point mutation when transformed in BL21 - (Mar/17/2010 )

Hi everyone,

I have a problem regarding the transformation into BL21. I have managed to clone my insert into pET15b in a DH5 alpha host cell. But because i need to express the protein, so i transformed my pET15b:insert into BL21. Here comes the problem, when i transformed my plasmid in to BL21 i always get point mutations and insertion mutation in my plasmid sequence. I am 100% sure that my plasmid sequence in DH5 alpha has no problem at all, but when i transformed the plasmid into BL21, my plasmid sequence contained alot of mutation. I have tried many times, but i always get mutation in my BL21 plasmid. But the insert im trying to clone is toxic to the cell, but what is the reason that my plasmid in BL21 also get mutation???

NEED HELP!!! Thank you very much...

-jason0429-

Need a little clarification...

jason0429 on Mar 17 2010, 04:48 AM said:

when i transformed my plasmid in to BL21 i always get point mutations and insertion mutation in my plasmid sequence.

Do you mean to say that there are always point mutations in both your vector and your insert? How do you know this? Do you sequence the entire plasmid (vector plus insert) every time? Are the mutations always the same, or are they always in the same place?

jason0429 on Mar 17 2010, 04:48 AM said:

I am 100% sure that my plasmid sequence in DH5 alpha has no problem at all...

How do you know this?

jason0429 on Mar 17 2010, 04:48 AM said:

But the insert im trying to clone is toxic to the cell, but what is the reason that my plasmid in BL21 also get mutation???

If your insert is toxic to E. coli, how is it toxic to BL21 but not to DH5 alpha?

If your insert is in fact toxic to E. coli, you'll always only recover mutants. The mutations could be in the insert, destroying protein function or full length transcription, or they could be in the plasmid, destroying the promoter or a copy number control gene (perhaps the plasmid copy number is reduced to the point where gene dosage drops to a level where the product of the gene is no longer toxic).

-HomeBrew-

hi, thanks for replying me...
1. first i screened my colonies. Those colonies with positive PCR reaction, i will only sequence them.
2. because my gene of interest is a type of endonuclease, so this might be toxic to the DNA.
3. DH5 alpha does not have T7 RNA polymerase, whereas BL21 has. Therefore, when i transform my plasmid from DH5 into BL21 the gene of interest my expressed.

I'm not sure whether i'm right or wrong, but please give me some suggestions.

-jason0429-