I am not getting colonies when I had transformed my construct in rosetta 2 cells whereas transformation worked in Bl21-pLyse cells. I had used the same heat shock protocol for both. The one change that I had observed every time with rosetta cells is that the turbidity after 45 min incubation (after the heat shock and addition of LB) is very less in comparison to BL21-pLyse cells. Apart from this the same stock of rosetta cells are been used by other members in my lab and their transformation is working. What could be the probable reason for the same?
is your protein toxic? why are you not using rosetta 2 DE3 plysS? Did you tried Bl21 DE3 (not pLysS) strain to see if your transformation is working?
No my protein isn't toxic. I had got my transformation in pLys but I am not getting in rosetta2 or codon. Even in BL21 DE3 the number of colonies are quite few.
Dear Shubashid
in my experience generally rosetta 2 shows low competence and sometimes trasformation was difficult. however if your colleagues with similar amount of different clones were able to obtain colonies is it possible, ad Dhiraj already suggest that your protein is toxic, you have some basal expression and it di not allow to the colony to growth well into the plates.
are yoy using rosetta2(de3) with vector carryng a t7 promlter? because of course rosetta 2 plyss is a possible solution but in case you do not have this strain before buy it you can simple try to plate the bacteria in lb plates supplemented with 1% of glucose ( t7 promoter is repressed by glucose) or incubate the plate at ed instead 37 for linker time (to slow down possible protein expression and reduce the toxicity for the cells ) and see if ine of this 2 conditions allow you to obtain some colonies. in both cases perform also a negative test with empty cells to ne sure that the colonies thay yiu will obtain are specific.
however increase at 1h the incubation after the lb (at 37C and at least 180 rpm shaling)
good luck
Manuele
Hello Manuele. Thanks for the suggestion. Well, I don't think my protein is toxic but yes it's under a T7 promoter.
Dear Shubhashish
if your protein contain many rare codons the fact that you got trasformant in bl21 but not in Rosetta not means that it is not toxic because it is possible that due to rare codon content in bl21 its expression is very low or absent but in Rosetta where it can be expressed you observe the toxicity.
i dont now the nature of your protein but toxicity can be more related to his function. eg proteases, hydrofobic proteins can be toxic for the cell growth.
ciao
Manuele
Well thanks Manuele. My protein is a SAM domain of a membrane protein and has been expressed prior in BL21 and rosetta cells (as per literature). I had checked for rare codons and yes the protein do have rare codons. However, I had got my transformation in BL21 pLyse so I suppose it's not a toxic protein.
the very fact that you are getting better transformatio/colony in plysS suggests that e. Coli don’t like your protein. As Manuele Martinelli suggested, toxicity can be for multiple reason and has nothing to do with its behavior in human/animal body once injected. Too much expression can also cause toxicity. Try plysS and glucose.
I used other approach. The Rosetta phenotype is based on pRare plasmid with chloramphenicol resistance. I isolated the plasmid. Then I transformed in single step the BL21(DE3) with the mixture of both my expression plasmid with kanamycin resistance and the pRare plasmid with chloramphenicol resistance. Spreading on Cm/Kan plate. Transformation protocol was supported by goodies like SOC medium and prolonged incubation after heat shock. Worked for me.
Peter Kysel Thanks. Can you tell for how long had you incubated the cells after heat shock?
When transforming with plasmid with kanamycin resistance I incubate the cells for 2 hours. With ampiciline resistance I wait only 1 hour.
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