The transformed cells are then plated onto a selective media for propagation and formation. Streaking of the inoculated transformed host cells is done on the media to ends single colony formation.
The culture plate with the inoculated transformed E. coli cells are incu at 37° C in an incubator for growth and formation of single colony clones. STEP9. Screening and selection of the correct host cell clone containing the recombi plasmids:
The construction of the recombinant DNA and the transformation of E. coli cells | recombinant DNA are very delicate and sensitive processes. The percentage of success in the processes is extremely low. Secondly the success of these processes cannot be monil under a microscope. There may be three possible outcomes from these manipulating proce
1. Some E. coli cells may not undergo transformation by the cloning plasmids.
2. Some E. coli cells may be transformed by unrecombined cloning plasmids.
3. Some E. coli cells may be transformed by correct recombinant plasmids.
In the event of the above-mentioned circumstances, it becomes imperative to locate the clones of E. coli having the incorporated recombinant plasmid. This process is known as screening of the clones for the right one. Following screening, the clones other than the right ones are discarded for further experimentation. The primary step in screening is replica plating.
Replica Plating: The culture plate containing the inoculated transformed E. coli cells is known as the master plate. This plate has to be conserved and propagated for future experiments involving the E. coli cells transformed by the recombinant plasmids.
Any screening has to be made on a copy / replica of the master plate. The process of raising a replica of the master plate is known as replica plating. A sterile muslin cloth is tied onto a Petri plate that can just fit to the master plate. It is gently placed on the master plate so that a few cells from each of the clones adhere to the muslin cloth at the same relative positions.
The muslin cloth is then gently placed on a freshly prepared culture plate. This transfers the cells of the clones to the same relative positions on the culture plate as those of the master plate. The new plate is a replica of the master plate and is known as a replica plate.
Screening and Selection: Many method of genetic screening of the recombinants are avail The type of screening depends upon the genetic markers present in the plasmid. Insert inactivation is the most common method of screening for the right clone of host cells.
Genes have their functional integrity as long as they intact. A gene can not express when it is interrupted by another segment of DNA. This phenome is called insertional inactivation.
For example, the gene insert is introduced into a restriction site in the tee gene of pub The amp’ gene is intact and therefore, it can express itself, whereas the tee is interrupted by gene insert and therefore cannot be expressed. The Table 5.1 explains the whole story. In first case, there is no plasmid.
Such a bacterial cell clone is sensitive both to ampicillin tetracycline. In the second case, the host cells contain the uncombined plasmid. Both them’ are intact. Bacteria of such a clone are resistant to both the antibiotic. In the third case, a insert is introduced into the tee gene.
This gene breaks apart and cannot synthesize tetras resistance factor. In such a case, the host cells harbouring them are resistant to ampicillin sensitive to tetracycline. The cell clones, which are resistant to ampicillin and sensitive to tetrac are selected, and other clones are discarded. All this screening procedure is conducted on re plates.
Mass screening of a population of clones by colony hybridization : This method is applied to the mass screening of a cloned gene library.
The bacterial colonies carrying the recombinant vectors are lyses on nitrocellulose filters or nylon membrane. The DNA is denatured and fixed on the membrane.
The filter is then hybridized with a radioactively labeled probe that represents the desired sequence. A colony, where it occurs, is seen as a dark spot by autoradiography.
The recombinant vectors can be retrieved from the corresponding bacterial colony for further analysis. Remember that the colony hybridization procedure is executed on a replica plate construct. The master plate is kept uncontaminated for future reference.