Re: The chemistry behind transformation

Mandy:

I think this is a fantastic question. There is a ton of information to cover here, so please be patient, and I will try and give you the basics and lead you to some places where you can flesh out your knowledge a little if you wish.

First of all, to discuss the chemistry of transformation would probably require a book in and of itself. Unlike the atomic world of chemical reactions you are studying now, this is a biological process involving dozens of proteins, each containing tens of thousands of carbon, nitrogen and oxygen atoms. I think it would be nearly impossible to dissect each chemical step in any of several transformations within the space or time available.

Transformation is the process by which organisms (In this case bacteria) take up nucleic acids. Transformation was first discovered by Griffith in 1928 in strains of avirulent and virulent Streptococcus pneumoniae that could kill mice. He observed that extracts of killed virulent bacteria would enable avirulent strains to cause disease. Some bacteria, such as Bacillus subtilis do this naturally, and others such as the E. coli used in the pGLO kit, must be made to take up DNA, which we call competence. E.g. "Those cells are competent." This means they are able to take in DNA/RNA.

The process by which this happens varies greatly, but the three major methods used are

1. conjugation
2. chemically induced competence and
3. electroporation, which uses a pulse of electricity to put the DNA in the cells.

In chemical competence, the cells to be transformed are treated with a series of chemicals that make their membranes 'leaky' to DNA. This is typically a divalent salt such as CaCl₂ and buffers such as phosphate. After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.

In electrocompetence, the cells are prepared under conditions to remove all salt from the media. Typically they are washed multiple times in glycerol baths and frozen away for use. The cells are thawed, placed in a tube that has metal sides and salt-free DNA is added. An electrical pulse is applied that decays very quickly (a few milliamps for a few milliseconds), and this causes the cells to very quickly shake open and become leaky so the DNA can enter the cell. Electroporation, as it is called, has the advantage of not being so abusive to the bacteria, so typically, more bacteria containing DNA are recovered, (The efficiency is higher) which is important for many applications where you have a limited quantity of DNA to transform, or would like good representation of a library of DNA you are transforming. After transformation, the cells are grown for a short while, and plated on selective media to recover transformed bacteria.

I would like to discuss selection now too, briefly. When we transform, we have millions of cells in a small volume, and we add DNA. We need some way to separate the cells that recieved DNA from those that did not. If we just plated the transformation reaction, cells with and without DNA would both grow and the plate would be covered. (Just like the -pGLO picture on the regular LB plate. So, in order to do this, we usually have something on the DNA we are transforming that is necessary for survival of the bacteria. Often times this is an antibiotic resistance gene, and if we grow the bacteria with the antibiotic, such as ampicillin, only those bacteria that actually got the DNA (And the resistance gene) will grow. One can also use a gene that corrects a nutrition defect or something similar, but the idea is the same.

In the case of pGLO, by transforming the plasmid containing the GPF protein on it, which can be turned on by growing the cells in the sugar arabinose, you can see your plasmid on and off, and the antibiotic is used to only grow those cells that recieved the DNA. You also asked if pGLO has been used in real life situations, and I can tell you probably not. pGLO is used as a teaching plasmid for the process of selection, expression and transformation, but all of these as well as GFP are used on a daily basis and have been absolutely crucial to modern molecular biology. These developments allow for the trnasfering, modification, and expression of proteins and genes and GFP allows us to look into a cell and see 'where' things are actually located. Most developments in molecular biology and medical research have been supported by laboratory work that utilizes transformation and expression. Transformation, PCR, restriction enzymes, and antibodies are the basis of molecular biology.

This process has become big business. Many companies essentially provide products for the exclusive use of transforming. They make better plasmids, more competent cells, simpler protocols etc. etc. I have listed their URL's below if you are interested.

If you plan on making your own competent cells, one of the easiest protocols is a chemical method developed by Chung et al. and published in PNAS (Proceedings of the National Academy of Science; reference below). This might be cheaper than ordering cells, but quality control is typically very good at companies so reproducibility and reliability are not as much of an issue. If you need plasmids, most scientists would be glad to send you something, or help you design something similar to pGLO, which is only $50 in the US. I hope this information was helpful, let us know if you need anything else.

-Matt Champion-

PNAS Reference: One-step preparation of competent Escherchia coli: Transformation and storage of bacterial cells in the same solution Chung, C.T. Niemela, S.L., Miller, R.H. PNAS v86 pp2172-2175 April, 1989.

Protocols online: http://www.protocol-online.net
Biorad http://www.biorad.com
Stratagene http://www.stratagene.com
Promega http://www.promega.com
Invitrogen http://www.invitrogen.com