Tuesday, 28 April 2015

GGS LIVE - Bacterial Transformation

Welcome Biofreakers,

Today, GGS TEAM is happy to present  the bacterial tranformation technique.

Method: Transformation of E.coli cells with DNA plasmid.

About: This technique allows for introduction of foreign DNA into bacteria cells in order to for example propagate the DNA or express a protein of interest.

 What: Transformation of E.coli strain TOP10 with pEGFPN1 plasmid containing MCM2 cDNA (pEGFPN1-MCM2).

Transforming plasmid DNA into bacteria cells is fast and easy. Of course we need bacteria cells and for that purpose different E. coli strains (such as TOP10, DH5alpha or others) are usually used. These cells are previously prepared in order to receive the DNA in the process that makes bacteria cells "competent". Such competency is nothing more than making the bacterial cell wall transiently penetrable (pores in the bacterial cell wall). Such condition of bacterial "coatings" allows for the take up of the DNA. Ok, lets start then. On the Figure below you can see an outline of the bacterial transformation process.

First competent bacteria are prepared using variety of different methods. These cells are then snap frozen using liquid nitrogen and stored at -80C to -150C to preserve to competent state. Once the cells are needed they are placed onto ice to defrost. On ice the cells are still competent but their ability to uptake DNA decreases with time and with increasing the temperature. Next, cells are mixed with DNA, in our case the DNA is the pEGFPN1-MCM2 plasmid. If the DNA that is used for transformation is a pure plasmid we do not need to use a lot but if the DNA we are using is for example DNA ligation reaction, we should use as much as possible to increase a chance of getting our DNA into the cells. After addition of the DNA the bacteria-DNA mix is incubated on ice for 20-30min in order to create "bacteria-DNA complexes". Next, the reaction is transfered to 42C (usually a water bath or hot plate) and incubated for a short time such as 90s. This step called heat shock opens previously introduced pores (these appeared when cells were made competent) and allows up-taking DNA that was previously in the contact with bacterial cell wall.After the heat shock there is a time for cold shock at 4C to close the pores and traps the DNA inside bacterial cells. After the "shocks" cells are allowed to recover by addition of fresh media and incubation at 37C shaking for approximately two cell cycles which in case of E coli is approximately 40min. Cells are then seeded onto plates containing appropriate antibiotic, in our case it is the Kanamycin and plates are the placed in the 37C incubator; and incubated over night. The amount of the cells seeded also depends on the DNA that was transformed. As previously mentioned, if the DNA was a pure plasmid we can seed very little (approximately 5-10% of the transformation) but if the DNA came from the ligation reaction we can seed all the transformation to be sure we get colonies back Next day, the colonies appear on the plates. Single colony is then picked up and expanded as a culture. Such culture can be then used to isolate bigger quantities of the DNA which later can be used for other purposes.

I hope you enjoyed my come back:)


1 comment:

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