Hi everyone
I am growing E. coli to express a novel heterologous protein. At first I was getting good expression with similar growth rates to my controls (protein expressed in the same E. coli strain). Recently, however my heterolog is not appearing on my PAGE gels and I have seen a dramatic difference in growth rate for my E. coli containing my modified protein (my controls are still growing at the same rate but I need to still check the protein quality as the control protein did not appear clearly on the gel either). The slow growth rate is present in E. coli that has be newly transformed with the original modifed plasmid.
My target protein does posses a tag that is meant to transport the heterolog across the periplasmic membrane. This has lead to a marginal reduction in growth rate in the past (compared to both + and - controls) but nothing like this before.
Some suggestions of the cause are:
Changes in tryptone or yeast extract composition (different batches) used to make up LB growth medium (i.e. there is a specific amino acid that is limited).
Preparation or degradation of glycerol stocks or plasmid mutation.
So far my plan of action is to isolate the plasmid and send it off for sequencing and in the meantime do a PCR of my construct as well as an RE digest to see if the region is still present and the right size. I will also test on a different growth medium, as well as remake the LB with a different batch of Tryptone or Yeast Extract.
Any other suggestions as to what I should do or possible explanations as to why my bacteria and construct would suddenly behave this way would be appreciated.
Dear Matthew
I suspect that you have basal expression (expression with out induction) and your specific gene result toxic for the e.coli and therefore it affect the growth of the strain.
which plasmid and e.coli strain ante you using? pet and bl21(de3) which growth media ? lb ?
if I'm right you can solve the growth issue by :
- changing the e.coli strain to one better repressed (e.g. plyss strain)
- change the growth media using a media thay thanks to the presence of the glucose may guarantee high repression (e.g. enpresso media)
you can find some more information about enpresso on my blog: ProteoCool https://proteocool.blogspot.com/
at page 2:Protein expression
ciao
Manuele
I don't quite get all details of the problem. From what I understand, you have an expression plasmid (is it T7-based?) which is derived from a previous expression plasmid. In this new plasmid, the heterologous gene was modified by fusion to a tag-coding sequence (a signal peptide, it seems?). When the expression strain is transformed with the new plasmid, its growth rate is slower than that of the same strain transformed in parallel with the old plasmid and grown under exactly the same conditions, and worse, it does not express the target protein.
Did I get it right?
Manuele Martinelli I will definitely check that out now, thank you. Sorry for the lack of detail but you are correct in most regards. I am using a pet expression system on BL21(DE3). I am retrying the growth on an enpresso growth medium now to see if my results reflect what I was getting previously.
Alejandro Martin The expression system is a pET expression system and I am inducing with IPTG as well as reducing the temperature after induction (growing up at 37C and reducing to 30C at induction). I have previous evidence that this is optimimal for temp and IPTG conc. (100uM or 1000uM IPTG). I am using a signal peptide to investigate protein transport of heterologous proteins.
Both my old glycerol stock and my newly transformed E. coli show a reduced growth rate compared to previous experiments and my controls (run in parallel). And this is only a recent observation. In March of this year my bacteria was growing fine alongside my controls and producing my target protein.
Thank you for the comments
-Matt
Matthew Fisher ,
Oh OK now I see, thanks. So, cultures made from the glycerol stocks of both the new plasmid and the old plasmid have stopped expressing and exhibit lower growth rates than they usually did.
Sometimes T7-based constructs stop expressing misteriously once their glycerol stocks have been stored long enough (This is gene-specific, it is not a random thing). However, they actually grow faster, not slower, once they're free from the burden of expressing a heterologous protein. So that's not the case here.
Signal peptides can be toxic if the sequence surrounding the cleavage site is less than optimal (the resulting protein may jam the protein export machinery). However your signal peptide-bearing clone was doing fine before the problem appeared, so this is not the issue either.
I agree with Manuele Martinelli that the problem is likely caused by poor repression of basal expression, maybe brought about by low-level lactose contamination of new batches of tryptone. High basal expression does select for non-expressing mutants during growth (this is specially true in T7 systems) so that would explain what you are seeing. Manuele it the nail on the head; I agree that switching to Enpresso will probably do the trick. An alternative that you may try in your situation is to add glucose in the 0.2 to 2% range to regular LB; this should help in repressing basal expression through inducer exclusion.
Hope it helps!
Dear MAtthew
just one more question
what do you mean for reduced growth rate?
Are the bacteria able to reach high OD but in longer time or that at one point the bacterial growth stop and that the OD start to decrease?
Ciao
Manuele
Dear Alejandro Martin
My supervisor mention that some sort of change to the glycerol stock may have occurred but I agree that it should show a higher growth rate not a reduced growth rate. I will try adding 0.2-2% glucose to help with the basal expression.
Dear Manuele Martinelli
I have grown up on enpresso medium but have not had time to analyse the protein. I have a presentation coming up so I will only get to that next week maybe.
The slow growth rate I observe is my cultures taking a long time to reach a high OD (they still continuously grow and I do not observe a decrease in OD). One thing I observed on enpresso is still a reduced growth rate but at a later endpoint (approx. 13hrs) my construct showed a very high OD (higher than my control bacterium). This indicates to me that it is likely a growth medium problem (and also a toxicity problem). But as I said before I havent anaylsed the protein content yet from the enpresso experiment. I will let you both know as soon as I have done a PAGE of my samples.
Plasmids are so fussy in yeast, that if I want reliable expression? I just use CRISPR and put the target in the genome with a strong constitutive promoter (yeast GDP promoter in my case) and a terminator sequence flanking the template. I use plasmids regularly, of course, for everyday cloning - but for a cell factory they are unreliable. I have had e. coli and yeast strains come out of glycerol storage and grow very slowly ie nearly dead. Since I added trehalose and dmso to the glycerol cryo protocol, this doesn't happen as much.
Hi Just an update to this thread:
PCR and sequence data are fine. So my genetic material in the plasmid itself is not the issue.
I still cant express my target protein to similar levels like I was getting before. I am seeing feint bands on my SDS PAGE gels where as before I was getting very strong bands under the same conditions (37C, 1000uM IPTG).
Growing up on Enpresso medium has been inconclusive. I am getting smeared gels so I think my protein has been going insoluble and my lysis buffer is not completely solubility my protein. I am going to try and redissolve my samples in 3M urea to see if that helps.
Growing on LB (under the standard conditions above) and seeing if my protein resided in the insoluble fraction (dissolved lysate pellet in 3M Urea and 1xPBS). It seems as though my protein is still not being expressed under these conditions.
I managed to fix my growth rate problem:
- Before I was adding 50uL of glycerol stock (1:1 mix of 50% glycerol and LB culture grown to about 0.8). This was the problem, too much starting culture.
- Solution: pick up a small amount of culture with a pippette tip.
- I only used the above method as a suggestion to fix my expression problems but it clearly can cause problems with my overnight cultures.
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