Gene Deletion in E.Coli Bl21 - (May/03/2008 )
Hi All,
I have benn having problems in deleting pgk gene from E.Coli Genome. I am using lambda red recombinase method for gene knockouts.
The way primers are designed are with 20 base pairs of chloramphenecol and 40 base of around the pgk gene. I am able to get the PCR product for the same. This PCR product is then transformed into Bl21 strain expressing recombinase by electroporation.
After transformation , I have tried plating the product on chloramphenecol plates with low concentration(25microl/ml) and Have also tried plating on pyruvic acid plates, glucose and chloramphenecol market plates. HOwever, I cudnt get any colonies in 37C condition.
It hard to find the literature for somebody who did. Can anyone explain me whats going on and how can i succefully knock out the gene?
How is the Lambda RED system put into the BL21? How are you inducing it?
Thanks alot for reply phage.
I am transforming it with pkD46 and inducing it with L-Arabinose when the OD is 0.2. Then let it grow till 0.5 and start washing for electrocompetent cells.
I hope this information helps you.
Metzz
Can you do control transformations of your BL21 cells after induction? BL21 is not especially competent, and it would be good to check the competence of the cells and compare against a strain that you have had success with. Can you knock this gene out of DH10B for example? How does the transformation efficiency of the cells compare when transformed with something like pUC19?
If you can't get the gene recombined in BL21, think about P1 transduction of the fragment from a strain you can get it working from. That's the way many of the mutations were introduced in the strains we use.
Alternatively, think about why you need BL21, and whether another strain would do. Can you knock out the proteases from a more user-friendly strain?
If you can't get the gene recombined in BL21, think about P1 transduction of the fragment from a strain you can get it working from. That's the way many of the mutations were introduced in the strains we use.
Alternatively, think about why you need BL21, and whether another strain would do. Can you knock out the proteases from a more user-friendly strain?
Thanks alot Phage,
I have succesfully worked on few single knock outs before by same strategy and same strain. Boss wudnt let me use P1 transduction. The problem arises for double knock-outs and pgk gene deletion.
I think one of the reason could be that pgk is lethal deletion for E.Coli. Therefore, after transformation, I am not getting any colonies on the plates. (THis plate are made up of pyruvic acid, glucose and chloramphenecol). Is there any strategies, I can use or any nutirent supplementation to keep the cells alive?
Thanks in advance.
MEtzz.
When I did Knock-out using the pkd46 plasmid, I normally grew cells (50ml) in 250ml flask to OD 0.5-0.6, and concentrated to 500 ul. For electroporation, I used 40 ul of cells with 500 ng DNA, and it has never failed. But when i used less DNA, sometimes i did get transformants.
Hope it helps
Hope it helps
You do not induce it with L- Arabinose?
Hope it helps
You do not induce it with L- Arabinose?
Sorry, I do. i add arabinose at 10mM to the LB media.
Hi,
I'm doing the same thing, and I can't extract pkd46 from my cells to confirm the transformation of my cells with pkd46!.
However I may have some advices, for what I'm told you need to PLATE A LOT to get some transformants!. You can also try to decrease the amount of antibiotics to the plate.
Please read: Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes
http://www.biomedcentral.com/1471-2199/7/31
They have to change almost everything to get it working.
Best regards,
Carla