I am trying to transform the BL21(DE3) pLysE Chemically Competent Cells with my bacterial plasmid showed bellow which is Ampicilin resistent and expressing my genes of interest:
https://www.addgene.org/21877/
Several weeks ago I tried to transform the same plasmid with my gene of interest in DH5-alpha and was very easy and fast. But now I am trying to transform the same plasmid in this BL21(DE3) pLysE bacteria and after several attempts still not working. I was following the recommended protocol of SIGMA for this bacteria. Plasmid (1ul (10 ng)) + cells (50 ul), 30 minutes in ice, heat-shock 45 seconds 42°C, 2 minutes in ice, add recovery medium 1 ml, incubate 37°C 1h shacking and plate bacteria in Agar + Ampicillin (1:1000). As an alternative, I also tried to transform adding 50 ng of plasmid, 1.7 ul of 1.42 M 2-Mercaptoethanol in 100 ul of cells. But It didn't work.
What could be the problem?
Thanks a lot,
Ivan
It's hard to tell *what* the problem is, but I can suggest two experiments to see *where* the problem is.
Try transforming that plasmid into another strain (DH5A, XL1-Blue, etc). In parallel transform a control plasmid (e.g. PUC19) into both the BL21 cells and the other strain. That should tell you whether the problem is plasmid or strain specific. If it is neither, then you may have a problem with your bacteriology reagents.
Naively, a failure to transform the plasmid into any strain seems most likely to be a culprit of poor plasmid quality (e.g. the plasmid was cut, or it is misidentified and you're using the wrong resistance marker). You could check these via agarose electrophoreis and a few control digestions, if you have enough sample. If it doesn't digest as expected, or if it doesn't appear to be supercoiled, you'll know you have a problem with the plasmid.
If it's a failure to transform into a particular strain, it may be poor stock quality (e.g. storage at -20*C, accidentally allowing the whole box to warm to RT, they're homemade cells with low transformation efficiency, etc). In such a case you could either try transforming more plasmid, plating more cells or finding a better stock to work with.
It may be that your construct expresses a toxic protein, even though the pLysE plasmid should mitigate the problem. You may try a host with a more stringent control, such as the arabinose-inducible strain BL21AI.
Hi Ivan
1. I think if the construct is toxic the cells must get transform and on the next day we get small / tiny size bacterial colonies - this is as per experience of some researches i came across.
For this change the inducible strain is best solution.
2. Did you purchase the chemically competant BL21(DE3) pLysE bacteria or you make them competant.
Try to prepare at your own and do not forget to use chloramphenicol to maintain BL21(DE3) pLysE bacteria
It's hard to tell *what* the problem is, but I can suggest two experiments to see *where* the problem is.
Try transforming that plasmid into another strain (DH5A, XL1-Blue, etc). In parallel transform a control plasmid (e.g. PUC19) into both the BL21 cells and the other strain. That should tell you whether the problem is plasmid or strain specific. If it is neither, then you may have a problem with your bacteriology reagents.
Naively, a failure to transform the plasmid into any strain seems most likely to be a culprit of poor plasmid quality (e.g. the plasmid was cut, or it is misidentified and you're using the wrong resistance marker). You could check these via agarose electrophoreis and a few control digestions, if you have enough sample. If it doesn't digest as expected, or if it doesn't appear to be supercoiled, you'll know you have a problem with the plasmid.
If it's a failure to transform into a particular strain, it may be poor stock quality (e.g. storage at -20*C, accidentally allowing the whole box to warm to RT, they're homemade cells with low transformation efficiency, etc). In such a case you could either try transforming more plasmid, plating more cells or finding a better stock to work with.
By not working, do you mean that you get no colonies growing, or no expression? These are two very different problems. In general, BL21s do better with shorter heat shock (20 sec), but it depends on how they are prepared. Are these home-made competent cells or from a commercial source? Also, why would you use a DE3 strain here- that's for T7-based promoters, not the pTac promoter in the plasmid you linked to. BL21(DE3) is the wrong host strain for this type of expression system.
Dear Daniel,
Thank you so much for your answer. I meant no colonies growing. I was using 45 sec for the heat shock. Now I tried with 15 seconds and it looks like I could have few colonies. You mean that my BL21(DE3) pLysE will not express in any case my protein of interest with this plasmid? Even if it's not a toxic protein for the bacteria?
What kind of host strain could I use in order to over express my protein of interest in this plasmid?
Thanks
Hi Ivan. Glad to hear the shorter heat shock worked. DE3 strains are for T7-based promoters such as the pET vectors. If I understand correctly, you have a pGEX vector. You can try expression in DH5a or any derivative thereof.
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