I have cloned B. subtilis endogenous genes in pHTO1 plasmid to overexpress them in B. subtilis. Real-time study is showing overexpression of the genes in B. subtilis, but there is no band corresponding to the overexpressed proteins when cell lysate is subjected to SDS-PAGE. Transformed cultures are induced with 1mM IPTG at an OD600 = 0.6-0.8. protein samples are taken out after 12 hrs of induction. Chloramphenicol 5mg/L is also used in all induction medium.
I would start by doing a western blot with lysates taken at different time points after induction as early as half hour after. Real-time PCR can be very sensitive so you can pick up even trace expression. SDS-PAGE on the othe hand needs a lot more protein for detection. Also check if there is a chance of protein dimerization or possible post translational modification which sometimes changes the way a protein runs on the gel despite SDS denaturation. Good luck!
RT-PCR is highly sensitive and traces can be detected. I would suggest that you should go for western blot by harvesting the cells at different time points. some proteins have very small life span and they get degraded very quickly and the way you are studying proteins after 12 hrs it may be the case that the expressed protein may get degraded.
Hi Suresh.
You can detect the Protein, but it is not overexpressed or stronger present as in the controls - is that right? Or dont you see the protein at all?
There are many "standard" things you can try.
1: Different inducing ODs like 0.4 or even 0.25 depending on the B.-subtilis-strain you use.
2: Try different amounts of IPTG, like a series from 5 mM to 0.2 mM.
3: Look at the amino-acid-sequenz of your protein. Maybe it is degraded by "special" proteases, so you have to use additionally protease-inhibitors like PMSF, metallo-proteases-Inhibitors etc... for the lysis-buffer of the bacterial slurry.
4: reduce antibiotica-conzentration in your main culture (for inducing) to 25% of the normal concentration.
5: take samples after different after inducing... like the others propose.
Do you reduce the temperature of the incubator, after inducing with IPTG?
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