KARAKTERISTIK DAN FUNGSI SEL KOMPETEN
Sel kompeten memiliki kemampuan untuk berikatan, dan mengambil DNA eksogen. Sel kompeten merupakan sel yang telah siap untuk ditransformasi menggunakan materi genetik asing. Sel kompeten pada umumnya terdapat pada bakteri, dan merupakan mekanisme penting untuk transfer gen secara horizontal. Sel kompeten dapat mengekspresikan protein terspesialisasi tertentu yang berasal dari pengambilan DNA luar ke dalam sel. Dalam banyak organisme, perkembangan kompetensi dan ekspresi dari pengambilan DNA luar merupakan regulasi yang diatur dari respon sinyal antar sel dan kondisi nutrisional (Solomon, 1999). Bakteri kompeten alami memiliki kemampuan untuk mengambil DNA eksogen dan undergo transformasi genetic (Chen,2004). Sel kompeten pada makhluk hidup dapat diperoleh secara alami ataupun buatan. Sel yang belum kompeten dapat dibuat dengan menggunakan metode kalsium-klorida (substansi yang terbuat dari ion organik) dan juga dengan metode heat shock (Anonim,2010). shock (Anonim,2010).
Gambar 1. Skema pemasukan DNA eksogen pada sel kompeten
Competent Cells: Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order to make bacteria take in the plasmid, they must first be made "competent" to take up DNA. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA together on ice, placing them briefly at 42oC (heat shock), and then putting them back on ice. This causes the bacteria to take in the DNA. The cells are then plated out on antibiotic containing media. For a short animation on E. coli transformation click here.
Competency The procedure to prepare competent cells can sometimes be tricky. Bacteria aren't very stable when they have holes put in them, and they die easily. A poorly performed procedure can result in cells that aren't very competent to take up DNA. A well performed procedure will result in very competent cells. The competency of a stock of competent cells is determined by calculating how many E. coli colonies are produced per microgram (10 -6 grams) of DNA added. An excellent preparation of competent cells will give ~108 colonies per ug. A poor preparation will be about 10 4 / ug or less. Our preps should be in the range of 10 5 to 10 6. In this experiment you will be making competent cells, transforming them with a plasmid and calculating their competency. There will be a lab report due for this lab.
Naturally competent bacteria are able to take up exogenous DNA and undergo genetic transformation. The transport of DNA from the extracellular milieu into the cytoplasm is a complex process, and requires proteins that are related to those involved in the assembly of type IV pili and type II secretion systems, as well as a DNA translocase complex at the cytoplasmic membrane. Here, we will review the current knowledge of DNA transport during transformation (Chen, 2004).
Natural genetic competence, the ability of cells to bind to and to take up exogenous DNA, is widespread among bacteria and might be an important mechanism for the horizontal transfer of genes. Competent cells express specialized proteins that assemble into a DNA-uptake complex. In many organisms, the development of competence and expression of the uptake machinery is regulated in response to cellcell signaling and/or nutritional conditions. Exciting new progress has been made in characterizing the signals and pathways that regulate the development of competence (Solomon, 1999).
Pembuatan sel kompeten bakteri secara buatan untuk transformasi dapat dilakukan dengan dua metode, yaitu menggunakan metoda kalsium-klorida, dan metode elektroporasi. (Sharma, 1996).
Rakesh C. Sharma and Robert T. Schimke, "Preparation of Electro-competent E. coli Using Salt-free Growth Medium", Biotechniques 20, 42-44 (1996). http://www.chem.uga.edu/scottgrp/grpprotocols/competent_cell_preparation.htm
Inês Chen & David Dubnau. DNA uptake during bacterial transformation. Nature Reviews Microbiology 2, 241-249 (March 2004) | doi:10.1038/nrmicro844. http://www.nature.com/nrmicro/journal/v2/n3/full/nrmicro844.html
Jonathan M. Solomon and Alan D. Grossman. Who's competent and when: regulation of natural genetic competence in bacteria Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, UK. Available online 19 Marc h 1999. Trends in Genetics Volume 12, Issue 4, April 1996, Pages 150-155. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCY-3W25BGJ4N&_user=10&_coverDate=04/30/1996&_rdoc=1&_fmt=high&_orig=search&_orig in=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_url Version=0&_userid=10&md5=dd0c391b975a6216d2e1e0c3132fae5d&searchtype=a
Brown, T.A; editor: Soemiati Ahmad Muhammad & Praseno. 1991. Pengantar Kloning Gena. Penerbit Yayasan Essentia Medica, Yogyakarta. Sel yang telah siap untuk ditransformasi menggunakan materi genetik asing
Competent cells are those that possess more easily altered cell walls that DNA can be passed through easily. These cells readily incorporate foreign DNA. On example of a competent cell is E. coli.
A cell that is not already competent can be made more competent through calcium chloride (a substance that creates organic ions) and heat shock. This is especially easy with cells undergoing very rapid growth.
This characteristic has an interesting potential application. With low levels of calcium chloride and heat shock that does not affect less-susceptible cells (in this case, those that do not have very rapid growth), it may be possible to make rapidlydividing cancer cells competent enough to take up DNA that will replicate with the cell. This DNA can have a variety of different characteristics: it may be easily traceable, or it may be very susceptible to a specific chemotherapy drug, for instance.
This sort of treatment is years away. It's made more complicated by the fact that the process of getting competent cells to take up trojan DNA is time consuming, a real problem when you're working with a real person. http://www.iscid.org/encyclopedia/Competent_Cells
www.dnalc.org/ddnalc/resources dari blog al-chemy.
Plasmid transformation into bacterial competent cells is a key technique in molecular cloning. In early 1970’s Cohen (Cohen et al. 1973) successfully transformed R-factor and recombinant plasmids into E. coli cells using a calcium chloride method. Since that time this method has been widely used due to its convenience. An alternative transformation method used is electroporation which results in a higher
transformation efficiencies of up to 109 - 1010transformants/μg DNA ( Ryu and Hartin, 1990). McCormac (McCormac et al. 1998) published a simple method for the production of highly competent cells of Agrobacterium tunrefaciers/rhizobium for transformation via electroporation. Okamoto (Okamoto et al. 1997) also reported high efficiency transformation of Bacillus brevis by electroporation, however, special equipment is required for electroporation that many laboratories cannot provide. Tsen has found certain strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies (Tsen et al. 2002). Kurien and Scofield have described a quick and moderately efficient method of bacterial colony transformation (Kurien and Scofield, 1995). More recently, Chen has proposed an alternative convenient and rapid method for the genetic transformation of E. coli with plasmids. By mixing the recipient cells and plasmid DNA and spreading them directly on selective medium plates containing Ca2+, the so-called 'plate transformation' could achieve almost the same transformation efficiency as the classical transformation method with calcium, yet the whole protocol takes only approximately 2 min ( Chen et al 2001). Based on this method, we have established an efficient system using E. colicompetent cells for transformation plasmids. Plasmids then can be stored as bacterial stocks in our China-UK joint laboratory, which allows amplification of plasmids for future experiments.
Pada umunya DNA supercoiled, dan plasmid berukuran kecil, dibandingkan dengan large or legated dna. . Before electroporation, ligated DNA to be transformed must be purified to remove DNA ligase, a potential inhibitor of electroporation. Our StrataClean™ resin dramatically simplifies this process
Sel kompeten berkembang dalam fase lag, tapi akan terhenti pada perkembangan eksponensial. Ketika plasmid ditambahkan dalam suspensi sel, jumlah transforman menjad meningkat. Laju transforman meningkat seiring dengan jumlah plasmid hingga mencapai fase plateu. Supercoiled monomer (Tsen et al ,2002) Tahap berikutnya dalam eksperimen pengklonan gen setelah molekul DNA rekombinan berhasil dikonstruksi adalah memasukkan molekul DNA rekombinan tersebut ke dalam sel hidup yang disebut sel inang. Sel inang yang paling umum digunakan adalah Escherichia coli. Tidak semua sel secara alami memiliki
kemampuan untuk menyerap DNA asing. Setiap sel memiliki efisiensi yang berbeda dalam menyerap molekul DNA. Beberapa spesies yang dapat menyerap DNA secara efisien adalah Bacillus dan Streptococcus. Eschercia coli tidak memiliki kemampuan transformasi secara alami, strain E. coli can incorporate ekstraseluler plasmiud ke dalam sitoplasma naturally dalam frekuensi yang rendah. Peningkatan kemampuan transformasi secara alami dilakukan dengan memanen sel pada fase stasioner dan penumbuhan kembali pada media (Tsen et al.2002). Tidak semua sel kompeten itu dapat melakukan transformasi. Bagian sel yang menyerap DNA plasmid sangatlah rendahg, sehngga diperlukan metode untuk membedakan antara sel transforman dengan sel nontransforman
Untuk membuat sel kompeten digunakan kultur yang telah mengalami fase log. Fase log diperlukan agar jumlah bakteri yang terisolasi untuk pemnuatan sel kompeten optimal.
The optimal optical density (OD600) range for competent cell preparation varied for each of the strains investigated, and for XL1 blue it was 0.15-0.45; for TG1 it was 0.2-0.5; and for DH5α it was 0.145-0.45. The storage time of competent cells and its correlation to transformation efficiency has been studied, and the result showed that competent cells can be stored at 20ºC for 7 days and at -70ºC for 15 days. .
Plasmid transformation into bacterial competent cells is a key technique in molecular cloning. In early 1970’s Cohen (Cohen et al. 1973) successfully transformed R-factor and recombinant plasmids into E. coli cells using a calcium chloride method. Since that time this method has been widely used due to its convenience. An alternative transformation method used is electroporation which results in a higher transformation efficiencies of up to 109 - 1010 transformants/μg DNA (
There are two main methods for transformation of competent bacterial cells, the calcium chloride and the electroporation method (Dargert et al. 1979; Okamoto et al. 1997; Topcu, 2000).
Bacteria that are able to take up DNA are called "competent" and competency can be induced by treatment with calcium chloride in the early log phase of growth. The bacterial cell membrane is permeable to chloride ions, but
is non-permeable to calcium ions. As the chloride ions enter the cell, water molecules accompany the charged particle. This influx of water causes the cells to swell and is necessary for the uptake of DNA; the exact mechanism of this uptake is unknown.
A second factor, which can have an impact on the transformation efficiency, is that the competent cells must be maintained in cold environment, both during storage and in use. Dargert (Dargert and Ehrlich, 1979) reported that competent cells could be stored at 4ºC in Calcium chloride for 24-48 hrs. In the prime 12-24 hrs, the transformation efficiency rise 3-5 times, then reduces to average level. Our experiments show that competent cells can be stored at 70ºC for 15 days without obviously reducing their transformation capacity. However, if the competent cells are stored at -20ºC, the highest transformation efficiencies appear at 2-7 days. But if the storage time was over 7 days, the transformation efficiency was dramatically reduced. If competent cells were stored at 4ºC, they will l ose their competency in only 3 days. Competent cell cannot be stored long term under liquid N2 and cannot be defrosted more than once.
The addition of DMSO or PEG8000 during bacterial transformation can also affect transformation efficiency. Hanahan (Hanahan et al. 1991) found the addition of DMSO greatly increased the transformation efficiency. Similarly, incubation of competent cells and plasmid DNA in a solution of polyethylene glycol/Calcium chloride (PEG/CaCl2) following by a brief incubation and heat shock resulted in efficient uptake of DNA (Kurien and Scofield, 1995). OurTu et al. 2005. An improved system for competent cell
preparation and high efficency plasmid transformation using different Escherichia coli strains. Electronic Journal of Biotechnology 8.
The competent cells can be used for many standard molecular biology applications
http://www.promega.com/tbs/tb095/tb095.pdf
Penambahan waktu kultur menjadi tiga jam dapat meningkatkan nilai OD namun tidak meningkatkan jumlah koloni (Haris et.al,2005)
Haris, Nurhaimi,et al.2005.Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Menara Perkebunan, 2005, 73(2), 44-62
http://microvet.arizona.edu/Courses/MIC205/Exams/05Exams/05Ex2key.htm
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