I have a desinged gene which is 1263 BP long inserted in pET32a(+) plasmid and transformed in BL21(DE3). I had difficulty in seeing its expression in BL21 and JM-109 Expression cell lines. So I started looking into plasmid stability test in both cell lines.
Couple of questions which i cant seem to understand after few trials:
1. The number of colonies of transformed cells which grow on LB+IPTG plate is significantly more than the number of colonies which grow on LB plates only,Wht can be interpreted from these results?
The Number of colonies of transformed cells which grow on LB+Antibiotic+IPTG in case of BL-21 transformants is zero but in JM_109 transformants the number is significantly much more than LB+antibiotic as well as LB plates only. Does this mean the plasmid is unstable in JM-109.
The instablitly of plasmid means its getting ejaculated from the bacteria or the expression is insignificant? what does this term translates to?
when we are looking for number of colonies in terms of percentage, what is the baseline we are meant to use. Is it the number of transformed cells colonies on LB plate?
I have performed the similar tests on untransofrmed cells and have observed that the number of colonies is significantly larger on LB+IPTG plate compared to LB plates only. why IPTG is facilitating the growth of colonies ?
If someone could help me with these qustions, it ll be great.
Thanks in advance!!
Did you perform the same for the empty vector, pET32(+)? Have you done all the necessary quality control (QC) for your plasmid materials? Did you verify cloning of your desired gene by using gene sequencing tools? Is your gene product (or target protein) hydrophobic?
First off, you will not have expression in JM109. It does not produce T7 RNA polymerase (which is required to drive transcription from the T7 promoter present in the plasmid).
Second, plating an expression strain (transformed with an expression construct) into medium containing the inductor is not a good idea. Heterologous protein expression places a huge metabolic burden on the cells, so the colonies will take longer to grow, you will select for fitter mutants that do not express the protein, etc. Sometimes the cells won't even grow (as you are observing).
Third, I have no idea what the increased number of JM109 colonies in the presence of IPTG represents. What is it exactly that you are plating here? Untransformed cells? A transformation mixture? A transformed strain?
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