Problem with construction of deletion mutant (Bacteria) - Deletion mutant, pK19mobsacB, Corynebacterium glutamicum (Apr/20/2011 )

Dear friends,

I am trying to construct a few deletion mutants of Corynebacterium glutamicum using the integration vector pK19mobsacB. I amplified the gene having the desired deletion using overlapping pcr, cloned it into pK19mobsacB and transformed into E. coli S17-1.
The recombinant plasmid was introduced into Corynebacterium glutamicum using either electroporation or conjugation. After electroporation, the cells were plated in LBHIS/Km10 plates and I was getting very few number of colonies compared to that in conjugation. In the case of conjugation, the cells were plated in LB/Km/Nx plates and I got quite number of colonies within the second day of incubation itself.
For the double cross over, I followed two different protocols.

Protocol 1
1. Picked up one of the kanamycin-resistant colonies and grew in BHI medium with 2% glucose and 5 mM sodium azide.
2. The cells were plated in LBHIS/Suc10 (10% sucrose) plates.

Protocol 2
1. Picked up‘6’ colonies and put each in LB and cultivated o/n at 30℃
2. Plating: LB/suc10 (10^-1 to 10^-4 dilution)
LB/km25 (10^-4)
LB/suc10/km25 ^(10-2)

Fifty colonies from LBHIS/Suc or LB/Suc10 were streak plated to LB plate and then to LB/Km10 plates. Unfortunately, all my colonies are kanamycin-resistant. It means that double cross over has not happened? Then how could the cells grow in sucrose medium? It is a bit cofusing to me (I have confirmed the sacB gene in the vector by sequencing it before starting the experiment). I did colony pcr to confirm the colonies and could know that all the colonies harbor the wild-type gene.I repeated the protocol many times, but the result is same.
Could anyone help me to solve this problem? I would really thankful to you.

Regards & Have a good day!!!
Bindu

Is the sacB gene driven by a promoter that Corynebacterium glutamicum recognizes?

Is the sacB gene lethal or inhibitory in C. glutamicum?

phage434 on Wed Apr 20 16:17:40 2011 said:

Yes, sacB is lethal to C. glutamicum in the presence of sucrose and the cells attain sucrose-resistance after second cross over.

HomeBrew on Wed Apr 20 14:55:32 2011 said:

Yes..SacB is expressed in C.glutamicum.