TRANSFORMATION

This is one of my favorite topics, I love doing transformation experiments. So lets begin first of all with, what is transformation?

Simply speaking transformation is uptake of exogenous DNA by the cells from its surroundings. This  uptake may lead to genetic alteration in the recipient cells. For an organism to take up the DNA is must be in a state of competence, now naturally this may occur due to starvation or as a response to any other environmental condition but in labs we can induce competence by using chemicals.

Natural transformation ,as I said, requires that the cells be competent, and also obviously the presence of a foreign DNA in the environment. Along with these it also requires a protein machinery to help DNA cross the cell membrane. The proteins that are required  are those that are involved in the assembly of type IV pili and type II secretin system as well as DNA translocase complex that is present at the cytoplasmic membrane. Another important factor is the type of cell. The method of DNA uptake is different in Gram positive and Gram negative bacteria. This difference arises due to the differences in the cell structure. Normally, the DNA first binds to the surface of the competent cells and then with the help of membrane protein translocase it is translocated to the inside of the cell. In case the foreign DNA is double stranded it is converted to single strand by degrading the other strand with the help of nucleases enzyme. Then this single stranded exogeneous DNA is incoporated into the host chromosome with the help of Rec- A dependent process.

This process differs in the case of Gram negative organism as they have an extra cell envelope. So here the DNA requires an extra protein channel formed by secretins for the movement of DNA into the cell.

Transformation in laboratories can be induced by artificially making the cell competent and giving the recipient cells heat shock in order to transfer the exogeneous DNA into the cell.artificially the cell are made competent using calcium chloride. When you treat the cells with calcium chloride it increases the chance of transformation as positively charged calcium chloride attracts the negatively charged DNA present in the environment. Another artificial technique used to introduce the DNA into the cell is heat shock method. Here the calcium chloride treated cells are for some seconds given a heat shock or kept at higher temperature(42°C ), this opens pores on the surface of the cells helping the DNA to enter the cell. All the other steps in transformation procedure are done on ice to prevent damage of the DNA or the cells , only the heat shock stage sees a raise in temperature.

To study transformation in laboraties usually a host E.coli strain (usually DH5α, JM109) that carries a lac z deletion ( but has ω peptide ) is used  and a plasmid that carries a the lac z α sequence is transformed into it . Only when these two sequences of the lac z gene come together does it produce a β galactosidase enzyme that can breakdown X-gal(an analog of lactose ) to form 5-bromo 4-chloro indoxyl which spontaneously dimerizes and oxidizes to form 5,5-dibromo 4,4-dichloro indigo which is a blue coloured compound. This method in which the two gene sequences only can work together but not alone is known as α complementation.

Here, the whole transformation screening works by disrupting the α complementation process. This is because the plasmid also carries within the lac Z α sequence an MCS or multiple cloning site which can be cut by restriction enzymes and foreign gene can be inserted in  these sites , thereby disrupting the function of the α lacZ gene(no blue colonies will be produced). So simply speaking those cells which have the inserts of foreign gene will not produce blue colonies and those would be the cells that are transformed (white colonies). Those cells that produce the blue pigment will not have any inserts in them and will be the ones that are non transformants. Another important chemical in the lab experiment of transformation is IPTG(Isopropyl β-d-1-thiogalactopyranoside) , which acts as an inducer of the lac operon should also be added to the media in order to facilitate the conversion of X – gal to the blue coloured pigment.

I know its a bit complicated but u just need to read and understand it step by step, the correct transformation protocol used in laboratories will be shared in the next blog.

Please follow and like us:

3 thoughts on “TRANSFORMATION”

Leave a Reply

Your email address will not be published. Required fields are marked *