cloning and activity problem

[denrul]

Hi there;
I have two question about my cloning attempt,

1) I have cloned three different genes (originated from bacillus) in to LacZ of pSK(-) (pbluescript) vector. After transformation E.coli DH5alpha and induce with 0.5 mM IPTG there is no difference on SDS gel between Host and Clones. Cloned genes has own promotor region and Shine-dalgarno sequence. I have tried different IPTG concentration, different time of induction, different media but still I see same SDS gel profile. (Cloned vector has been checked for right orientation). What do you think about this situation. Unfortunately for my protein there is no antibody and I coudn’t make western blot.

2) As you know pSK vector has LacZ promoter region and Shine-dalgarno sequence of B-galactosidase and Cloned genes has own promotor region and Shine-dalgarno sequence. Is this situation create any problem for activity.

[JackBean]

1) I did you sequence the insert after transformation?
II if zou have pBLUESCRIPT, zou should be able to perform white/blue test, shouldn’t you?

2) sure, the cloned gene is driven by itselve promoter, so the IPTG can’t induce expression

[denrul]

1) Yes I have sequenced my fragment and of course I selected my clone via white/blue screen.
2) When inducing with IPTG, Is quantity of mRNA increase?? Basically; increasing mRNA quantity cause increasing protein quantity so increasing activity of protein. I want to ask you is ribosome use SD sequence of B-gal ??

[JackBean]

It would increase, if it has been driven by the b-glu promotor, but if the gene has its own promoter, than probably not.
BTW how large is your insert? It must be really long, if it contains also the promotor.

[denrul]

inserts long 2 kb, 2.5 kb and 4,5 kb respectivelly.

[JackBean]

Nice, but anyway, for IPTG induction, you must remove the natural promoter and keep the gene under b-glu promotor control 😉

[Author]

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