Hi there,
I tried to express a protein thought to be a kind of acetyltransferase by using Rosetta-receptive E. coli.The 1L bacterial solution was very thick before induction.And I added 300 ul 1M ITPG in it.16 hours later, the bacterial solution became somewhat clearer compared to the state at induction.After I centrifuged the bacterial solution at 4000 rpm, it appeared that there was a large amount of cellular debris hanging on the centrifuge bottle. It appears that maybe 1/3 of the expected E. coli could be collected.This occurred three times.What are the reasons for this situation？Phage contamination or protein toxicity?Plz help me.
In addition：
This protein is belong to GNAT super family.Induction OD was 0.7 and the induction temperature was 18℃.
It is hard to state without a lot more information. But you can do a few controls, for example the same strain but with a control plasmid that has no insert, and also treat your test strain the same way but without added IPTG and let grow the extra 16 hours.
Dear Tim Lee
Cell lisys during the induction may suggest presence of phage but it is something that may happen also due to gene toxicity so it is not so simple to undertand which is the problema.
just some question to undertand:
what do you mean for very thick before induction?"
Can you provide some more details about. Induction OD, induction temperature?
Phage contamination could be assessed performing Plaque assay (https://microbeonline.com/phage-plaque-assay-principle-procedure-results/) it is labororious but not impossible protocol.)
good luck
Manuele
As you told about the cell reduction after induction it may be caused by gene toxicity. In that case you can try PlyS BL21 E.coli variant which can tightly express toxic genes too. And one another thing you can do is change those parameters as temp , IPTG concentration to check the induction . Do it alternatively in small culture first then go for large culture. And one thing what's is your protein size and PI that also important in case of expression.
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