BL21DE3-PLysS cells give a slimy and sticky pellet...and have a pale wood yellow color? Is it ok to have a slimy cell pellet? I suspect probably a few cells (may be around 10 % or so) lyse during spin down @4 degree C causing a slimy cell texture. Has anyone else observed it? I have performed the cell culture thrice and observed the same slimy texture....Also my labmate observed the same texture for C41(DE3) PLysS cells? Is this something to do with the centrifuge bottle material? Or something more serious such as contamination or something common for PlysS cells
One probable explanation could be that a significant amount of your cells are getting lysed during harvesting by centrifugation and the release of deoxyribonucleic acid is the cause for the slimy texture. You could try to optimize your cell harvesting protocol and see if any changes occur. If you are working with protein over expression then just add some DNase with Magnesium ions and go for sonication to completely rupture the cells and break up the deoxynucleic acid.
@Aditya Jyoti Basak Thank you for your input....I feared that the cells may be lysing....I do not know if harvesting at room temperature will avoid it...but I shall try..
thank you.
Could be a range of reasons, but most reasonable explanation is that the cells are lysing and, as mentioned before, releasing DNA, which often makes the prep appear 'slimy'.
pLysS plasmid does reduce the structural rigidity of cells, allowing for easier release of cytoplasmic protein. Any Gram-negative cells carrying this plasmid will have reduced rigidity and may lyse during prolonged induction of protein production or freeze-thaw procedures. Not sure if centrifugation would also lyse the cells, but I would not be surprised if that was the case.
If you are trying to over-produce a protein, this should not be a problem, as following re-suspension and lysis using sonication the texture should be normal. Also, if protein production is the end goal, would not recommend spinning at room temperature.
If you are trying to produce competent cells, then you just need to grow the culture for a short time from a fresh overnight/colony (to OD600 ~ 0.4) and make sure to keep it on ice. Make sure you harvest at 4oC and keep the cells on ice at ALL time. Low temperature is key for making good competent cells. Lastly, even if you get some cell lysis, it shouldn't be a problem as the amount of cells are usually more than enough for efficient transformation.
@Rokas and @Aditya Thank you for the detailed answer...Goal is to express protein.I have now switched to BL21DE3 cells. Will see if the pellet will be ok this time. Will update about it. As I read in following article too that the PLysS cells lyse during centrifugtion at low temperatures. Thank you for your time and input.....Was very helpful....
Selleck, W., & Tan, S. (2008). Recombinant protein complex expression in E. coli. Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et Al.], Chapter 5, Unit 5.21. http://doi.org/10.1002/0471140864.ps0521s52
In pLYSE cell you can see some structural disruption, due to lysozyme amount produced by this stain. But for pLYSS, I had several experiences with this cell an there was not such a problem.
The contamination with environmental bacteria, specially bacteria from the skin such as Staphylococcus may occur or some times the growth ability reduce due to several passage of the cell.
The better way is to change your bacterial sources (preferably from another lab.)
@Ali hatef Salmanian, Thank you for your valuable answer. I was away for 10 days hence the late reply.
I shall keep your suggestion into consideration.
Thank you again.
Hey Chandrakala, I have similar problems at the moment an just wanted to ask if you figured out what had happend?
@Nadine Gerlach, Apparently thats how the pellet comes out to be.
I have continued my expression with the same cells. I do not pellet them at very low temperature like 4C. Very low temperature can lyse plysS cells which can in turn cause them to be too slimy. While pelleting I use 15C to 20C temperature with 4000g for 30 to 40 minutes....The pellet is still slimy and sticky to some extent but apparently that is how it seems to be for these cells. I did not have any problem in purifying protein since past 2 years and hence did not bother to check what causes the slimyness..
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