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).

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 cell-

cell 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 March 1999. Trends in Genetics

Volume 12, Issue 4, April 1996, Pages 150-155.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCY-3W25BGJ-

4N&_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 rapidly-

dividing 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 intoE. 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 adalahBacillus 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 competentcell preparation varied for each of the strainsinvestigated, and for XL1 blue it was 0.15-0.45; for TG1it was 0.2-0.5; and for DH5α it was 0.145-0.45. Thestorage time of competent cells and its correlation totransformation efficiency has been studied, and theresult 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 akey technique in molecular cloning. In early 1970’s Cohen(Cohen et al. 1973) successfully transformed R-factor andrecombinant plasmids into E. coli cells using a calciumchloride method. Since that time this method has beenwidely used due to its convenience. An alternative

transformation method used is electroporation which resultsin a higher transformation efficiencies of up to 109 - 1010

transformants/μg DNA (

There are two main methods for transformation ofcompetent bacterial cells, the calcium chloride and theelectroporation 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. Thebacterial cell membrane is permeable to chloride ions, but

is non-permeable to calcium ions. As the chloride ions enterthe cell, water molecules accompany the charged particle.This influx of water causes the cells to swell and isnecessary for the uptake of DNA; the exact mechanism ofthis uptake is unknown.

A second factor, which can have an impact on thetransformation efficiency, is that the competent cells mustbe maintained in cold environment, both during storage andin use. Dargert (Dargert and Ehrlich, 1979) reported thatcompetent cells could be stored at 4ºC in Calcium chloridefor 24-48 hrs. In the prime 12-24 hrs, the transformationefficiency rise 3-5 times, then reduces to average level. Ourexperiments show that competent cells can be stored at -70ºC for 15 days without obviously reducing theirtransformation capacity. However, if the competent cellsare stored at -20ºC, the highest transformation efficienciesappear at 2-7 days. But if the storage time was over 7 days,the transformation efficiency was dramatically reduced. Ifcompetent cells were stored at 4ºC, they will lose theircompetency in only 3 days. Competent cell cannot bestored long term under liquid N2 and cannot be defrostedmore than once.

The addition of DMSO or PEG8000 during bacterialtransformation can also affect transformation efficiency.Hanahan (Hanahan et al. 1991) found the addition ofDMSO greatly increased the transformation efficiency.Similarly, incubation of competent cells and plasmid DNAin a solution of polyethylene glycol/Calcium chloride(PEG/CaCl2) following by a brief incubation and heatshock resulted in efficient uptake of DNA (Kurien andScofield, 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 jamdapat meningkatkan nilai OD namun tidakmeningkatkan jumlah koloni (Haris et.al,2005)

Haris, Nurhaimi,et al.2005.Konstruksi pustaka cDNA dari daun klon karet AVROS 2037yang 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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