I successfully cloned a gene and checked it by colony pcr and restriction digestion. There after I transformed the construct in E.coli DH5alpha for plasmid prep and isolated sufficient amount of plasmid whose quality has been checked through agarose gel electrophoresis.
When i tried to clone the same expression construct in BL21DE3 i am not getting any colonies.
My results are-
control A- competent cells bl21de3 plated on LB without antibiotic- got lawn of bacteria
Control-B competent cells bl21de3 transformed with pEt23b null vector- a lot of colonies are visible 0-0 transformation successful
Actual Transformation-competent cells BL21de3+PET23b based construct- got no colonies.
Antibiotic used is ampicillin and concentration in plates are 50ug/ml .
Help me to sort out the issue.
The competent cells looks good according with the Control B result. Maybe the problem is the amount of plasmid DNA used in the transformation or the size (bp) of your plasmid construction. Big amounts of DNA or big plasmid sizes could inhibit or reduce transformation efficiency.
I assume that your insert gene is out of the ampicillin resistance gene.
Since the transformation of the controls obviously works i have two guesses.
1. The protein you are trying to express is toxic for E.coli (expression should be blocked, but in my experience the pET Vector system is always leaky), although I don't think this is very likely
or 2. How did you purify the plasmid DNA? Maybe you have residues of phenol or ethanol in the DNA which inhibits transformation.
i used Qiagen spin mini prep kit for purifying plasmids so i dont think it should have chances of phenol or etanol. As i mentioned above the plasmid is working fine in case of DH5-alpha...
Assuming that you used equal amounts of plasmid DNA, the most likely explanation is that the protein is toxic. You should limit basal (uninduced) expression as much as possible. Things you can try:
- Include glucose (0.2 to 2%, whatever) in the LBA plates to further repress expression of T7 RNA pol through catabolite repression/inducir exclusion
- Use pLysS or pLysE to inhibit T7 RNA pol in the uninduced state.
- Try the Walker strains (C41 and C43(DE3)).
Hope it helps!
Aljandro i will try all your steps.
i decided to go through following route.
1- check the integrity of my constructs through agarose gel electrophoresis.
2- dilute it and transform on LB plates having 0.5% glucose.
3- if no results then going to use walker strains.
There's also the Lemo21(DE3) strain from -I think- NEB. Essentially BL21(DE3) with a pLys plasmid where T7Lys is under control of a tunable rhaBAD promoter.
Hi Vishal
I think you are either transforming too less DNA or too excess. As per my experience, a typical BL21(DE3) host gives best results when transformed with DNA concentrations ranging from 5ng to 50ng. I usually transform 5ng. Also, a toxic gene rarely affects transformation process. Chances of not getting even a single colony are very less even if your gene is toxic.
Hope that helps...
Good Luck... :)
Vishal Srivastava Alejandro Martin Sascha Röth Shridhar Kinkar I am also facing this.
My experimental conditions are
0- Kanamycin (50mg/ul)
1- Plasmid (pET29a+ PlantGene (1kb))
2- Plasmid conc. 125 ng/ul and 1ul for transformation
3. Bacterial cells (BL21-DE3)
4- Negative control: BL21-DE3 without plasmid DNA + kanamycin agar plate
5- Test control: Plasmid (1ul) + kanamycin agar plate
Results: No colonies
Can you guide me on what should I try to get colonies?
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