We are working with a species of Lactobacillus that is difficult to manipulate genetically. A mutant in a gene was made and we are trying to rescue the change in phenotype using complementation. We tried several expression vectors and none of them worked well in terms of level of gene expression. I was wondering if there is another way besides complementation to rule out polar effect and confirm that the change in phenotype is a result of a mutation in bacteria.
You can do the complementation in your bacteria by using the same strategy like you did knock out. Just select an operon from your bacteria that is putative or unknown function and replace it with your intact complete gene along with promoter+antibiotic resistant cassette. you can use another known constitutive promoter instead of original promoter.It is a bit tedious, but it works. If your gene could be expressed in E. coli and you can verify the function, such as enzymatic activities? binding properties etc...! This will be the acquirement of phenotypic characteristic in E. coli in comparison to E. coli containing empty vector. This is how it is proved.
Since you know the mutated gene, Your work is easy.
1. Transform the wild type gene into your mutant and see. It should recover the phenotype.
2. Knock out the gene from your WT Lactobacillus and see. It should loose the phenotype.
If they don't happen your gene and phenotype are not for each other.
If they happen partially, your gene is one of the many factors causing the phenotype.
If they work out. You are lucky.
Best of Luck
I would also try 2) of Salvatore Mandalà.
Perhaps it also makes sense to complement your mutant "chemically" (e.g. by the addition of the product of the catalysed reaction) or to use an known/specific inhibitor for your target protein.
There are still other possibilities (Antisense), but I would first try the easiest ones...
All the best!
Can you recommend any shuttel vectors for Lactobacillus spc.?
I worked on Mycobacterium tuberculosis, where compementation is possible but very slow, and have thought about alternatives. It depends on the reason(s) for complementation. We are usually required by journals to complement a defined mutation to make 'sure' that the phenotype is not due to a secondary mutation elsewhere on the chromosome - very necessary in the old days of chemical mutagenesis, and less so now, although there are probably instances where it occurs. We have on occasions constructed the same mutant twice, independently, and shown the phenotype is present in both, making a second-site mutation highly unlikely, and used that as our evidence. A better way these days might be just to completely sequence the genome.
This doesn't help you if you are working with an operon and trying to exclude downstream effects, though it will often be possible to be fairly certain is a mutation is likely to have polar effects, and the solutions above would be more suitable.
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