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..)

2. A problem with ORI studies is that placing bacterial ORI on plasmids is usually lethal since plasmids occur in high copy number inside a cell. Why would this be? (3 points)

So, let’s say you need another way to get your altered ORI in side a cell, such that following entry the DNA will integrate itself into the host genome so that it can be maintained in the cell.

Answer 1

Answer to 2)

Plasmids can replicate autonomously leading to a high copy number if provided with an origin of replication.

1) The problem which arises on placing bacterial ORI on plasmids is usually that the plasmids utilise the bacterial replication machinery to the full potential to initiate and carry out replication from this ORI. Certain bacterial ORIs require DNA polymerase in large quantitities to generate plasmids in high copy number, therefore depriving the bacterial chromosome of DNA polymerase when required for replication.

2) Considering E.coli genome size to be 5.44 x 10⁶base pairs, it takes around 20 minutes for a bacterial cell to replicate its genome and divide into two daughter cells. Plasmids range from 2-16 x 10³ base pairs, accounting for only a fraction of a minute to get replicated. So in 20 mins E. coli generation time, there will be several thousand copies of the same plasmid. So the major polymerase activity will be focussed on the replication of the plasmids.

3) Additionally the expression of undesired gene products from the plasmid may turn out to be lethal for the bacteria. A high copy number of plasmid is also equivalent to multiple copies of the gene of interest. Deregulated transcription of the gene may lead to large quantities of the undesired gene product which may be lethal to the bacterial cell.

So the altered ORI can be introduced inside a bacterial cell with the help of the Lambda bacteriophage. The bacteriophage can be genetically modified such that the DNA sequence of interest carrying the ORI is positioned within the att site of the phage. During integration, the phage DNA, carrying the altered ORI, recombines in a site specific manner into the bacterial att site, located between the gal and bio operons. Now this altered ORI is integrated into the bacterial host genome and is maintained within the cell.