Question

In an experiment, E.coli cells have been genetically modified through site directed mutagenesis. This is a process through which specific locations in an organism’s genome can be altered in order to study the function of such locations, or proteins coded for by genes found in a location. There are many techniques available for site directed mutagenesis. (You have access to PCR ingredients, any primer you wish, laboratory equipment, Lambda phage, and the ability to alter Lambda genome, a commercially available plasmid, such as Puc19, E.coli K-12, plates, media, etc..)

1. Design an experiment where you can synthesize an altered origin of replication of your target cell in-vitro.(4 points)

Answer 1

1. One need to first decide what is the sequence of the origin of replication that needs to be introduced. Correspondingly a vector and the insertional origin of replication need to be found out. One can design primers that includes this insertion of origin of replication at defined point. Then use original plasmid as template and insert the origin using either a Phusion polymerase and specific primers with origin. One can introduce the origin of replication either at multiple cloning site region, making sure that frame of the gene does not change. Or one can insert the origin immediately after a particular selection marker (should have restriction sites at both ends) such that both altered origin of replication and selection can take place parallely.

If the difference in the altered origin of replication is not much in terms of sequence, one can go on with Agilent s quick change protocol or NEBs site directed mutagensis kit. One need to design primer in such a way that the mutation is single/double but the primers should have some region of overlap with the vector so that when Phusion polymerase polymerizes the entire plasmid, the mutation is also included. Although this will show lower transformation efficiency, it is an effective way of obtaining a point mutation. Dpn1 needs to be used for template removal and then transformation of the mix can be perfomed.