terrible clonning - (Feb/23/2008 )
I've been trying to clone gene E (E-lysis) from bacteriophage phix174 under the control of thermoinducible promoter. my plasmid is chloramphenicle resistant. I want to know wheather this antibiotic leads to bacterial prelysis before thermal upshift or not.
Chloroamphenicol inhibits protein expression. It does not cause cell lysis. An old trick in molecular biology is to thrown in some chloroamphenol just prior to harversting. The cells strop growing but plasmid replication continues, increasing plasmid yeilds.
The cmR gene should be under a constituitive promoter in your plasmids. Thus thermal upshift should not play a role at all.
In this problem I think you have to examine
- how confident you are at the your cloning abilities. Cloning failure might be cause by regular cloning problems.
- how tightly regulated is your promoter. From the name of your gene, it sounds like you are expressing a lysis protein. If the thermal sensitive promoter is leaky you might be experience low levels of expression, resulting in cell lysis.