I have successfully intergated several genes into the chromosome of E.coli to produce an oligosccharide. Theoretically, this oligosaccharide can be produced in a scale of at least 0.3g/L, and this is enough for TLC. However, it turns out that it doesn't exsit through TLC. What may cause this oligosaccride not to be detected through TLC?
Next step, I will detect it through HPLC to verify, and do reverse transcription to see whether these genes are transcribed.These genes are all in a cassete of T7 promoter、T7 terminater. I guess this put too much pressure on poor E.coli, so maybe not all these genes are succssfully expressed, or they are expressed but not functional. Is that possible?
I presume you have done this in a strain that is expressing the T7 polymerase as well? Did your construct express the oligosaccharide while on a plasmid?
However expression of T7 polymerase does have physiological consequences on the bacterial cell. You might be better off using a more normal promoter that uses the E. coli RNA polymerase.
@ Michael J. Benedik , thank you very much for answering my question, Dr. Benedik. As you persumed, BL21star(DE3) is used. I didn't construct these genes on plasmids, while it is reported in some literatures, and T7 promoters are used in these plasmids. Your suggestions are very helpful.I will verify my pathway on plasmids If it is neccessary.
There is one research got very high production using Ptac. While I choose T7 because I think maybe strong promoter will produce much more oligosaccharide.
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