I need to construct a conditional mutant for an essential gene in PA14. At first, I knocked in a copy under pBAD promoter into chromosome via attB site using integration plasmid. Gene expression was confirmed by WB. So, I go ahead to transfer knock-out plasmid by electroporation. This plasmid contains upstream, Gm cassette, and downstream homologous arms. I can get single crossover mutant, which is confirmed by colony PCR with upstream screening primer and Gm internal primer. This PCR product was confirmed by sequencing. This strain was GmrCBCr.
I am very happy to continue to use 10% sucrose+0.2% arabinose to screen double crossover mutant. I got many GmrCBCs colonies. Unfortunately, the gene seemed to restore to wild type. Colony PCR using screening primers showed the same size as wild type, which should be 500 bp shorter.
I am confused. How can a single crossover go back to wild type, but still with Gm resistance? I repeated several times. The same thing happened. what should I do? can anybody give me some suggestions? I appreciate your nice help.
Wei,
Sometimes for whatever reasons, the homologous recombination works better for one of the two 500bp region you clone (upstream or downstream). So what I would do is checking if the first recombination event occurs half upstream and half downsteam. I bet this is not the case. If it occurs mostly upstream, you should select a clone with a first recombination event that occurred downstream. Then, you will increase your chance to make a mutant for the second event!
Also, did you sequence the gene you cloned at the attB site? Because the fact that you detect it by WB doens't proove that it is indeed functionnal. It might have a deleterious mutation in an essential amino acid for example.
Good luck with that,
Best,
Thibault
Wei,
Sometimes for whatever reasons, the homologous recombination works better for one of the two 500bp region you clone (upstream or downstream). So what I would do is checking if the first recombination event occurs half upstream and half downsteam. I bet this is not the case. If it occurs mostly upstream, you should select a clone with a first recombination event that occurred downstream. Then, you will increase your chance to make a mutant for the second event!
Also, did you sequence the gene you cloned at the attB site? Because the fact that you detect it by WB doens't proove that it is indeed functionnal. It might have a deleterious mutation in an essential amino acid for example.
Good luck with that,
Best,
Thibault
try doing a double jointed pcr to incorporate the mutations instead of colony with primers that have overhangs and rectrictions so that you know the joints worked out when you digest
Something else you can try is to increase the arabinose concentration when doing the second homologous recombination... You can go up to 1%.
Thibault
Hi, Thibault,
Thank you for your good suggestion. In fact, this experiment makes me exhausted and frustrated. I do not know who I should discuss with, as I think I am very familiar with the whole process of gene knockout in P.a. And this situation looks very strange. I am stuck and can't move forward. I am very glad to see your reply. I know that you must be very good at this field.
I also noticed that the recombination had a bias between two arms. I will try to get single crossover with the other side. Also, 1% arabinose should help.
To troubleshoot, I inoculated single crossover mutant with or without integrated copy in LB+10% sucrose+0.2% ara. After O/N incubation, I diluted and spread culture on LB+30Gm+0.2%. Then I randomly picked up 16 colonies and test their resistance, and found that with integrated copy, all 16 colonies were GmrCBCs, but without integrated copy, all of them are GmrCBCr. This indicated that integrated copy is functional and made this difference. Unfortunately, PCR showed that GmrCBCs were not right mutants, but wild type. I am hesitating to figure out the reason why they became wild type but still contained Gm resistance.
I really doubt that PCR are not right. But when I double check the primers, they are right. Should I construct new plasmid with longer arms?
thank you again for your reply.
Hi Wei,
Making a deletion mutant in Pseudomonas might be very complicated sometimes and trust me, I really understand your frustration!
Before making a new plasmid, I would first sequence the gene that you inserted at the attB site to make sure there is no (deleterious) mutation. Because if it is the case, your mutant will not be complemented and because you are making an essential gene deletion, it would be fatal for the bacteria. That may be why you don't get it!
When you inoculate single crossover mutant, do you inoculate 8 clones with left recombination event and 8 clones with right recombination event?
Indeed, if the sequence of your gene at the attB site is right, and if you inoculate both types of clones, another option for you would be to construct plasmid with longer arms.
You can also order new primers for checking your double recombination event, just in case yours have any kind of problem. I guess these are "outside" primers? Meaning they are outside of the region cloned in your mutation plasmid (on the chromosome right after the region cloned on your plasmid)?
Good luck with that!
Thibault
Another thing I'm thinking of... That's probably a stupid question but : Does your primers match the region you cloned at the attB site?
Thibault
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