Question

a.) In conjugation, if E. coli is the donor strain and S. marcescens is the the recipient strain, why is it important that the recipient strain does not contain the pir gene.

b.) typically, transposase gene (tnp) are inside the transposon. Why is it important that the tnp gene fall outside of the transposon?

c.) You want to use the pRL27 plasmid and the E. coli donor strain to mutagenize another strain of E.coli. What genetic/ phenotypic characteristics would need to be different between the E.coli donor and an E.coli recipient strain, and why ? Hint: think about characteristics of the S. marcescens that are required to male this experiment work.

Answer 1

a) pir gene, encodes pi protein , necessary for replication of R6K plasmid (a plasmid used in construction of conditionally replicative cloning vectors). pi protein and gamma ori origin of replication constitute the R6K minimal replicon. pi protein is a replication initiator at low gamma ori levels as well as a replication inhibitor at high gamma ori levels.It is important for maintenance and replication of these conditionally replicative plasmids that pir protein is expressed in trans (from a lambda phage or another plasmid), which is possible if recipient strain doesn't contain pir gene. If the recipient strains harbors a wild type pir gene, the copy number of plasmid comprising oriR6kgamma would be quite less.

b) Transposase gene encodes transposase enzyme which in turn binds to the end of a transposon and enables its movement to another part of the genome. Transposons like Tn5 contain two sequences, OE (outside end) and IE (Inside end) to which transposases bind and facilitate their movement. IE if often methylated blocking recognition by transposase. Some transposons like Tn5 have a preference to act on cis sites to the sites of their synthesius, thus, these should fall outside the transposon.

c) Conjugation can enable donor E.coli strain to mutagenize another E.coli strain via plasmid pRL27. For this, the donor strain should harbor an F factor (fertility factor) while the recipient E.coli strain should not harbor it. pRL27 contains transposon Tn5 which harbors kanamycin resistance gene. The recipient strain should be kanamycin sensitive. It would develop kanamycin resistance upon conjugational transfer of pRL27 plasmid.