00(! 1#(#D ?4,34 3$( %4#( .# BA#& E)" &)?(A%"#$* 3-)(,(1 $++-,3$%,)(AF S4,-# %4#A# %?) A%"$,(A B-%,*$%#-/ +#"E)"* %4# A$*# EB(3%,)(D %4#"# ,A A)*# #N,&#(3# %4$% 00(! Survival™ can be more difcult to transform, depending upon the specic plasmids being used.

S# ?)B-& $-A) -,C# %) ()%# %4$% ?4,-# GJ:F7 $(& 00(! Survival strains have been developed specically with 00(!X3)(%$,(,(1 N#3%)"A ,( *,(&D $(/ +-$A*,& %4$% 3)(%$,(A %4# ` +-$A*,& 9`l A%"$,(A< ?,-- $-A) .# "#A,A%$(% %) !3&JD $A %4# ($%,N# 00(' ?,-- .# +"#A#(%F 2)A% 3)**)( 3-)(,(1 A%"$,(A )E >? 0;@8  &) ()% 3)(%$,( %4# ` +-$A*,& 9$(& $"# 3)(A,&#"#& `X$1#

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,''&0&1:#" D"1:&:9 3.$1456.$  0A 3-)(,(1 3)(%,(B#A %) #N)-N#D B(&#"A%$(&,(1 $-- )E %4# +-$/#"A .#3)*#A #N#" *)"# ,*+)"%$(% %) #(AB"# %4$% /)B 34))A# %4# *)A% $++")+",$%# *#%4)& E)" /)B" )?( #Z+#",*#(%AF >-#$A# 34#3C )B% 0&&1#(#lA !4))A,(1 $ 2)-#3B-$" !-)(,(1 M#34(,[B# +$1# $(& %4# 0&&1#(# J-)1 E)" $ N$",#%/ )E BA#EB- "#A)B"3#A $(& +")%)3)-Ap S# ,(N,%# E##&.$3C E")* $-- *#*.#"A )E %4# "#A#$"34 3)**B(,%/ %) 4#-+ BA &#N#-)+ %4#A# "#A)B"3#A EB"%4#"F `##E"## %) )EE#" /)B" 3)**#(%A $(& AB11#A%,)(AD $(& #N#( %) AB.*,% $ 1B#A% .-)1 #(%"/ )E /)B" )?(p

Further Reading 1. J#"($"&D >F i>)A,%,N# V#-#3%,)( )E L#3)*.,($(% GH0 ./ !3&JFj J,)%#34(,[B#AF 7gge 0B1o5795B.2#& >2OGK ffe5f7gF 2. J$4$AA,D ;2FD #% $-F i` +-$A*,& !3&J C,--#" +")%#,(K 33&J 1#(# *B%$(%A 3)&,(1 E)" ()(X3/%)%)Z,3 +")%#,(A ?4,34 "#%$,( %4#," "#1B-$%)"/ EB(3%,)(AFj 2)- 2,3").,)-F 7ggc 2$"o7c9eB.2#& >2OGK 8e5:ecgF 3. J#"($"&D >FD #% $-F i>)A,%,N#XA#-#3%,)( N#3%)"A BA,(1 %4# ` +-$A*,& 33&J C,--#" 1#(#jF U#(#F 7gg\ I3% 77o7\f97B.2#& >2OGK 8g5ef\7F 4. Bernard, P., et al. “The F plasmid CcdB protein induces efcient ATP-dependent DNA cleavage by 1/"$A#Fj h 2)- J,)-F 7gg: G#3 co5:\9:B.2#& >2OGK f5c\ecfF 5. J#"($"&D >F $(& !)B%B",#"D 2F i!#-- C,--,(1 ./ %4# ` +-$A*,& !3&J +")%#,( ,(N)-N#A +),A)(,(1 )E GH0X %)+),A)*#"$A# OO 3)*+-#Z#AFj h 2)- J,)-F 7gg5 0B1 co55e9:B.2#& >2OGK 7:5\:5\F 6. M$*D h;F $(& d-,(#D J!F i!)(%")- )E %4# 33& )+#")( ,( +-$A*,& `Fj h J$3%#",)-F 7gfg 2$/o7879cB.2#& >2OGK 5ec7:ggF >B.2#& !#(%"$- >2!OGK >2!56gg6fF

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S4#( E$3,(1 $ 3-)(,(1 +")@#3%D A3,#(%,A%A $"# () -)(1#" -,*,%#& %) %"$&,%,)($- "#A%",3%,)( #(W/*# 3-)(,(1F O(A%#$&D %4#/ 3$( 34))A# $ *)-#3B-$" 3-)(,(1 %#34(,[B# %4$% ?,-- ?)"C ?#-- ?,%4 $ 1,N#( A#% )E "#A)B"3#AD %,*#D $(& #Z+#",*#(%$- (##&AF V,(3# ,%A ,(N#(%,)( ,( %4# -$%# 7gg6AD U $%#?$/ 3-)(,(1 %#34()-)1/ 4$A .#3)*# N#"/ +)+B-$" as a rapid and highly efcient way to move DNA sequences into multiple vector systems. With the appropriate

#(%"/ $(& &#A%,($%,)( N#3%)"AD )(# 3$( BA# U$%#?$/ %) 3-)(# $ 1#(# )E ,(%#"#A% ,(%) $ N$",#%/ )E #Z+"#AA,)( A/A%#*AF d##+ "#$&,(1 %) -#$"( *)"# $.)B% %4# U$%#?$/ 3-)(,(1 *#%4)& $(& ,%A $&N$(%$1#AF

,: ?:051'460&1: 01 [#0.7#X A.6N:1"19X M4# U$%#?$/ 3-)(,(1 *#%4)&D &#N#-)+#& ./ O(N,%")1#(D ,A $( 8* O87/; N#"A,)( )E %4# ,(%#1"$%,)( $(& #Z3,A,)( "#3)*.,($%,)( "#$3%,)(A %4$% %$C# +-$3# ?4#( -$*.&$ +4$1# ,(E#3%A .$3%#",$F U* O8O;D %4#A# "#3)*.,($%,)( "#$3%,)(A $"# E$3,-,%$%#& ./ %4# "#3)*.,($%,)( )E $%%$34*#(% A,%#A E")* %4# +4$1# 9$%%>< $(& %4# .$3%#",$ 9$%%J $(& $%%J A,%#AD %4# +4$1# ,(%#1"$%#A ,(%) %4# .$3%#",$- 1#()*# anked by two new recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL

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[,A=O,\ DEB@?@[ ;DB@AW-> Gateway vectors contain modied versions of the att sites so that scientists can easily clone in their desired DNA

A#[B#(3#AF U$%#?$/ %#34()-)1/ "#-,#A )( %4# %?) "#$3%,)(A &#A3",.#& .#-)?K The BP Reaction takes place between the attB sites anking the insert and the attP sites of the donor vector.

M4,A "#$3%,)( ,A 3$%$-/W#& ./ %4# J> !-)($A# #(W/*# *,Z $(& 1#(#"$%#A %4# #(%"/ 3-)(# 3)(%$,(,(1 %4# GH0 )E interest anked by attL sites. As a byproduct of the reaction, t he 33&J 1#(# ,A #Z3,A#& E")* %4# &)()" N#3%)" F

Figure 2: The Gateway system adapts phage integration into the BP and LR reactions. The BP reaction creates an attL-anked entry clone. The LR /&+078;* 0/&+7&C +* &AB/&CC8;* 0@;*& E87% +@@ ;G 7%& 0;,B;*&*7C *&0&CC+/" G;/ )&*& &AB/&CC8;*?

M4# YL L#$3%,)( %$C#A +-$3# .#%?##( %4# $%%Y A,%#A )E %4# 1#(#"$%#& #(%"/ 3-)(# $(& %4# $%%L A,%#A )E %4# &#A%,($%,)( N#3%)"F M4,A "#$3%,)( ,A 3$%$-/W#& ./ %4# YL !-)($A# #(W/*# *,ZF 0A $ "#AB-%D $( #Z+"#AA,)( 3-)(# with the DNA of interest anked by attB sites is generated. As in the BP reaction, a DNA fragment containing the 00(! 1#(# ,A #Z3,A#& E")* %4# &#A%,($%,)( N#3%)"F

I(3# %4# J> $(&Q)" YL "#$3%,)(A $"# +#"E)"*#&D %4# (#Z% A%#+ ,A %) %"$(AE)"* 3)*+#%#(% >? 0;@8  3#--A $(& A#-#3% %4# +)A,%,N# 3-)(#AF M4# #(%"/ 3-)(# $(& &#A%,($%,)( N#3%)" 3$""/ &,EE#"#(% $(%,.,)%,3 "#A,A%$(3# *$"C#"A 9,(&,3$%#& 4#"# ./ +-$A*,& 3)-)"? 0;@8  strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The 00(! 1#(# ,A +"#A#(% ,( %4# &)()" N#3%)"A $(& %4# &#A%,($%,)( N#3%)"A +",)" %) "#3)*.,($%,)(D $(& ,% ,A #Z34$(1#& ?,%4 %4# 1#(# )E ,(%#"#A% &B",(1 %4# J> )" YL "#$3%,)(AF V,(3# %4# !3&J +")%#,( ,(4,.,%A %4# 1")?%4 )E !3&J A#(A,%,N# >? 0;@8  A%"$,(AD *)A% 3)-)(,#A A4)B-&  ec = >$1#

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`17 01 D"1:. C$&:9 [#0.7#X A.6N:1"19X M) .#%%#" B(&#"A%$(& %4# +")3#AAD ?#l-- ?$-C %4")B14 $( #Z$*+-# #Z+#",*#(% ?4#"# ?# *,14% BA# U$%#?$/ 3-)(,(1 %) 1#(#"$%# )B" &#A,"#& 3)(A%"B3%AK -#(%,N,"$- #Z+"#AA,)( )E %4# 4B*$( dL0V 1#(# ,( *$**$-,$( 3#--AF FA=! )+ [.:.5#0. #: =:05X D"1:. There are a few different ways to generate our desired entry clone - human KRAS anked by attL sites.

2#%4)& 0K "#3)*.,($%,)( )E $( $%%JX>!L +")&B3% )" +-$A*,& ?,%4 $( $%%> &)()" N#3%)"F O( %4,A 3$A#D ?# ?)B-& BA# >!L %) $&& $%%J A,%#A %) #,%4#" #(& )E %4# dL0V 3)&,(1 A#[B#(3#F OE /)B 34))A# %4,A A%"$%#1/D ,%lA ,*+)"%$(% %) ,(3-B&# %4# +")+#" +")%#,( #Z+"#AA,)( #-#*#(%A 9",.)A)*# "#3)1(,%,)( A#[B#(3#AD A%$"% 3)&)(D A%)+ 3)&)(AD "#$&,(1 E"$*# 3)(A,&#"$%,)(AD #%3
Figure 3: Method A to create an entry clone: recombination of an attB-anked PCR product with an attP-containing donor vector.

2#%4)& JK MI>IX3-)(,(1 )E %4# &#A,"#& ,(A#"% ,(%) $( $%%YX#(%"/XMI>I N#3%)"F MI>I 3-)(,(1 $&&A A4)"% #(&9A< %) E$3,-,%$%# 3-)(,(1 ,(%) $( $%%YX3)(%$,(,(1 #(%"/ N#3%)"F

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/)B" #Z+#",*#(%F M4,A 34),3# ?,-- &#+#(& )( $ (B*.#" )E E $3%)"AD -,C# /)B" )"1$(,A*D &#A,"#& #Z+"#AA,)( -#N#-D $(& #Z+#",*#(%$- +B"+)A#F `)" *$**$-,$( -#(%,N,"$- #Z+"#AA,)(D ?# 3)B-& BA# $ N#3%)" -,C# +Y#(%, !2] >B") G;VM 9?77fX7< )" %4# &)Z/3/3-,(#X,(&B3,.-# +YOt•\65F M4# 34)A#( $%%L &#A%,($%,)( N#3%)" ?,-"#3)*.,(# ?,%4 %4# $%%YX#(%"/ 3-)(# %) 3"#$%# %4# #Z+"#AA,)( 3-)(#F FA=! <+ =Y25.$$ X145 [.:. 1I ?:0.5.$0c

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,']#:0#9.$ 1I 0N. [#0.7#X D"1:&:9 G.0N1' Compatibility and exibility: Once you generate the entry clone with your DNA sequence of interest, you can

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L8)F/& MX g&*&/+78*) +* &AB/&CC8;* 0@;*&? $%& /&+078;* <&7E&&* 7%& &*7/" 0@;*& +*( (&C78*+78;* O&07;/ 0/&+7&C 7 E; B/;(F07CX 7%& (&C8/&( &AB/&CC8;* 0@;*& and a byproduct containing the ccdB gene. Since the ccdB product is toxic to the cell, Gateway cloning efciency can reach >99%.

V+##&K M4# U$%#?$/ A/A%#* #($.-#A %4# 1#(#"$%,)( )E %4# #Z+"#AA,)( 3)(A%"B3% ,( )(-/ 7 &$/D $A )++)A#& %) 5v &$/A ?,%4 %"$&,%,)($- "#A%",3%,)( $(& -,1$%,)( 3-)(,(1F O% ,A $-A) +)AA,.-# %) A#% B+ %4# J> $(& YL "#$3%,)(A ,( %4# A$*# %B.#D A+##&,(1 B+ %4# 3-)(,(1 )E %4# $%%JX>!L +")&B3%A &,"#3%-/ ,(%) &#A%,($%,)( N#3%)"AF M4# 3-)(,(1 process is simple - no restriction, ligation or gel purication steps are required!

2B-%,+-# E"$1*#(% 3-)(,(1K ^)B 3$( BA# U$%#?$/ 3-)(,(1 %) ,(A#"% *B-%,+-# GH0 E"$1*#(%A ,(%) *$(/ N#3%)"A at once in the same tube. You can clone up to 4 DNA fragments, in a specic order and orientation, in one

%B.#D ,(%) )(# U$%#?$/ N#3%)" %) +")&B3# %4# &#A,"#& #Z+"#AA,)( 3-)(#F M4,A ,A +)AA,.-# %4$(CA %) %4# U$%#?$/ vectors’ design. They have modied versions of the attB, P, L and R sites that recombine very specically and

&,"#3%,)($--/K $%%J7 A,%#A "#$3% )(-/ ?,%4 $%%>7 A,%#Ao $%%J5 )(-/ ?,%4 $%%>5D $%%Y7 )(-/ ?,%4 $%%L7o $%%Y5 )(-/ ?,%4 $%%L5D $(& A) )(F M$C# $ -))C $% A)*# )E %4# U$%#?$/ 2B-%,A,%# +-$A*,&A $N$,-$.-# $% 0&&1#(#D ,(3-B&,(1 %4# `"#? Y$. 2B-%,+-# Y#(%,N,"$- ;Z+"#AA,)( V/A%#*A 92BY;< d,% D %4# 2B-%,V,%# U$%#?$/ 3-)(,(1 C,%D $(& 2B-%,V,%# U$%#?$/ +-$A*,&AF !)(A%$(% "#$&,(1 E"$*#K S4#( /)B *)N# $ GH0 E"$1*#(% E")* )(# U$%#?$/ N#3%)" %) $()%4#"D %4# ,(A#"%#& GH0 E"$1*#(% A%$/A ,( E"$*#F High efciency: The positive (antibiotic) and negative (CcdB) selection markers used for Gateway Cloning can increase cloning efciency to >99%.

b(,N#"A$-,%/K 0-- %/+#A )E GH0 E"$1*#(%A *$/ .# 3-)(#&K >!L E"$1*#(%AD 3GH0 )" U#()*,3 GH0 $(& ,A $N$,-$.-# E)" $-- C,(& )E )"1$(,A*A E")* *$**$-A %) >? 0;@8 F 3.#'X 01 05X 140 [#0.7#X 6"1:&:9P 2$(/ A3,#(%,A%A $")B(& %4# ?)"-& 4$N# 1#(#"$%#& $(& &#+)A,%#& %4#,"

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Over the past decade, scientists have developed and ne tuned many different ways to clone DNA fragments

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The Gibson assembly technique was rst described by Dr. Daniel Gibson and colleagues at the J. Craig Venter

O(A%,%B%# ,( 566gF R#"# $% 0&&1#(# ?# $&&#& %4,A )+%,)( %) )B" &")+ &)?( *#(B )E 3)**)( 3-)(,(1 )+%,)(A ,( %4# &#+)A,% +")3#AA ,( 567\ .#3$BA# )E ,%A 1$,( ,( +)+B-$",%/F U,.A)( $AA#*.-/ ,A ?#-- C()?( E)" $--)?,(1 #$A/ $AA#*.-/ )E *B-%,+-# -,(#$" GH0 E"$1*#(%AD .B% 3$( $-A) .# BA#& ,( .$A,3 3-)(,(1 )E $( ,(A#"% ,(%) /)B" N#3%)" )E choice as shown in the gure below. To start, you need to have DNA fragments with regions of homology at their

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`17 ?0 O15/$ The required homology between neighboring fragments can be created via PCR amplication with primers that

3)(%$,( %4# $++")+",$%# 4)*)-)1)BA A#[B#(3#AF H;J "#3)**#(&A $( )N#"-$+ )E 7cX\6 .+D ?,%4 $ +",*#" *#-%,(1 %#*+#"$%B"# 1"#$%#" %4$( \f |!F J)%4 V($+1#(# $(& H;J 4$N# %))-A %4$% 4#-+ /)B &#A,1( +",*#"A E)" >!L amplication of fragments to incorporate such regions of homology. M4,A N,&#) 1,N#A $ 4#-+EB- &#*)(A%"$%,)( )E 4)? %) BA# V($+1#(#lA +")1"$* %) &#A,1( +",*#"A E)" U,.A)( 0AA#*.-/F `)" $ A,*+-# #Z$*+-# )E BA,(1 U,.A)( $AA#*.-/D ,*$1,(# %4$% /)B ?$(% %) ,(A#"% /)B" 1#(# )E ,(%#"#A% ,(%) $ N#3%)" ?,%4 $ -$"1# %$1 $% %4# HX%#"*,(BAD .B% /)B &)(l% 4$N# %4# %$1 $-"#$&/ ,(3-B&#& ,( %4# N#3%)" /)B ?$(% %) BA#F OE /)B ,(A#"%#& %4#A# %?) +,#3#A )E GH0 ./ "#A%",3%,)( #(W/*# 3-)(,(1D /)B ?)B-& 4$N# %) &) ,% ,( %?) A%#+AD $(& $ A3$" ?)B-& -,C#-/ "#*$,( .#%?##( %4# %?) E"$1*#(%AF R)?#N#"D ./ BA,(1 U,.A)( $AA#*.-/D /)B 3$( ,(A#"% .)%4 %4# 1#(# )E ,(%#"#A% $(& %4# %$1 A#[B#(3#A ,(%) %4# N#3%)" ,( )(# A%#+ ?,%4)B% A3$"A $A &#+,3%#& .#-)?F `,"A%D /)B (##& %) &#A,1( +",*#"A %) $*+-,E/ %4# %?) E"$1*#(%A ?4,-# $-A) ,(3-B&,(1 "#1,)(A )E 4)*)-)1/ %) %4# N#3%)" )" (#,14.)",(1 E"$1*#(%F M4#( /)B ?)B-& $*+-,E/ %4# E"$1*#(%A $(& N#3%)" ./ >!LD N#",E/ %4$% /)B 4$N# .$(&A )E %4# 3)""#3% A,W#D $(& +B",E/ %4# GH0 E"$1*#(%AF `,($--/D /)B A,*+-/ ,(3B.$%# %4#A# %4"## E"$1*#(%A %)1#%4#" $-)(1 ?,%4 %4# U,.A)( $AA#*.-/ *$A%#" *,Z E)" 7 4)B"D $(& %4#( %"$(AE)"* ,(%) 3)*+#%#(% 3#--AF M4# AB33#AA "$%# )E %4,A "#$3%,)( ,A %/+,3$--/ E$,"-/ 4,14D A) %4#"# ,A BAB$--/ () (##& %) A3"##( $ -$"1# $*)B(% )E 3)-)(,#AF ;Z3-B&,(1 %4# %,*# ,% %$C#A %) 1#% +",*#"AD /)B 3$( 4$N# /)B" 3)(A%"B3% *$&# ,( c &$/AF

I(# &"$?.$3C %) %4# U,.A)( $AA#*.-/ %#34(,[B# ,A %4$% %4# +")3#AA ?)"CA .#A% ?,%4 E"$1*#(%A )N#" 566 (B3-#)%,&#AF M4,A *$/ .# .#3$BA# %4# #Z)(B3-#$A# 3)B-& 34#? %4")B14 $( #(%,"# E"$1*#(% A4)"%#" %4$( 566 (B3-#)%,&#A .#E)"# %4# $((#$-,(1 $(& +)-/*#",W$%,)( A%#+A 3)B-& )33B"F V#3)(&-/D ,% &)#A ()% ?)"C ?#-- ,E %4# #(&A )E %4# E"$1*#(%A 4$N# A%$.-# A,(1-# A%"$(&#& GH0 A#3)(&$"/ A%"B3%B"#D AB34 $A $ 4$,"+,( )" $ A%#* -))+ 9$A *,14% .# $(%,3,+$%#& %) )33B" ?,%4,( $ %#"*,($%)" A#[B#(3#$1#

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[?JFB@ ,FF=GJE\ ;DB@AW-> U,.A)( $AA#*.-/ ,A 3)**)(-/ BA#& ,( A/(%4#%,3 .,)-)1/D *$,(-/ .#3$BA# )E %4# #$A# ,( $AA#*.-,(1 *B-%,+-# fragments in one step with no scar sequences remaining in the nal product. The long overlapping region

.#%?##( E"$1*#(%A $-A) .#%%#" #(AB"#A 3)""#3% $AA#*.-/ )"&#" )E %4# E"$1*#(%A $A 3)*+$"#& %) %4# A*$--#" )N#"-$++,(1 A#[B#(3#A -#E% ?,%4 $ "#A%",3%,)( &,1#A%F 0 A%B&/ ,( 567: E)B(& %4$% U,.A)( $AA#*.-/ ?$A )(# )E %4# *)A% 3)**)(-/ BA#& $AA#*.-/ *#%4)&A 9d$4- 567:
?$(% (#Z% %) ,%D A) %4,A &)#A(l% $--)? E)" *,Z $(& *$%34 )E *$(/ &,EE#"#(% E"$1*#(%AF I(# ?$/ $")B(& %4,A ,A %) BA# $ 3)*.,($%,)( )E A%$(&$"& A#[B#(3#A ?,%4 )N#"-$+ "#1,)(AD AB34 $A ,( %4# 3$A# )E 2IG0Y 9*)&B-$" )N#"-$+X &,"#3%#& $AA#*.-/ ?,%4 -,(C#"A< ?4,34 .",(1A *)&B-$",%/ %) )N#"-$+X&,"#3%#& *#%4)&A 9 !$A,(, 567\
[&H$1: ,$$.%H"X G..0$ D3?F!3 U,.A)( 3$( .# $&$+%#& %) *)"# 3)*+-,3$%#& 3-)(,(1 A34#*#AD AB34 $A %4)A# ?4#"# %4# N#3%)" %4$% /)B ?$(% %) BA# ,A N#"/ -$"1#D 4$A $ 4,14 U! 3)(%#(%D 3)(%$,(A $ -)% )E "#+#$%AXX$(/ )E ?4,34 3)B-& *$C# %4# >!L A%#+ difcult--OR there is no convenient restriction site for linearization. This is a perfect case for t he use of Gibson

$AA#*.-/ 3)*.,(#& ?,%4 %4# +)+B-$" !LOV>L %#34()-)1/ $(& ,A &#A3",.#& ,( $ "#3#(% +B.-,3$%,)( ./ %4# Y)3C#/ -$. 9S$(1D #% $-F 567c!L %) *$C# -,(#$",W#& N#3%)"D %4# Cas9 enzyme along with a specic gRNA was used to cleave the 22kb vector. When followed by the standard

U,.A)( $AA#*.-/ %#34(,[B#A &#A3",.#& $.)N#D %4,A "#AB-%#& ,( %4# &,"#3% $(& A#$*-#AA 3-)(,(1 ,(%) $ N#3%)" ?4#"# () )%4#" *#%4)&A ?#"# $N$,-$.-#F 0 A#3)(& #Z$*+-# )E BA,(1 U,.A)( 0AA#*.-/ $-)(1 ?,%4 !LOV>L ?$A $-A) &#A3",.#& "#3#(%-/ 9h,$(1 #% $-F 567c
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B0N.5 `1%1"19X J#$.' A.6N:1"19&.$ S#lN# &#A3",.#& V#[B#(3# $(& Y,1$%,)( O(&#+#(&#(% !-)(,(1 9VYO!< ,( $ +"#N,)BA A#3%,)(F 0-%4)B14 VYO! *$/ .# *)"# 3)A% #EE#3%,N#D U,.A)( $AA#*.-/ ,*+")N#A )( %?) $A+#3%A )E %4# VYO! *#%4)&AF `,"A%D ,% BA#A $ &#&,3$%#& cl #Z)(B3-#$A# ,(A%#$& )E BA,(1 %4# #Z)(B3-#$A# E#$%B"# )E M\ GH0 +)-/*#"$A#D ?4,34 *BA% .# 3)(%")--#& ./ %4# +"#A#(3# )" $.A#(3# )E &HM>AF V#3)(&-/D ,( U,.A)( $AA#*.-/ $ -,1$A# ,A $&&#& %) "#+$," %4# (,3CA ,( N,%")D ?4#"#$A ,( VYO! %4#A# 3)(A%"B3%A $"# "#+$,"#& 8* O8O;, which ends up being much less efcient. O( $&&,%,)( %) VYO! $(& U,.A)(D %4#"# $"# /#% *)"# 4)*)-)1/ .$A#& $AA#*.-/ *#%4)&A %4$% 4$N# .##( &#A3",.#&XX!>;! 93,"3B-$" +)-/*#"$A# #Z%#(A,)( 3-)(,(1< $(& VY,!; 9V#$*-#AA Y,1$%,)( !-)(,(1 ;Z%"$3%< %) ($*# %?) *)"#F Y,C#?,A#D %4#"# $"# A#N#"$- A%)"#X.)B14% 3-)(,(1 C,%A $N$,-$.-# ?4,34 $"# $-- .$A#& )( -)(1X )N#"-$++,(1 "#1,)(AD 4$N# () "#[B,"#*#(%A E)" "#A%",3%,)( #(W/*#AD $(& () A3$" A#[B#(3#A .#%?##( E"$1*#(%AF V)*# )E %4#A# +")&B3%A ,(3-B&#K • • • •

GeneArt® Seamless Cloning and Assembly (Thermo Fisher Scientic)

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[?JFB@ ,FF=GJE\ ;DB@AW->  Although these kits may come at a high price, their manufacturers tout great efciency and low time commitments. These kits also come with specic protocols, suggestions for ratios of product to insert, and tools

E)" +",*#" &#A,1(D A) ,%lA $-?$/A .#A% %) 34#3C ,( ?,%4 %4# ,(A%"B3%,)(A E")* %4# +$"%,3B-$" *$(BE$3%B"#" )E %4# C,% /)B ?,-- .# BA,(1F V)*# )E %4#A# +")&B3%A *$/ )EE#" $&N$(%$1#A %4$% U,.A)( $AA#*.-/ &)#A ()% AB34 $A increased efciency, shorter incubation times, or the ability to accommodate smaller fragments.

f:17 \145 D"1:&:9 G.0N1'$ M4#"# $"# %?) )%4#" 3)**)( 3-)(,(1 *#%4)&A %4$% $"# #$A/ %) 1#% *,Z#& B+ ?,%4 U,.A)( $AA#*.-/ 9%4#/ $-- A%$"% with a G!) but actually work by signicantly different methods. U)-&#( U$%# 3-)(,(1 &)#A "#AB-% ,( %4# A#$*-#AA  joining of fragments, but uses site specic restriction sites (Type IIS restriction endonucleases) to cleave DNA

)B%A,&# )E %4# "#3)1(,%,)( A#[B#(3#F M4,A "#[B,"#A %4$% %4# N#3%)"A $(& GH0 E"$1*#(%A 3)(%$,( %4#A# A,%#A $% %4# correct location and NOT in the middle of your insert. Gateway cloning utilizes λ integrase to catalyze directional cloning of DNA parts that are anked by orthogonal versions of the attB and attP sites recognized by the

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p

Further Reading 1. U,.A)( GUD ^)B(1 YD !4B$(1 L^D ]#(%#" h!D RB%34,A)( !0 :"&D V*,%4 RIF ;(W/*$%,3 $AA#*.-/ )E GH0 *)-#3B-#A B+ %) A#N#"$- 4B(&"#& C,-).$A#AF H$%F 2#%4)&A 566go eD :\:X:\cF >B.2#& >2OGK 7g:e:\gcF 2. S$(1 hSD S$(1 0D Y, dD S$(1 JD h,( VD L#,A#" 2D Y)3C#/ L`F !LOV>LQ!$Ag (B3-#$A# 3-#$N$1# 3)*.,(#& ?,%4 U,.A)( $AA#*.-/ E)" A#$*-#AA 3-)(,(1F J,)M#34(,[B#A 567co cfK7e7X786F >B.2#& >2OGK 5cfe7g5fF 3. h,$(1 SD y4$) tD U$.",#-, MD Y)B !D ;.#(A%#,( ^D y4B M`F !$AgX0AA,A%#& M$"1#%,(1 )E !R")*)A)*# A#1*#(%A !0M!R #($.-#A )(#XA%#+ %$"1#%#& 3-)(,(1 )E -$"1# 1#(# 3-BA%#"AF H$% !)**B(F 567co V#+ 7oeKf767F >B.2#& >2OGK 5e:5::c\F >B.2#& !#(%"$- >2!OGK >2!\ceg868F 4. d$4-D YF hF q ;(&/D GF 0 AB"N#/ )E #($.-,(1 %#34()-)1,#A ,( A/(%4#%,3 .,)-)1/F hF J,)-F ;(1F 567:o 8D 7:F >B.2#& >2OGK 5:ee:\\8F >B.2#& !#(%"$- >2!OGK >2!:ef\c7eF 5. !$A,(,D 0"%B")D #% $-F iI(#X+)% GH0 3)(A%"B3%,)( E)" A/(%4#%,3 .,)-)1/K %4# 2)&B-$" IN#"-$+XG,"#3%#&  0AA#*.-/ ?,%4 Y,(C#"A 92IG0Y< A%"$%#1/Fj HB3-#,3 $3,&A "#A#$"34 \5F7 9567\B.2#& >2OGK 5\7c:776F >B.2#& !#(%"$- >2!OGK >2!:f8\56fF

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3)B"A#D %4#A# *$**$-,$( N#3%)"A *BA% .# 3)*+$%,.-# ?,%4 %4# 3#-- %/+# %4#/ $"# %"$(AE#3%#& ,(%) _ $ .$3%#",$- )",1,( )E "#+-,3$%,)( 9ILO< ?,-- ()% $--)? E)" +-$A*,& "#+-,3$%,)( ,( *$**$-,$( 3#--AD E)" #Z$*+-#D $(& $ %)Z,( %4$% C,--A .$3%#",$ *$/ ()% 4$N# $(/ &,A3#"($.-# #EE#3% )( *$**$-,$( 3#--AF V) 4)? &) *$**$-,$( +-$A*,&A &,EE#" E")* %4#," .$3%#",$- 3)B(%#"+$"%AT R)? &)#A "#+-,3$%,)( )33B"T OA A#-#3%,)( (#3#AA$"/ E)" %"$(AE#3%#& 3#--AT

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b(-,C# .$3%#",$- %"$(AE)"*$%,)(D A3,#(%,A%A &) ()% iA#-#3%j E)" 3#--A %4$% 4$N# %$C#( B+ %4# +-$A*,& ,( %4# A$*# ?$/F V#-#3%,)( *#%4)&AD &#A3",.#& .#-)?D $"# %/+,3$--/ #*+-)/#& ?4#( 3"#$%,(1 A%$.-# 3#-- -,(#A $(& $"# ()% BA#& E)" 1#(#"$- +-$A*,& A#-#3%,)(F O(A%#$&D reporter genes are often employed to easily monitor transfection efciencies and expression levels in the

3#--AF O&#$--/D %4# 34)A#( "#+)"%#" ,A B(,[B# %) %4# 3#--D ,A #Z+"#AA#& E")* %4# +-$A*,&D $(& 3$( .# $AA$/#& 3)(N#(,#(%-/F 0 &,"#3% %#A% E)" /)B" 1#(# )E ,(%#"#A% *$/ .# $()%4#" *#%4)& %) $AA#AA %"$(AE#3%,)( AB33#AAF U`> ,A )E%#( BA#& $A $ "#+)"%#" F !-,3C 4#"# to skip ahead in this eBook to uorescent tagging.

A5#:$&.:0 A5#:$I.60&1: #:' 0N. ="4$&]. jG#%%#"&#: B3?k For many experiments, it is sufcient for the transfected plasmid to be expressed transiently. Since the DNA

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"$&#& )" &,-B%#& )N#" AB.A#[B#(% 1#(#"$%,)(AF M4,AD 4)?#N#"D *$/ ()% .# $ +").-#* &#+#(&,(1 )( %4# &B"$%,)( )" )%4#" +$"$*#%#"A )E /)B" #Z+#",*#(%F 2$**$-,$( 3#--A &)B.-# $% $ *B34 A-)?#" "$%# %4$( %4$% )E .$3%#",$ 9P5\ 4 NA 56 *,(D "#A+#3%,N#-/
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"#A+#3%,N#-/F M4# +"#A#(3# )E %4#A# N,"$- 3)*+)(#(%A 1"#$%-/ "#&B3#A %4# "$%# )E +-$A*,& &,-B%,)( .B% &)#A ()% guarantee 100% transfection efciency.

F0#H". A5#:$I.60&1:  0 A%$.-# %"$(AE#3%,)( ,A BA#& %) 3"#$%# $ +)+B-$%,)( )E 3#--A %4$% 4$N# EB--/ $(& AB33#AAEB--/ ,(3)"+)"$%#& E)"#,1( 1#(#%,3 *$%#",$- ,(%) %4#," 1#()*#AF b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

!1$&0&]. F.".60&1: &: G#%%#"&#: D.""$ M) $34,#N# A%$.-# %"$(AE#3%,)(D %4#"# A4)B-& .# $ A#-#3%,N# +"#AAB"# %) E)"3# 3#--A %) ,(3)"+)"$%# %4# +-$A*,& DNA into the genome. For the purposes of this post, we will dene positive selection as t he means of picking

B+ +)A,%,N# %"$,%A 9,F#F %4# +-$A*,& 3)(%$,(A $ 3$AA#%%# %4$% ?,-- *$C# 3#--A "#A,A%$(% %) $ %)Z,(
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A)*# E")* 3#--B-$" *LH0AF S4$% %4#/ 4$N# ,( 3)**)( ,A %4# $.,-,%/ %) A+$"C %"$(A-$%,)( ,(,%,$%,)( ,(&#+#(&#(% )E %4# cl 3$+F OL;V #-#*#(%A $"# N#"/ BA#EB- $(& 3)**)(-/ E)B(& ,( .,3,A%")(,3 N#3%)"Ao 4)?#N#"D %4#/ &) 4$N# A)*# &,A$&N$(%$1#AF M4#A# #-#*#(%A $"# [B,%# -$"1# 9c66Xe66 .+< $(& *$/ %$C# B+ +"#3,)BA A+$3# ,( N,"$- %"$(AE#" N#3%)"A ?,%4 -,*,%#& +$3C$1,(1 3$+$3,%/F 0&&,%,)($--/D ,% *$/ ()% .# E#$A,.-# %) #Z+"#AA *)"# %4$( %?) 1#(#A $% $ %,*# BA,(1 OL;V #-#*#(%AF `B"%4#"D A3,#(%,A%A 4$N# "#+)"%#& -)?#" #Z+"#AA,)( )E %4# &)?(A%"#$* 3,A%")( &B# %) factors such as the experimental cell type and the specic genes cloned into t he vector.:

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* (GSG) residues can be added to the 5’ end of the peptide to improve cleavage efciency.

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,'.:1_#$$16�.' Z&54$ ;,,Z>  0&#()X$AA)3,$%#& N,"BA )" 00] ,A $ A*$-- +$"N)N,"BA %4$% ,(E#3%A 4B*$(A $(& A)*# )%4#" +",*$%# A+#3,#AD .B% ,A ()% 3B""#(%-/ C()?( %) 3$BA# &,A#$A#F OE 00] ,A #Z3-BA,N#-/ BA#& %) ,(E#3% 4B*$( 3#--AD ,% A%$.-/ ,(%#1"$%#A at a specic chromosomal site as part of its lysogenic life cycle; however, the addition of a helper virus such as adenovirus or a helper plasmid containing the specic viral proteins E1A, E1B, E2A, E4, and VA will cause

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fF7e 95667B.2#& >2OGK 77c6ggcfF 4. Vandenberghe, Luk H., et al. “Efcient serotype-dependent release of functional vector into the culture *#&,B* &B",(1 $&#()X$AA)3,$%#& N,"BA *$(BE$3%B",(1Fj RB*$( 1#(# %4#"$+/ 57F76 95676B.2#& >2OGK 56e\g\8cF >B.2#& !#(%"$- >2!OGK >2!5gc85:8F 5. ]#-&?,@CD 2$"-)( LFD #% $-F iG#N#-)+*#(% $(& )+%,*,W$%,)( )E $ "#$-X%,*# [B$(%,%$%,N# >!LX.$A#& *#%4)& E)" %4# %,%"$%,)( )E 00]X5 N#3%)" A%)3CAFj2)-#3B-$" M4#"$+/ eF5 95665B.2#& >2OGK 75:\gf5eF 6. t,)(1D S#(@B(D $(& !)(A%$(3# !#+C)F iG,A%,(3% #Z+"#AA,)( +$%%#"(A )E 00]f N#3%)"A ?,%4 .")$&-/ $3%,N# +")*)%#"A E")* AB."#%,($- ,(@#3%,)(A )E (#)($%$- *)BA# #/#A $% %?) &,EE#"#(% $1#AFj L#%,($- G#1#(#"$%,N# G,A#$A#AF V+",(1#" O(%#"($%,)($- >B.-,A4,(1D 567eF c67Xc68F >B.2#& >2OGK 5e\58\c5F 7. Okada, Takashi. Efcient AAV Vector Production System: Towards Gene Therapy For Duchenne 2BA3B-$" G/A%")+4/F OHM;!R I+#( 033#AA >B.-,A4#"D 567:F O(%#34 S#.+$1#F 8. S)@()D 0&$* >FD ;",3 0F >,#"3#D $(& h#$( J#((#%%F iV##,(1 %4# -,14%Fj V3,#(3# %"$(A-$%,)($- *#&,3,(# cF78c 9567:B.2#& >2OGK 5:\e8ccgF

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YB*,(#A3#(% 9)" i-,14%X#*,%%,(1j< *)-#3B-#A $"# $ N#"/ BA#EB- 3-$AA )E %))-A %) A3,#(%,A%A .#3$BA# -,14% ,A A)*#%4,(1 %4$% ?# 3$( #$A,-/ &#%#3% $(& *#$AB"#F >")%#,(A %4$% 1,N# )EE -,14% ,(3-B&# uorescent proteinsD -,C# U"##( `-B)"#A3#(% >")%#,( 9U`>
&#",N#& E")* ,%< 3$*# %) .# BA#& $A $ "#A#$"34 %))- ,A +$"%,3B-$"-/ ,(%#"#A%,(1F 0-%4)B14 4B*$(A 4$N# C()?( $.)B% .,)-B*,(#A3#(% )"1$(,A*AD $(& %4#," 34$"$3%#",A%,3 "$&,$(3#D E)" A)*# %,*# _ there are records describing them dating back to the rst century  A.D. – the rst time anyone truly examined what caused this

1-)?,(1 +4#()*#()( ?$A(l% B(%,- *B34 -$%#"F O( %4# 7ge6AD IA$*B V4,*)*B"$ A%B&,#& %4# .,)-B*,(#A3#(% +")+#"%,#A observed in the crystal jellysh '&QF;/&+ O807;/8+ 9-#E%< ,( *)-#3B-$" &#%$,-F  '&QF;/&+ O807;/8+? '77/8
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glimmer, Shimomura collected many, many jellysh specimens from Puget Sound in the Pacic Ocean, off of the coast of Washington state. Using these samples, he was able to isolate two proteins from the jellysh’s photoorgans; the rst, which he called aequorin, gives off a faint blue

-,14% ?4#( ,% .,(&A 3$-3,B* ,)(AD $(& %4# A#3)(&D ?4,34 ?# ()? 3$-- U`>D $.A)".A %4$% .-B# -,14% $(& 1-)?A 1"##(F O( %4# -$%# 7gf6AD $()%4#" "#A#$"34#"D G)B1-$A >"$A4#"D 1)% %4# ,&#$ %4$% %4,A (#? 1-)?,(1 1"##( +")%#,( 3)B-& .# BA#& %) *#$AB"# 1#(# %"$(A3",+%,)( $A ?#-- $A %) %"$3C +")%#,( -)3$-,W$%,)(F 0(& $A ,% %B"(A )B%D 4# ?$A ",14%p R# .#1$( %) A%B&/ %4# '? O807;/8+ 1#(# "#A+)(A,.-# E)" #(3)&,(1 U`>D $(&D ,( 7gg5D 4# "#+)"%#& ,%A A#[B#(3#F Soon after that, in 1994, Prasher’s collaborator Martin Chale expressed GFP in exogenous organisms (>? 0;@8 D $(& -$%#" a? &@&)+*C) for the rst time. It was then that scientists began to truly realize the potential of GFP

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Bioluminescence and uorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellysh for millions of years; however, it took until the 1960s for scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its uorescent derivatives are a staple in the lab. GFP

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ONX [5..: K"415.$6.:0 !510.&:P U`> ,A $ P58 CG$ +")%#,( 3)(A,A%,(1 )E 5:f $*,() $3,&A &#",N#& from the crystal jellysh '&QF;/&+ O807;/8+. It has a uorescent

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The protein structure, rst reported in 1996, is an eleven β-sheet-

3)(%$,(,(1 i.$""#-j A4$+#D ?,%4 %4# 34")*)+4)"# 3)(3#$-#& $% %4# 3#(%#" )E %4# A%"B3%B"#D A4,#-&#& E")* [B#(34,(1 ./ $[B#)BA A)-N#(%F M4,A %,14%-/X +$3C#& A%"B3%B"# #Z+-$,(A %4# ,*+)"%$(3# )E %4# #(%,"# U`> +")%#,(D ?4,34 is almost completely required to maintain uorescent activity; very little

%"B(3$%,)( ,A %)-#"$%#&D 4)?#N#"D +),(% *B%$%,)(A $"# $33#+%$.-#F U`>lA main advantage over conventional uorescent dyes of the time was the

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 0-*)A% $A A))( $A ,%A A#[B#(3# ?$A #-B3,&$%#&D A3,#(%,A%A .#1$( +00&CC&(X =c4=c234I? 6%;7; +F7%;/X .+CF/? #(1,(##",(1 (#? N#"A,)(A )E U`> %4")B14 *B%$1#(#A,A ,( )"&#" %) ,*+")N# ,%A +4/A,3$- $(& .,)34#*,3$- +")+#"%,#AF O( 7ggcD L)1#" ^F MA,#( &#A3",.#& $( VecM +),(% *B%$%,)( %4$% ,(3"#$A#& the uorescence intensity and photostability of GFP. This also shifted its major excitation peak from 395 nm to 488 nm, effectively ameliorating the deciencies found in the wildtype protein and facilitating its widespread use in research. Many other mutations have since been introduced to GFP and new iterations of uorophores are

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Table 1: The Specic Mutations Comprising Common Fluorophores K"415.$6.:0 !510.&:

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M.%,*% 7)"% that many uorescent proteins found on the red side of the spectrum are not GFP derivatives, but $"# ,(A%#$& "#-$%#& %) %4# &AL#& +")%#,( ,A)-$%#& E")* V8C0;C;,+ CBF V,*,-$" ?)"C 4$A .##( &)(# %) #Z+$(& %4# red-uorescent protein repertoire; however, these proteins are unique from GFP and the mutation denitions

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Table 2: Functional Role of Specic Mutations in GFP Derivatives G40#0&1:

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f:17: K4:60&1: Increased uorescence, photostability, and a shift of the major excitation peak to 488 nm Increased folding efciency at 37C

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J&1$.:$15$: A wide array of GFP-based uorescent biosensors has been designed to detect a

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K"415.$6.:6._,60&]#0.' D."" F150&:9 ;K,DF>: This is a type of ow cytometry that separates mixtures of cells into distinct populations based on uorescent signal. Thus, FACS can be used to separate cells

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FPs Classied by the Emission Color (Emission Wavelength Range) FPs are usually classied by emission color as outlined below (or emission wavelength range). By mutating GFP, %4# N$",$(%A H"4. K! 9J`>< ?#"# &#",N#&F 2$(/ )E %4# 15#:9. K!$ $(& %4# 3.' K!$ E")* ?4,34 %4#/ ?#"# &#",N#& 3)*# E")* %4# 3)"$- V8C0;C;,+ CBF D1"15

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;$34 `> 4$A ,%A B(,[B# #ZQ#* +#$CF M4#"#E)"#D 34))A# `>A %4$% /)B" A/A%#* 3$( #Z3,%#D $(& &#%#3% %4# #*,AA,)(F `)" #Z$*+-#D ,E /)B" *,3")A3)+# 4$A )(-/ %?) -$A#"AD $% \ff(* $(& ce7(*D you will not be able to use far-red FPs. If you do not have a lter that will pass blue light to the

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S8 A.%2.5#045.: FPs maturation times and uorescent intensity can be affected by the temperature. For ,(A%$(3#D #(4$(3#& U`> 9;U`>< ?$A )+%,*,W#& E)" :8s!D $(& ,A %4#"#E)"# *)A% AB,%#& E)" *$**$-,$( )" .$3%#",$ A%B&,#AD ?4#"#$A U`> VecM ,A .#%%#" AB,%#& E)" /#$A% A%B&,#A 95\X:6s!
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Q8 2` F0#H&"&0XK M4,A +$"$*#%#" ,A ,*+)"%$(% ,E /)B $"# +-$((,(1 %) #Z+"#AA %4# `> ,( $3,&,3 #(N,")(*#(%A 9#F1F /#$A% 3/%)A)-D ?4,34 ,A A-,14%-/ $3,&,3D )" A/($+%,3 N#A,3-#AA 4$N# &,EE#"#(% #ZQ#* A+#3%"$ (e.g. mKeima) or change uorescent intensity upon pH changes (e.g. pHluorin, pHTomato).

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Further Reading 1. `-B)"#A3#(% +")%#,(A $A .,)*$"C#"A $(& J,)A#(A)"AK M4")?,(1 3)-)" -,14%A )( *)-#3B-$" $(& 3#--B-$" +")3#AA#AF V%#+$(#C) #%F $-F 9566f" ;7&8*? 6&B7? P08 F g9\B.2#& >2OGK 7feg775\F 2. `-B)"#A3#(% +")%#,(A $(& %4#," $++-,3$%,)(A ,( ,*$1,(1 -,N,(1 3#--A $(& %,AAB#AF !4B&$C)N #%F $-F 956764/A,)-F L#NF g6K776:F >B.2#& >2OGK 56ee\6f6F 3. Modern uorescent proteins and imaging technologies to study gene expression, nuclear localization, $(& &/($*,3AF SB #%F $-F 95677B.2#& >2OGK 575\568fF 4. `> 1B,&# $% 0&&1#(# 5. `-B)"#A3#(3# V+#3%"B* ],#?#"  E")* JG J,)A3,#(3# 6. `-B)"#A3#(3# V+#3%"$],#?#"   $% O(N,%")1#( 9Y,E# M#34()-)1,#A< A,%# 7. ;N")1#( V+#3%"$ ],#?#"  $% ;N")1#( 8. ,-)N#1E+ _ $ A,%# ?,%4 N#"/ 3)*+"#4#(A,N# &$%$ A4##%A )( *$(/ `> N$",$(%A

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D`BBF?@[ \BC3 KECB3=FD=@A !3BA=?@F KB3 GCEA?_DBEB3 ?G,[?@[ By Kurt Thorn of the Nikon Imaging Center at UCSF | Oct 9, 2014

 A common requirement for live cell imaging experiments is the ability to follow multiple uorescently tagged species simultaneously. To do so with uorescent protein labels requires multiple uorescent proteins ?4)A# excitation and emission spectra differ sufciently for them to be imaged in distinct uorescent channels on the microscope. With the proliferation of uorescent proteins in recent years, there are many uorescent protein combinations that can be imaged together, but this also means that the choice of uorescent proteins requires

A)*# %4)B14%F The rst step in choosing uorescent proteins for your multi-color imaging experiment is to be aware of what uorescent proteins are available. With new uorescent proteins being published every month, deciding on the best protein for a given application is a challenge. To help keep you abreast of the latest uorescent proteins, I *$,(%$,( $( interactive graph and table of the best uorescent proteins currently availableF

DN11$&:9 D1%2#0&H". K"415.$6.:0 !510.&:$ To choose a set of uorescent proteins to be imaged together, you will need to consider the same factors as when choosing an individual uorescent protein (brightness, photostability, and so on; see the +"#N,)BA A#3%,)( for more discussion of these factors). In addition, you will also need to choose uorescent proteins that can

.# &,A%,(1B,A4#& E")* )(# $()%4#" $(& %4$% 3$( .# ,*$1#& ?,%4 %4# )+%,3A )( %4# *,3")A3)+#9A< /)B ,(%#(& %) use. An accurate determination of whether two uorescent proteins can be separated from each other requires C()?-#&1# )E %4#," #Z3,%$%,)( $(& #*,AA,)( A+#3%"$D .B% # 911' 54". 1I 0N4%H &$ 0N#0 H10N 0N. 2.#/ .Y6&0#0&1: 7#].".:90N #:' 2.#/ .%&$$&1: 7#].".:90N 1I 0N. 071 2510.&:$ $N14"' H. $.2#5#0.' HX S*_V* :%F `)"

#Z$*+-#D !`> 9#Z \:6 (* Q #* \8\ (*< $(& ^`> 9#Z c7\ (* Q #* c58 (*< 3$( .# ,*$1#& %)1#%4#" .B% !`> and GFP (ex 488 nm / em 507 nm) show some crosstalk between the two uorescent proteins. If you must image uorescent proteins whose spectra overlap, there are techniques, like A+#3%"$- B(*,Z,(1D ?4,34 3$( .# used to separate the uorescent proteins, but these are beyond the scope of this eBook.

,5. \145 K"415.$6.:0 !510.&:$ D1%2#0&H". 7&0N \145 G&651$612.W$ B20&6$P To determine if the uorescent proteins you are interested in are compatible with your microscope optics, you will want to compare the excitation and emission spectra of your protein with the lter sets or lasers on your microscope. Ideally, you would like to have substantial overlap between the excitation and emission lters

$(& %4# #Z3,%$%,)( $(& #*,AA,)( A+#3%"$ )E %4# +")%#,(D A) %4$% %4# +")%#,( ,A ?#-- #Z3,%#& ./ /)B" *,3")A3)+# and the uorescence emission of the protein is efciently collected by the microscope. To compare the match between a uorescent protein and a lter set, many lter set vendors provide tools to plot the uorescence spectra of proteins and dyes and their lters (see !4")*$lAD V#*")3ClAD )" I*#1$lA
/)B" +")%#,( )E ,(%#"#A% 4$A $ A,*,-$" A+#3%"B*F `)" #Z$*+-#D 4#"#lA $ A3"##(A4)% E")* %4# !4")*$ V+#3%"$ Viewer comparing a standard Cy3 or Rhodamine lter set (Chroma #49004) to the spectra of both mCherry and

M$1L`>F

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R#"#D %4# M$1L`> A+#3%"B* ,A A4)?( ,( %4# &$"C#" 3)-)"A $(& %4# *!4#""/ A+#3%"B* ,A A4)?( ,( %4# -,14%#" colors; excitation spectra are blue and emission spectra are red. Neither is a perfect match to the lter set but the excitation lter excites more of the peak of the TagRFP and the emission lter collects a larger fraction of the TagRFP emission than the mCherry emission. For this lter set, we would expect TagRFP to give a brighter signal than mCherry. In general, lter sets designed for Rhodamine / Cy3 will work better with shorter wavelength red uorescent proteins like TagRFP or mRuby2 than longer wavelength proteins like mCherry. For background on uorescence and lter sets, see the O(%")&B3%,)( %) `-B)"#A3#(3# 2,3")A3)+/ -#3%B"# $% ,J,)-)1/F

D1%%1:"X C$.' K&"0.5 F.0$ #:' 3.".]#:0 K"415.$6.:0 !510.&:$ Commonly used lter sets for multicolor imaging include ones designed for CFP, YFP, and RFP or the Sedat Quad lter set, designed for DAPI / Fluorescein / Rhodamine / Cy5 (e.g. V#*")3ClA< $(& %4# A,*,-$" \X-$A#" combination on a confocal (405 / 488 / 561 / 640 nm). In our hands the best uorescent proteins for imaging

?,%4 %4,A A#% $"# *M$1J`>5D ;U`> )" )(# )E %4# ,*+")N#& U`> N$",$(%AD *LB./5 )" M$1L`>XMD $(& $( ,(E"$"#& uorescent protein such as iFP1.4 or iFP2.0. Beware that these infrared uorescent proteins require biliverdin as

$ 3)E$3%)" $(& A) /)B *$/ (##& %) AB++-#*#(% /)B" 3#--A ?,%4 .,-,N#"&,( E)" *$Z,*$- .",14%(#AAF O( *$**$-,$( 3#--AD )(# )E %4# ,*+")N#& E)-&,(1 N$",$(%A )E ;U`> -,C# *;*#"$-& )" !-)N#" ,A +").$.-/ .#A%o *H#)(U"##( ,A an even newer green uorescent protein that is supposed to be extremely bright. In P? 0&/&O8C8+&D ?#lN# %#A%#& a number of green and red uorescent proteins with this lter set and have "#+)"%#& .",14%(#AA *#$AB"#*#(%AF R#"#D ;U`> )B%+#"E)"*A %4# ,*+")N#& E)-&,(1 N$",$(%AD +"#AB*$.-/ &B# %) %4# -)?#" 1")?%4 %#*+#"$%B"#F

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D`BBF?@[ \BC3 KECB3=FD=@A !3BA=?@F KB3 GCEA?_DBEB3 ?G,[?@[ ;DB@AW-> This also suggests, however, that there is no single uorescent protein optimal for all organisms and that if you

?$(% %4# .",14%#A% A,1($-D /)B *$/ (##& %) %"/ A#N#"$- +")%#,(A ,( /)B" A/A%#* )E ,(%#"#A%F `,($--/D ,( %4,A A#% )E +")%#,(AD %4# 1"##( $(& "#& +")%#,(A $"# 1#(#"$--/ %4# *)A% &#%#3%$.-# $(& A) A4)B-& .# BA#& %) %$1 /)B" -#$A% $.B(&$(% +")%#,(AD ?,%4 %4# .-B# $(& ,(E"$"#& 34$((#-A BA#& E)" *)"# $.B(&$(% +")%#,(A )" *$"C,(1 AB.3#--B-$" 3)*+$"%*#(%AF I hope this sheds some light on multicolor imaging with uorescent proteins. With the right microscope and the right choice of uorescent proteins, imaging four colors simultaneously should be pretty straightforward.

Further Reading 1. V+#3%"$- ,*$1,(1 $(& ,%A $++-,3$%,)(A ,( -,N# 3#-- *,3")A3)+/F y,**#"*$((D M,*)D h#(A L,#%&)"ED $(& L$,(#" >#++#"C)CF L>!P @&77&/C c\eF7 9566:B.2#& >2OGK 75f5g5\7F 2. Improved blue, green, and red uorescent protein tagging vectors for S. cerevisiae. Lee, Sidae, Wendell  0F Y,*D $(& dB"% VF M4)"(F 6@;P ;*& fF8 9567:B.2#& >2OGK 5:f\\75:F 3.  0&&1#(#lA `-B)"#A3#(% >")%#,( UB,&#

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The rst time I heard about FRET during a journal club, my guitarist brain automatically thought about the raised

#-#*#(% E)B(& )( %4# (#3C )E */ 1B,%$" FFF ()% "#$--/ BA#EB- E)" $ .,)-)1,A% /)B ?)B-& A$/F M4# A%B&#(% ?$A )E 3)B"A# %$-C,(1 $.)B% %4# ()? ?#--XC()?( %#34(,[B# 3$--#& `L;MD )" `-B)"#A3#(3# 9`‚"A%#"< L#A)($(3# ;(#"1/ M"$(AE#" , which allows the detection of molecules’ interactions, modications or dissociations 8* C87FF bA#& A,(3# %4# *,&Xg6AD %4,A %#34(,[B# 4$A "#N)-B%,)(,W#& %4# ?$/ ?# $++"#4#(& *)-#3B-$" 3)*+-#Z#A $(& ,A A%,-- $ N#"/ BA#EB- %))-F Like a guitar hero (that I’m not), FRET loves playing “live”. Indeed, FRET was one of the rst techniques

%4$% #($.-#& %4# *#$AB"#*#(% )E A,(1-# *)-#3B-# ,(%#"$3%,)(A ,( -,N,(1 3#--A BA,(1 $ *,3")A3)+#F R,A%)",3$--/D *)-#3B-$" ,(%#"$3%,)(A ?#"# &#%#3%#& ./ ,(&,"#3% *#$(A )E%#( BA,(1 +").#A ?,%4 %4# +)%#(%,$- %) %$"1#% A#N#"$*)-#3B-#AF J/ $($-)1/D ,% ?$A -,C# +),(%,(1 )B% $ 1")B+ )E A%B&#(%A ,( $ B(,N#"A,%/ 4$-- .B% ()% C()?,(1 ,E %4#A# A%B&#(%A C(#? )" ,(%#"$3%#& ?,%4 #$34 )%4#"F `L;M #(4$(3#& )B" +#"3#+%,)( $.)B% *)-#3B-$" ,(%#"$3%,)(AF

ON#0 &$ K3=AP O( %4#," 566: h!J +$+#" , Sekar and Periasamy dened FRET as “a distance-dependent physical process by which energy is transferred non-radiatively from an excited molecular uorophore (the donor) to another uorophore (the acceptor) by means of intermolecular long-range dipole-dipole coupling.” The emission of the acceptor uorophore can be measured using microscopy techniques. FRET measurement sensitivity makes it suitable for studying interactions within living cells. By coupling uorophores to proteins, pioneers of this

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ON#0 ,5. 0N. !#5#%.0.5$ 0N#0 ,II.60 K3=AP FRET occurs when the two uorophores used are in close vicinity. Thus, the '&$0#:6. H.07..: 0N. 071 uorophores and their orientation to one another  3$( $EE#3% `L;MF S4#( ,% 3)*#A %) A%B&/,(1 $( B(C()?( interaction between two proteins, these parameters are difcult to overcome but they have to be considered when analyzing data from your FRET experiments. You may have to design several different constructs to nd

)(# %4$% 3)B-& .# BA#& E)" %4# +B"+)A# )E /)B" A%B&,#AF M4#A# +$"$*#%#"A $"# -#AA ,*+)"%$(% ?4#( &#A,1(,(1 $ biosensor, as the distance between the two uorophores and their spatial orientation will be xed.

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)N#"3)*# ./ 34))A,(1 $ 3)*+-#*#(%$"/ +$," )E uorophoresF M) *$Z,*,W# %4# `L;M A,1($-D /)B A4)B-& 34))A# %4# 4,14#A% [B$(%B* /,#-& &)()"D the highest absorbing acceptor, and uorophores with signicant overlap in their spectra. The pair CFP-YFP was the rst to be used to study

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d()?( 3#--B-$" A,1($-,(1 +$%4?$/A 3$( .# ,(N#A%,1$%#& ,( $ 4,14X%4")B14+B% A3"##( BA,(1 -B3,E#"$A# $3%,N,%/ $A $ *#$AB"# )E A,1($-,(1 $3%,N,%/F 2B-%,+-# "#A+)(A# #-#*#(%A $"# 3-)(#& ,(%) $ +-$A*,& B+A%"#$* )E $ *,(,*$- +")*)%#" -,(C#& %) -B3,E#"$A#F 03%,N$%,)( )E %4# +$%4?$/ ?,-- "#AB-% ,( -B3,E#"$A# #Z+"#AA,)( $(& *)"# 3)*+-#Z 3#-- %"#$%*#(%A 3$( .# &#A,1(#& %) EB"%4#" #Z+-)"# %4# +$%4?$/F

 77g = >$1#

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 756 = >$1#

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!3BA=?@ A,[F !" >/80 H? 6&/98*C1 '(()&*& - V&0 441 234I

M')"%&7 ",:* ,'% 3*3,..8 *(,..&*$ 1%1"&2%* &7-)'1)',"%2 &7") , "',7*.,"%2 1')"%&7F 0A &#+,3%#& ,( %4# accompanying cartoon, they have a multitude of uses including (but not limited to) purication, detection, A)-B.,-,W$%,)(D -)3$-,W$%,)(D )" +")%#$A# +")%#3%,)(F S#lN# $-"#$&/ 3)N#"#& GFP and its related uorescent

+")%#,(AD ?4,34 $"# A)*#%,*#A BA#& $A %$1A E)" &#%#3%,)(o 4)?#N#"D %4)A# 3)(A%,%B%# @BA% )(# 9$&*,%%#&-/ -$"1#< 3-$AA )E 3)**)( EBA,)( +")%#,( %$1AF J,)34#*,A%A $(& *)-#3B-$" .,)-)1,A%A ?4) (##& %) )N#"#Z+"#AA $(& +B",E/ +")%#,(A 3$( E$3# $(/ (B*.#" )E %#34(,3$- 34$--#(1#A &#+#(&,(1 )( %4#," +")%#,( )E ,(%#"#A%F 0E%#" A#N#"$$&#A )E %"/,(1 %) $&&"#AA %4#A# 34$--#(1#AD "#A#$"34#"A 4$N# $*$AA#& $ 3)(A,&#"$.-# *)-#3B-$" %))- .)Z )E tags and fusion proteins to aid in the expression and purication of recombinant proteins.

Different Uses for Protein tags. Left: Afnity tags. Middle: Solubility tags. Right: Cleavage tags. Image by Eric Perkins.

 757 = >$1#

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!3BA=?@ A,[F ;DB@AW-> A#9$ I15 F0#H&"&0X #:' F1"4H&"&0X S4$% $"# A)*# )E %4# 4B"&-#A %) )N#"3)*# ,( )"&#" %) )N#"#Z+"#AA $ "#3)*.,($(% +")%#,(T O% ,A ()% 1#(#"$--/ ,( $ 3#--lA .#A% ,(%#"#A% %) )N#"#Z+"#AA $ +")%#,(o #(#"1/ $(& 3#--B-$" "#A)B"3#A $"# A+#(% %) *$C# A)*#%4,(1 %4# 3#-- &)#A(l% (##& %) *$C#F ;BC$"/)%#A $(& A)*# .$3%#",$ &#+-)/ +")%#)A)*#A %)
"$&# ?4$% %4# 3#-*,14% 3)(A,&#" @B(C +")%#,(F M4)B14 %4#"# $"# $ (B*.#" )E 34#*,3$- $(& +#+%,&#X.$A#& +")%#)A)*# ,(4,.,%)"AD glutathione S-transferase (GST), which can be fused to recombinant proteins for one-step purication with

1-B%$%4,)(#D 3$( $-A) +")%#3% $1$,(A% +")%#)-/A,AF M4$%lA )(# E)"* )E ,(A%$.,-,%/F >")C$"/)%#A 3$( $-A) 4$N# $ 4$"& %,*# E)-&,(1 #BC$"/)%,3 +")%#,(AF ^)B 3$( 1#% /)B" .$3%#",$ %) +")&B3# *$AA,N# $*)B(%A )E +")%#,(D .B% ,E ,%lA ()% E)-&#& 3)""#3%-/D %4#"#lA () +),(% ,( 3"/A%$--,W,(1 it or testing its function. Small ubiquitin-related modier (Vb2I< 3$( 4#-+ ?,%4 E)-&,(1 $(& A%$.,-,W$%,)(D $A 3$( *$-%)A#X.,(&,(1 +")%#,( 92J> %$1A 3$( 4#-+ ?,%4 A)-B.,-,%/ ,AAB#AD .B% A3,#(%,A%A *$/ $-A) 34))A# %) $&& A*$--#" +")%#,(AD AB34 $A M4,)"#&)Z,( 0 9M"Z0<  that improve disulde bond formation in order to help keep a protein of interest soluble.

Tags for Afnity and Purication  An afnity tag, generally a relatively small sequence of amino acids, is basically a molecular leash for your

+")%#,(F OE /)Bl"# ?)"C,(1 ?,%4 $( B(34$"$3%#",W#& +")%#,(D )" $ +")%#,( E)" ?4,34 $ 1))& $(%,.)&/ 4$A ()% .##( &#N#-)+#& 9$(& @BA% .#3$BA# /)B" +")%#,( 4$A $ 3)**#"3,$--/ $N$,-$.-# $(%,.)&/D %4$% &)#A(l% *#$( ,%lA $ );;(  one), then your rst step towards detecting, immunoprecipitating, or purifying that protein may be to fuse an afnity tag to it. The `Y0UD 4#*$1-B%,(,( $(%,1#( 9R0
%$1 ?)"-& E)" /#$"AD $(& ,&,(1 )( ?4,34 )(# %) BA# ?,-- &#+#(& )( /)B" $++-,3$%,)( 9A## %$.-# .#-)?
%$1AD $ R,A %$1 3$( .# EBA#& %) #,%4#" %4# HX )" !X%#"*,(BA )E $ +")%#,(F b(-,C# )%4#" #+,%)+# %$1A _ ?4,34 ?4#( &)B.-#& )" %",+-#& ,(3"#$A# %4# %$1 A,W# [B,3C-/ _ *)&,E/,(1 %4# -#(1%4 $ +)-/4,A%,&,(# %"$3% &)#A ()% 1"#$%-/ $-%#" %4# A,W# )E %4# %$1F

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 Afnity

`"#[B#(%-/ BA#& E)" ?#A%#"( blots, IP, co-IP, IF, ow-

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]#"/ A*$-- A,W#D "$"#-/ $EE#3%A EB(3%,)(

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0% HX%#"*,(BAD +")*)%#A E)-&,(1 $(& A%"B3%B"$- ,(%#1",%/o 3-#$N$.-#F H)% 1"#$% E)" purication; too cleavable in

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ULLO>UYOH> SdLLSddH`O 0]V00HL`dd OVVVU0YG^G O>MM0V;HY^ `aU;`UY0aR G;0]GHd`Hd ;aaH0`^;OY RY>HYH;;aL H0`OaVYdGG >VaV0HYY0; 0ddYHG0a0> d]GHd`Hd;a aH0`^;OYRY >HYH;;aLH0 `OaVYdGG>V aV0HYY0;0d dYHG0a0>d] G0HRa 2VGdOORYMG GV`GMG]Yd0 GU0OY]G`S0 ;S!U>!d2O0 >OYG;O0G;^ aUdYM]0dYH OGaH>UM0>d ^UOLUO>MYY Y`dHU;]00M d]U0YVdUaY d;`YG0HY0U VUVUR2RRRR RRVVUY]>LU

57

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U))& E)" $(%,.)&/X.$A#& purication

D1%H1 #:' D".#]#9. A#9$ `"#[B#(%-/D $ A,(1-# %$1 ,A ()% #()B14F S4$% ,E /)B (##& )(# %$1 %) ,(3"#$A# A)-B.,-,%/ $(& )(# %$1 E)" purication? Or you want to combine a uorophore with a tag that localizes your protein to the nucleus? Or you want multiple rounds of purication to get your protein as pure as possible? Vectors that offer different

3)*.,($%,)(A )E %$1A $"# "#$&,-/ $N$,-$.-#D $(& %4)B14 $&&,(1 %)) *$(/ %$1A $(& EBA,)( +")%#,(A %) /)B" +")%#,( )E ,(%#"#A% ?,-- #N#(%B$--/ 1#% ",&,3B-)BA 9/)B 1#(#"$--/ &)(l% ?$(% *)"# %$1 %4$( +")%#,(
.,(&,(1 +#+%,&# 9!J>")%0 O1UX.,(&,(1 &)*$,(AF M0> 4$A A,(3# 3)*# %) #(3)*+$AA A#N#"$- )%4#" %$1 3)*.,($%,)(AD %4)B14 E"#[B#(%-/ %4)A# 3)*.,($%,)(A A%,-- ,(3-B&# $% -#$A% )(# #-#*#(% E")* %4# )",1,($- M0> %$1F M4# %#"*A &B$-X-$.#-,(1 $(& &B$-X%$11,(1 $"# $-A) BA#&F GB#  75\ = >$1#

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!3BA=?@ A,[F ;DB@AW-> to their small size and the ease with which they can be added to a purication scheme, His tags are frequently

3)*.,(#& ?,%4 )%4#" %$1A E)" &B$-X-$.#-,(1F M4# +").-#* ?,%4 $-- %4#A# %$1A ,A %4$% *$(/ )E %4#* A#"N# $ )(#X%,*# +B"+)A#D $(& /)B &)(l% (#3#AA$",-/ ?$(% %4#* %) A%,3C $")B(& $E%#" %4$% +B"+)A# 4$A .##( A#"N#&F 0% %4,A +),(%D +")%#$A#A 3$( .# /)B" E",#(& "$%4#" %4$( your enemy. Two common tags (SUMO and FLAG) are cleaved by specic proteases without requiring the

$&&,%,)( )E $( ,(&#+#(&#(% 3-#$N$1# "#3)1(,%,)( A,%#F O( E$3%D Vb2I 3$(()% .# BA#& ,( #BC$"/)%#A .#3$BA# there is already too much SUMO protease around, but it is convenient when used with puried protein since the

#(W/*# 3-#$N#A %4# Vb2I %$1 ,( %4# A$*# *$((#" $A ,% ?)B-& 4$N# ,( %4# 3)(%#Z% )E $ 3#--F `Y0U %$1A 3$( .# cleaved by enterokinase, which recognizes DDDDK^X, cleaving after the lysine. The efciency of this cleavage

&#+#(&A )( %4# ,&#(%,%/ )E tF  0 (B*.#" )E )%4#" +")%#$A#A $"# $N$,-$.-#D .B% A3,#(%,A%A ?)B-& (##& %) ,(3)"+)"$%# %4#," "#3)1(,%,)( A,%#A ,(%) %4#," +")%#,( %$1 ,( )"&#" %) BA# %4#* #EE#3%,N#-/F I(# )E %4# .#A% )+%,*,W#& ,A %4# %).$33) #%34 N,"BA 9M;]< +")%#$A#F 0 M;] +")%#$A# 3-#$N$1# A,%# ,A E"#[B#(%-/ +-$3#& .#%?##( %?) %$1A .#,(1 BA#& E)" %?) ")B(&A )E purication, with the cleavage reaction taking place between column runs. The TEV protease itself, with various mutations used to increase its stability activity, can be readily puried using plasmids E)B(& ,( %4,A +$+#" 

9$N$,-$.-# $% 0&&1#(#
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Further Reading 1. Lecombinant protein expression and purication: A comprehensive review of afnity tags and microbial $++-,3$%,)(AF ^)B(1 !YD J",%%)( yMD L).,(A)( 0VF L#3)* !8;7&0%*;@;)" H;F/*+@  5675 D 98B.2#& >2OGK 55\\56:\F 2.  A complete list of free handbooks for protein purication is provided by U; R#$-%43$"# Y,E# V3,#(3#AF

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A,[ \BC3 K,ZB3?A= \=,FA [=@=F O?A` =,F= !" HF@8+* $+"@;/T6+/9&/1 '(()&*& E87% 0;*7/8
O)().):)3* '%-)(+&7,"&)7 &* "$% 1')-%** +8 ;$&-$ 7%,'.8 ,.. 2)(,&7* )4        F L#A#$"34#"A

4$N# -)(1 %$C#( $&N$(%$1# )E %4,A ($%B"$- +")3#AA %) ,(%#1"$%# +")%#,( %$1A ,(%) %4# 1#()*#A )E P? 0&/&O8C8+& $(& P? B;,<&F M4# +")%)3)- ,A AB"+",A,(1-/ A,*+-#D requiring only a PCR product containing the modifying sequence anked by

$++")Z,*$%#-/ c6 .$A# +$,"A )E A#[B#(3# 4)*)-)1)BA %) %4# 34")*)A)*$A,%# )E ,(A#"%,)(F M4# -,(#$" >!L +")&B3% ,A ,(%")&B3#& ,(%) %4# 3#-- ./ &,"#3% transformation. A given insert will typically contain both a protein modication

A#[B#(3# $(& $ A#-#3%$.-# 1#(# +")&B3% E)" ,A)-$%,)( )E AB33#AAEB- %"$(AE)"*$(%AF Modied from Lee S, et al. (2013) 6^;P :W> kR5SX &M5`32?

 0&&1#(# &,A%",.B%#A A#N#"$- "#$&/X%)XBA#D *)&B-$" +-$A*,&AD 3)*.,(,(1 uorescent tags, epitope tags, protease sites, and selection markers. These are

#A+#3,$--/ BA#EB- ,( +")%#,( 3)*+-#Z A%B&,#A ?4#"# %$11,(1 )E *B-%,+-# +")%#,( +")&B3%A ,A &#A,"#&D $A *B-%,+-# A#-#3%,)( *$"C#"A 3$( #(AB"# %4$% $-- &#A,"#& %$1A 4$N# .##( ,(%#1"$%#&F V,*+-/ design your amplication primers with the desired targeting homology—in frame, of course—and start tagging!

 \.#$0_B20&%&e.' K"41512N15.$ I15 ?%#9&:9 2$(/ ,*$1,(1 A%B&,#A "#-/ )( &,"#3% EBA,)( )E uorescent proteins (FPs) to a yeast gene of interest. These uorescently tagged

1#(#A $"# #Z+"#AA#& B(&#" ($%,N# 3)(&,%,)(A $(& $--)? A3,#(%,A%A %) ()% )(-/ %"$3C %4# $.B(&$(3#D *)N#*#(%D $(& -)3$-,W$%,)( )E ,(&,N,&B$- +")%#,(AD .B% $-A) ,(N#A%,1$%# +")%#,(X+")%#,( ,(%#"$3%,)(A N,$ `L;MF V,&$# Y##D S#(&#-- Y,*D $(& dB"% M4)"( $% b!V` 4$N# "#3#(%-/ &#N#-)+#& $ A#",#A )E .-B#D 1"##(D $(& "#& `>A %4$% $"# codon optimized specically for expression in yeast. These tagging

N#3%)"A $"# .$A#& )( +"#N,)BA-/ &#A3",.#& +`0e$X-,(C N#3%)"A $(& ,(3-B&# $ d$(D V+ROVcD )" !$bL0: A#-#3%,)( *$"C#"F Y## &7 +@? assessed many of these uorescent tags in P? 0&/&O8C8+&D -))C,(1 $% %4#," +#"E)"*$(3# ,( 3$%#1)",#A AB34 $A .",14%(#AAD A%$.,-,%/D and disruption of the tagged protein. Based on their ndings, the

$B%4)"A "#3)**#(& )+%,*$- `> 3)*.,($%,)(A E)" BA# ,( /#$A% imaging, categorized by specic lter sets and experimental output "#[B,"#*#(%AF V#-#3% E")* %4#A# yeast-optimized uorophore

%$11,(1 N#3%)"A E)" /)B" A,(1-#X )" *B-%,X3)-)" ,*$1,(1 #Z+#",*#(%AF OE /)Bl"# -))C,(1 E)" $ 1"#$% "#A)B"3# $.)B% ,*$1,(1 %#34(,[B#AD 34#3C )B% dB"% M4)"(lA *,3")A3)+/ .-)1F  A variety of uorescent proteins expressed in yeast.  '(+B7&( G/;, ^&& P1 &7 +@? R234JS? 6^;P :W> kR5SX &M5`32 

 75e = >$1#

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