Hi Els, Have you tried to use low temperature after induction by IPTG such as 15-28C, or air availability by increasing or decreasing the rotation and medium  to flask volume ratio?

Good luck 

I may be completely wrong, but this erratic behaviour makes me wonder if the cell lysis is in fact not because of the DNA polymerase expression, especially since it sometimes occurs even before you induce. What comes to my mind when I read this is that it reminds me a lot of problems I faced in the past that were caused by bacteriophage infection in my cultures. Growth proceeded nicely up until around OD 1, and then rapidly dropped (within 0.5-1h), either before or just after addition of inducer. Have you tried looking for phages, for instance using a plaque forming agar plate assay?

Hi Els

I would try to grow your culture at lower temperatures as Paul suggested. I have a protein that is toxic to the cells and i express a small amount overnight at 37 degrees then when i inoculate in the morning i drop it to 32 degrees then when it reaches OD600 at 0.8 i induce with IPTG i drop it further to 28 degrees. it works every time for me.

Thank you for thinking with me! I try the lower temperature option and when this doesn't help, I may switch to a pLacI system.

Hi Els, Tryptone may contain lactose which can cause the promotor leakage before induction. This can be stopped by adding 0.3% glucose to the growing medium. MIght also do the trick.

Lower temps, eliminating phage, and pLacI are all good suggestions. I also have heard many complaints about using pLysS, which itself can be fairly detrimental to cells. Lucigen supplies a BL21 strain that contains a LacI plasmid, if you decide to try that approach.

Martin Gustavsson, your answer about phage contamination worries me a bit. Where can I find a protocol to check if I have bacteriophage infection?

I understand your concern, since dealing with phage contamination is not fun. Luckily, testing if you do indeed have lytic phages is quite simple and quick, provided you have a culture that shows the lysis you describe. Unfortunately I don't have a detailed protocol for this, but the traditional way is to do a plaque forming test on agar plates. I think if you search for phage plaque test you will find suitable protocols.

In brief what you do is you take your culture that you suspect have been lysed by phages and make a dilution series of the supernatant, similarly to how you would do if you where plating to check the number of colony forming units. Then you mix the dilution series with a healthy cell sample (either a growing culture or if you prefer you can use cells from a glycerol stock), put this mixture into soft nutrient agar at 50°C (I don't remember the percent agar you need to have in the soft agar, but lower than you use for normal agar plates), and spread the sofa agar with your cell + lysate mixture on standard LB or nutrient agar plates. Samples without phages are expected to make the entire soft agar overlay cloudy as the cells grow. If you have lytic phage contamination you will observe clear zones, with each clear zone theoretically corresponding to 1 lytic phage in your sample. By making the dilution series you can count these clear zones in the same way you would normally count the formation of bacterial colonies, thereby giving you an estimate of the number of phages. Hopefully your observations are not due to phage contamination, in which case you should see no zones of lysis in this assay.

I would recommend you to try synthetic media instead of rich media to prevent promotor leakage.

There are good suggestion above

1. virus contamination is an issue that I have had previously and cells were lysing in culture- Prepare fresh plasmid using thoroughly clean labware 

2. Leaky promoters are a problem with T7: you can achieve tighter control using 0.3% glucose on plates and in culture media as suggested. When ready to induce spin cells and refresh media (- glucose)  or pLacI as suggested (I have not used this)

3. I take it that you transform your plasmid each time into BL21 cells otherwise expression will be variable

good luck

Hi Els, I am also expressing a toxic (transmembrane) protein in E. coli. I am trying a number of different media at low temperature (20oC) overnight induction as I type this. I will definitely let you know which medium worked best for me. I doubt its phage contamination, I think it has more to do with the toxicity of the protein and the fast rate of production at 37, as I am experiencing this as well. Also, do you use glucose to prevent leaky expression from your expression vector?

We tried a new expression at lower temperature (and as a reference a culture without IPTG induction). As the non-expressing culture is continuing growing, the expressing culture is having a constant OD after about 3 hours induction. We harvest at this point.

Maybe the glucose option in the medium is an option we can try. How does this work, do you have to add fresh medium (so without glucose) when you start induction with IPTG?

Hi,

Glucose needs to be in the agar when plating transformations as with a leaky promoter your product (if toxic) will slow the growth of cells. It is possible that you are selecting cells from the agar plate that produce less product (less toxicity) and therefore grow quicker than other higher expressors. You could set up a control plate with no glucose to see differences in colony numbers.

Pick 3 isolated colonies and grow on in media + 0.3% glucose (+antibiotic). Pellet cells and resuspend in warmed media +/- glucose (OD ~0.5).  What you ideally need to do is grow your cells to a high density in glucose before induction since your product is toxic (OD ~1.5). Pellet the cells then resuspend in media (- glucose/+ IPTG).  Test expression over time.

The other option is not to change the media but just to grow to high OD + glucose then add IPTG. The glucose will be used up by the cells and concentration will fall, the high IPTG levels will eventually overcome the inhibition by glucose:  It is reported that you should still get expression even in the presence of glucose with this system so it is worth a try.

Controls -IPTG are a good idea and need to be run

Be aware that glucose may not be the answer since control of catabolite repression is supposed to be tight in this system But worth a go. Good luck

Hi again Els, I tried 2 minimal mediums (M9 minimal medium and Davis MM) and 2 rich mediums (LB and Terrific broth). I started growth in all mediums with glucose added (2mM final conc)  at 37 for 2 h, followed by IPTG addition (to growing cultures) then lowered growth temp to 20oC for 16 h when I harvest and assay for expression. I got the best results in M9 followed by Davis MM. In your case since the protein may be more toxic than mine, it may help to grow them to .5 or .6 OD600 before induction.

I also do add glucose to the medium at all prior steps (transformation, o/n cultures etc) as suggested above by Ian.

Hi Els,

All above suggestions are very good! Media really matters and must have no lactose whatsoever. Addition of Glucose is not enough although bugs would prefer glucose over lactose as an energy source but not 100%. I had best luck with Sigma's Vegitone media. Both LB broth Vegitone or Tryptic Soy Vegitone worked equally well when I was expressing ribosome inactivating toxin that generally kills the bacteria and prevents any expression. Avoid freezing the transfectants between your growth/induction attempts. The recovery of viable bacteria with intact toxic insert from frozen stocks is generally very low, so plan on producing numerous bug pellets from one successful transformation and prepare fresh transformants for a new production cycle. They are good for about 2 weeks on the plates at +4C. pLysS is not nearly as good as pLacI to control T7 promoter and at 37C not any lower as pLacI also needs to be expressed to suppress T7. Lowering the temp will lower the expression of pLacI and thus promoter control. Also I had much better yields from k12/pLacI strain than BL21/pLacI. Good luck.

Hi, we will definitely try the glucose for a better selection. The minimal media really sound promising. I keep it in mind. Thanks again! 

Andrew, thank you for your answer. However, I don't completely understand your explanation of pLacI. Do you prefer this strain over pLysS?

Hi Els,

The DE3 pLacI hosts have a chromosomal copy of pLacI gene and its expression is driven by lacUV5 promoter constitutively and independently from IPTG induction. As lacI protein is being constantly synthesized it binds and blocks lac operon in your plasmid. Blocking lac operon prevents activity of the T7 promoter that in turn blocks expression of your target sequence. The higher the temperature (up to 37C, higher temp (42C) is stressful and will decrease the growth rate of bacteria), the more lacI is being  synthesized, the better the control of the lac operon and the lower T7 promoter activation. When IPTG (analogue of lactose) is added to induce the T7 promoter, it overrides lacI and T7 promoter becomes active -> the expression of your target sequence starts.

pLysS is very different  - it is also a separate add-on plasmid coding for an antisense copy of Lysozyme. Despite of antisense orientation Lysozyme is still being produced in low quantities and it is a natural inhibitor of T7 RNA polymerase. It does work to keep the basal expression to a minimum, but just not as well as placI does. The difference between these two ways to control T7 promoter will not be noticeable if your target gene is not toxic to the host but will become crucially important if is is toxic and any premature expression must be prevented for the host to grow uninhibited up until IPTG induction. So the choice of pLacI host over pLysS is not a preference but a necessity in "toxic"cases.

Andrew

Adding glucose and expression at 30deg did the trick for us. Thank you for your contributions!