Question

6. The cloning strategy outlined below has been proposed as a means of moving the CHMO gene out of pMM2 and into pVector. Plasmids pVector and pMM2 will be individually digested with restriction enzymes BamHI and SphI, then the resulting mixtures will be combined so that a 2 : 1 molar ratio of the CHMO gene and digested pVector is obtained, then this mixture will be treated with T4 DNA ligase plus ATP and subsequently used to transform E. coli cells with selection for resistance to the antibiotic ampicillin (without prior digestion with EcoRI). What plasmid(s) would you expect to find in the transformed E. coli cells? (5 points). Why would treating the post-ligase mixture with EcoRI prior to transforming E. coli cells increase the probability of obtaining the desired plasmid (one in which the CHMO gene has been inserted into pVector)? (5 points). Be specific in your answer.

Answer 1

• If the E. coli cells are transformed without treating the ligase mixture with EcoRI than we would expect to find E. coli cells that contain either the pVector that contains its own fragment of DNA which was earlier released due to treatment with EcoRI and SphI or the pVector plasmid containing the CHMO gene.
• Few cells may also contain self-ligated vector, although this occurs at a very low rate since one site is BamHIdigested while the other is SphI digested. Also, pMM2 plasmid will not be present in any of the cells because this plasmid lacks the ampicillin resistance marker and therefore cells possessing this plasmid will not be able to grow on ampicillin.
• Treating post ligase mixture with EcoRI will increase the probability of obtaining pVector plasmids containing CHMO gene because when EcoRI is added it will further chop the fragment of DNA that has been released from pVector after treatment with BamHI and SphI.
• This is because this fragment contains a site for EcoRI and thus the fragment will be cleaved to generate two fragments which contains one BamHI or SphI compatible end and other EcoRI compatible end.
• In view of this the probability of ligation of these fragments back into the cleaved pVector plasmid is greatly reduced. Thus, the probability of obtaining transformed E. coli cells containing pVector with CHMO gene increases significantly.