Cloning and Sequencing of AmilCP and from Acropora millepora millepora and Rfp from Discosoma striata in pTTQ18. CBNS836 Elizabet E!pinoza "illega! #$ %3%&%86%
'b!tract In the present experiment the gene AimilCP encoding for the blue chromoprotein from Acropora millespora, millespora, and and the the gen gene Rfp enc encodin oding g a muta mutant nt of the the red uoresce uorescent nt protein protein from from the coral coral Discosoma striat a were cloned into a pTTQ1 plasmid !ector" This cloning experiment started with the PCR ampli#cation of the $%A fragments containing the AimilCP and Rfp genes" &oth the plasmid and the $%A fragments fragments encoding encoding the interes interestt genes genes were were digested digested with the restric restriction tion en'(mes )pnI and *maI" The $%A fragments were +oin together with $%A ligase" The constructed $%A plasmid containing the genes of interest either AimilCP or Rfp were were transfor transformed med into E. coli strain coli strain -1.1 cells, allowing the expression of these repor reporter ter genes genes and showi showing ng blue blue or red red uore uoresce scent nt color color"" An addit addition ional al $%A $%A se/uencing step was carried out in order to con#rm the correct insertion of the genes of interest in the !ector" )e( words0 words0 blue blue chromo chromopr prote otein, in, red red uore uoresc scent ent prote protein in,, rest restric rictio tion n en'(me en'(mes, s, reporter genes
#ntroduction $%A cloning is an essential techni/ue in molecular biolog( research" *ome reach areas such as proteomics, genomics and s(nthetic biolog( use this techni/ue to assembl( assembl( $%A construc constructs ts containin containing g genes genes in stud( stud(" $%A cloning cloning and plasmid plasmid construction use restriction en'(mes and ligases lo cut and re+oin a target $%A fragment and the !ector" *!ein 2 Rahmi, 3.145" 6!olutionar( processes has led to the di!ersi#cation of color di!ersit( in corals" The uorescent proteins from coral ha!e been used as reporter genes due to the( can be easil( detected therefore these pigment ser!e as optical reporters" The most comm common on uor uores esce cent nt prot protei ein n is 78P 78P has has been been wide widel( l( used used in as a pote potent ntia iall geneticall( encoded encoded uorescent uorescent label" The chromoprotein chromoprotein from from the coral Acropora millespora is millespora is blue because two mutations *94C and *1:T" Alie!a, et al", 3..5" In the present present stud( a plasmid plasmid containing containing the Rfp Rfp gene from from Discosoma striata was cloned in the plasmid pTTQ1 and transformed into E. coli -1.1 coli -1.1 cells"
(aterial! and metod 1. )e!triction dige!t! and agaro!e gel electropore!i! In this step the plasmid !ector Ptt/1 and the PCR amplication product encoding for the AimilCP or Rfp gene were digested whit the restriction en'(me )pnI and *maI" The digestion was carried out a :; )b which correspond to the PCR product" The presence of the clear bands in the gel electrophoresis con#rmed the good /ualit( of the $%A, therefore this $%A is good enough for the use in the further steps of cloning"
Figure 1. Gel electrophoresis (1.0% agarose) of the digested plasmid DA p!!"1# a$d the PCR product or the reporter ge$e. The transformation in 6"coli cells strain -1.1 was successfull( carried out" It is possible to obser!e in the culture in agar @agar ampicillin 8igure 35 the presence of red colonies that are containing the reporter gene Rfp for the red uorescent protein from the coral $iscosoma striata" Bther groups wor?ed with the blue chromoprotein for the gene and also obtain positi!e result" The result for these experiments are shown in Table 1.
Fig&'agar ampicilli$ culture of E. coli *101 co$tai$i$g a$d e+pressi$g the Rfp reporter ge$e
7roup 1 3 : 4 > 9 ; D 1 . 11 13 1: Total Percentage
red 1> . 1 1; . 19 . . 3: . . . 1; 1> 131 1: E
blue . 93 . . D; . 133 D1 . 4; 143 13. . . 91 49"E
Table 1.
hite 9; 43 >3 : > 94 ;1 ;> 4. >. D. 94 44 1 ;;: ;E >:";E
E+perime$tal data of the tra$sformed colo$ies o,tai$ed e+pressi$g ,lue a$d red -uoresce$t protei$s.
In table 1 are shown the experimental results obtained for the groups that wor?ed with the red and the blue uorescent proteins" A 1:". E correspond to the red protein and ;E to the white colonies that were not transformed" In contrast for the blue uorescent protein the transformation percentage was 49"E, this percentage is higher than the transformation eFcienc( for the red protein"
$i!cu!!ion Cloning and transformation of the uorescent proteins gene AimilCP encoding for the blue chromoprotein from Acropora millespora and the gene Rfp, encoding a mutant of the red uorescent protein from the coral $iscosoma striata were was successful" This methodolog( can be successfull( used when cloning a single $%A insert in a plasmid" Plasmid construction and cloning has been used as a important tools in molecular biolog(" Cloning and plasmid construction has been performed using restriction en'(mes and ligases to cut and re+oin the $%A fragments" This techni/ue is suitable
when treating with two fragment $%A insert and plasmid5, but this methodolog( becomes ineFcient when the $%A fragments to arrange and assemble increase" This occurs because of the stic?( end caused for the restriction en'(mes, this sort $%A molecules does not pro!ide enough stabilit( or specif( for eFcient plasmid construction when using three or more fragments" 8urthermore when ha!ing more $%A fragment is diFcult to #nd uni/ue restriction site in the plasmid" The cloning of multifragments in a plasmid !ia con!entional cloning implicate more subcloning steps that can be time consuming ta?ing e!en months to reach the #nal desired plasmid" Gill 2 6atonR(e, 3.145 8luorescent proteins are exceptional tools for li!ecell imaging in research" The use of uorescent protein in molecular biolog( has enable the !isuali'ation of d(namic process within the cells, and also ma?e faster the de!elopment of new cell imaging techni/ues" The red uorescent protein has been used in I !i!o imaging because of the longer wa!e light penetrates tissues more easil(" -HllerTaubenberger 2 Anderson, 3..;5
)eference!
Alie!a, %", )on'en, )", 8ield, *", -elesh?e!itc, 6", 6" Gunt, -", &eltranRamire', =", " " " -at', -" 3..5" $i!ersit( and 6!olution of Coral 8luorescent Proteins" P'o/E ;5, e393" Gill, R" 6", 2 6atonR(e, " " 3.145" Cloning, Plasmid Construction b( *@IC or *e/uence and @igation Independent" In =" *!ein, 2 R" @ale, DA Clo$i$g a$d Assem,l2 *ethods pp" 3>:95" %ew or?0 humana Press" -HllerTaubenberger, A", 2 Anderson, )" 3..;5" Recent ad!ances using green and red uorescent" Appl *icro,iol 3iotech$ol 44, 1J13" *!ein, =", 2 Rahmi, @" 3.145" DA Clo$i$g a$d Assem,l2 *ethods. %ew or?0 Gumana Press"
