Hi everyone,
I am now trying to delete some genes of the Agrobacterium Tumefaciens K84 strain. I decided to use the in-frame deletion method using the pk18mobsacB plasmid which harbours sac gene as a marker.
However, the resistance gene of pk18mobsacB is kanamycin, while kanamycin could not kill the Agrobacterium Tumefaciens K84 strain. So I screened several antibiotics and found that gentamicin was able to kill the strain and could be selected as a marker.
As there was no commercially available gene deletion plasmids with gentamicin resistance, I had to do a plasmid engineering of pk18mobsacB. I used PCR to remove the kanamycin resistance gene of the plasmid and replaced it with the sequence of gentamicin resistance gene from the pRL662 plasmid.
However, after infusion cloning, the competent cells could not grow on a gentamicin added LB plate. I think the resistance exchange engineering was not successful but I do not know why...
If you can give any result, I would be more than grateful.
(I attached the pk18mobsacB and GenR-pk18mobsacB plasmid map)
Best regards,
Zhongtian
1 Incorrect PCR amplification: It's possible that the PCR amplification intended to swap out the gentamicin resistance gene for the kanamycin resistance gene failed. The primers used for PCR amplification must be accurately designed and specific to the target regions. To get the desired result, the PCR conditions (such as annealing temperature and extension time) should also be optimised.
2. Ineffective ligation: During the infusion cloning process, ineffective ligation is another reason that could exist. The resultant plasmid would lack the necessary resistance marker if the linearized pk18mobsacB vector and the PCR product carrying the gentamicin resistance gene were not successfully ligated. The ratio of insert to vector, DNA concentrations, and incubation periods should all be optimised for ligation.
3. Loss of plasmid integrity: It's possible that the cloning process damaged the plasmid integrity. Particularly when subjected to repeated manipulations or freeze-thaw cycles, plasmids are prone to deterioration. Make that the plasmid DNA used for engineering is of excellent quality and has not been deteriorated.
4. Incompatibility issues : Agrobacterium strains can be difficult to work with since they contain a variety of plasmids with distinct replication sources and compatibility groups. The plasmid you designed might not replicate well and end up being lost if the Agrobacterium Tumefaciens K84 strain is incompatible with it.
The following actions are what I would advise taking to troubleshoot the problem:
Use PCR or a restriction digest analysis to confirm the gentamicin resistance gene is present in the modified plasmid. If the resistance exchange was successful, this will demonstrate it.
Think about reintroducing the modified plasmid into new competent Agrobacterium Tumefaciens K84 cells and measuring the growth of those cells on LB plates with gentamicin. Cell viability might have been impacted by the initial transformation's low efficiency or by other causes.
Consider verifying your experimental techniques again and doing the cloning steps again, paying particular attention to primer design, PCR settings, and ligation optimisation, if the aforementioned processes do not produce the intended results.
- Badges
- Science topic