We are experiencing a decrease in protein expression in our BL21 DE3 strain. So far we were able to rule out an influence of i.e. IPTG (prepared new fresh stock), however we have no clue what else might lead to our issue.
We always freshly transform a BL21 aliquot, make a pre-culture, add this one to the main culture with a starting OD around 0.1-0.15, let them grow till OD 0.6-0.8, and induce with IPTG. However, when we control the expression after the usual expression period at 37°C, we realised that our bacteria aren't producing anymore as much protein as half a year ago. Does anyone has an idea which factors might influence the expression ability of bacteria?
The media is a self-prepared modified LB with Potassium Phosphate buffer at pH=6.8, always made max. 1-2 weeks in advance.
Best, Chris
Yes, it is common. If all other conditions are maintained correctly then it is most probably due to chromosomal mutation that is diminishing the level of functional T7 RNA polymerase, hence further causing the decreased expression of target protein from the plasmid. Solution: Purify your plasmid DNA from non-expressing strains and reintroduce your plasmid into fresh BL21 DE3. Do not use stored transformed cells over and over again (use within 2 weeks). Every time you plan to purify, use freshly transformed cells. Please let me know if it helps.
Mutation of the cloned Gene/ Gene responsible for the desired activity is the prominent case of decline of protein expression. Check the cloned Gene for mutation.
Dear Christoph,
We have faced similar difficulties while we tried using BL 21 DE3 cells from a frozen stock, transformed them, and attempted expression. As Ritu Rathi says, I assumed it be the T7 RNA polymerase's issue. But, when we used another expression strain (which was in use currently, unlike the frozen BL21), enhanced expression yields were observed. If you are sure about your plasmid and IPTG, I would recommend you to use a fresh sample of BL21.
- Badges
- Science method