I PCR amplified my gene of interest, both the WT form and a mutant form with a missense mutation. The gene encodes an ABC transporter and is about 4kb. Both PCR products looked good on the gel and had similar concentrations. I did TA cloning using the TOPO kit and plated 100ul bacteria. The mutant plate had ~30 colonies. I sequenced 4 of them and they all had the correct insert. However, there were no colonies on the wt plate. I repeated the experiment and still got nothing. Is it possible that the wt gene is toxic to the TOP10 competent cells? If so, what should I do?
Have you tried to use other competent cell such as DH5-alpha?
In this study, they cloned PxABCH1 with top10 and seems without any problem.
"and subcloned into the pEASY-T1 vector (Transgen, Beijing, China) before transformation into Escherichia coli TOP10 competent cells (Transgen, Beijing, China) for sequencing."
There is a list of strain for competent cell:
https://www.thermofisher.com/hk/en/home/life-science/cloning/competent-cells-for-transformation.html
Before you have tried on other strains, there is no enough information to know if the ABC transporter can kill the bacteria.
It certainly is possible that an ABC transporter could be lethal. However it could also be that your PCR product just has some contaminant in it that is stopping you from successfully cloning. I would first repeat your pair of PCRs and try the cloning a second time. You might also incubate your selection plates not at 37 but at lower temperature for a longer time period. Sometimes partially toxic products can survive at slower growth conditions.
Based on molecular weight, Your ABC operon might be an ion ABC transporter, probably phosphate ABC (4.1 kb).Look to our publication Mol. Microbiol. 2006, https://www.researchgate.net/publication/7454612_A_proteomic_analysis_of_penicillin_resistance_in_Streptococcus_pneumoniae_reveals_a_novel_role_for_PstS_a_subunit_of_the_phosphate_ABC_transporter
The ABC transporter can be associated with antibiotic resistance/susceptibility.
I guess it is a problem of your antibiotic selection, check the concentration and incubate in lower concentration of selective drug and incubate more time. If your mutant induces a differencial expression of your ABC it can explains why you have a positive colonies and not in the WT.
Article A proteomic analysis of penicillin resistance in Streptococc...
- Badges
- Science method