1. What would happen to the plasmid copy number of the F plasmid and ColE1 plasmid if you added the following antibiotics to an E. coli culture (the strain is sensitive to the antibiotics, but the question is not what happens if the culture dies or stops growing - it's how does the antibiotic affect the plasmid). In a sentence explain your answer. Hint - at one time one of these treatments was used to aid in plasmid extraction.

A. ColE1 - Rifampin
Since rifampin will stop transcription and since RNA-II is essential for initiation of DNA replication for ColE1, copy number will decrease.

B. F plasmid - Rifampin
Even though F plasmid replication is protein based (RepE), where do proteins come from? mRNA! So rifampin would decrease the copy number of F, although in a less direct manner than for ColE1.

C. ColE1 - Chloramphenicol
There will be no detrimental effect. In fact, since de novo protein synthesis is not required for DNA replication (the polymerases, etc., are stable proteins) and since the Rop protein is inhibitory, the copy number will increase. This is where chloramphenicol can be used to increase plasmid yields for extraction and purification.

D. F plasmid - Chloramphenicol
Since the RepE protein is essential for plasmid replication, copy number will decrease.

2. For the F plasmid describe what would happen to copy number of the plasmids if the binding site for RepE at the promoter for repE and [not the ori] was mutated to possess lower affinity for the protein.
Since RepE binds to and activates its own promoter, less affinity and less binding would cause less RepE to be made and copy number would decrease.

3. Which of the following loci are necessarily cis-active? (Check all boxes that are correct.)
copA/incC of F (since coupling involves joining plasmids, this must be on the plasmid)
RNA-II of ColE1 (I find it hard to believe that RNA-II would be able to base-pair with the plasmid DNA if it was provided in trans.  I don't know if the experiment has been done.)
rop of ColE1 
oriV of F

4. What factors would determine the host range of an RNA regulated vs. protein regulated plasmid?

For RNA-regulated and initiated plasmids, it would primarily be the recognition of the promoter for the RNA-II equivalent.  This would also assume that RNase H would also work from the host cell.  For protein-requiring plasmids such as F, not only does the promoter for the repE equivalent have to be recognized by the host RNA polymerase, but the translation for the RepE equivalent must also be functional in the host cell.  These are not necessarily givens.  You could also take into consideration if you can get the plasmid into the recipient cell in the first place.  Not all bacteria are transformable and not all can act as recipients for conjugation with all plasmids.

5.  So called allelic exchange suicide plasmids are based on lack of an essential protein for replication on the plasmid.  How can you construct a suicide plasmid using the RNA regulated model?

You simply would put a very tightly regulated promoter in place of the RNA-II equivalent promoter so that it is not expressed unless induced.  We will discuss tightly regulated promoters.  It is difficult to get some that are absolutely zero, even when not induced.  You would induce expression in your donor strain and not induce in the recipient.  You could consider using an alternative sigma factor in your donor strain that would not be present in the recipient, or you could use a phage RNA polymerase in the donor that would not be present in the recipient.