different colonies produce different amount of protein... - (Feb/21/2006 )

i have presented my data in the seminar (playing with IPTG concentration and timing temp etc to get more protein) and my professor has told me to rather do different colonies to check which one produces more protein... i always thought that they all should produce the same amount....am I wrong? please explain this to me.....your help is always much appreciated....

i'm not very familiar with bacteria expression, but it's a fact : in eukaryotic cells, expression differs from one clone to an other. In bacteria, the copy number of plasmid may change which explain basical changes.

I agree with Fred.
You should check for the clone that express the best.
if you check in qiagen website, there are handboodks, you can find interesting information how to transfer some colonies from agar plate to nitrocellulose membrane, add IPTG, lyse, and do western-blot directly, then you directly see which clone produces the best, instead checking which clone is well transformed, culture miniprep, check which one has the right plasmid with the right insert...

thanx a lot everyone! ill try that membrane methods it sound great!

Dear Katty,

i work since 6 years on bacterial expression protein, i never seen any more in litterarure any paper refering to copy number role in bacterial expression, in contrasr with eucaryotr microorganisms the plasmid containing the cDNA of your target gen dosen't integrate the bacteria genome, so for all bacteria clones you have the same recombinant plasmid yield! if you want to check hwhat i'm saying, you can run a production step of 10 recombiant clones, but you must stop expression at an identical D.O600nm (exp: 2=O.D600nm) for the all 10 clones.
good luk