pET-16b vector in DH5alpha E. coli - (Oct/15/2008 )
I have got a gene cloned in pET-16b vector which carries an N-terminal His•Tag® sequence; the vector is in DH5alpha E. coli cells;
I think pET-16b is good to express a protein of interest in larger amounts in E.coli.
My questions:
which medium should I use to culture? will LB do?
Has pET-16b an antibiotica resistance gene? There is a region in the vector map designated "Ap (4646-5503)"; what does it mean? Is it ampicillin resistance gene?
Do I have to add an inductor to start expression of my protein from pET-16b?
I will be pleased for any hint...
I think pET-16b is good to express a protein of interest in larger amounts in E.coli.
My questions:
which medium should I use to culture? will LB do?
Has pET-16b an antibiotica resistance gene? There is a region in the vector map designated "Ap (4646-5503)"; what does it mean? Is it ampicillin resistance gene?
Do I have to add an inductor to start expression of my protein from pET-16b?
I will be pleased for any hint...
1. Can't use it in DH5 alpha, it has no T7 polymerase gene embedded. BL(DE3) is what ppl normally use. The host should have an IPTG-inducible T7 polymerase gene.
2. LB will do because there's no lactose or glucose.
3. antibiotic is Amp. Ap normally means amp.
4.please ensure the your protein sequence is in frame when designing the primer to do PCR>
Hi,
Do you guys know if the dam-/dcm- strains from NEB such as ER2925 can be used to express proteins from pET vectors? I'm looking for a good strain for protein expression but has to be dam-/dcm-, so I cannot use BL21
Thanks
i doubt it...
because this guy got no T7 polymerase gene embedded in its chromosomal DNA.
I think pET-16b is good to express a protein of interest in larger amounts in E.coli.
My questions:
which medium should I use to culture? will LB do?
Has pET-16b an antibiotica resistance gene? There is a region in the vector map designated "Ap (4646-5503)"; what does it mean? Is it ampicillin resistance gene?
Do I have to add an inductor to start expression of my protein from pET-16b?
I will be pleased for any hint...
1. Can't use it in DH5 alpha, it has no T7 polymerase gene embedded. BL(DE3) is what ppl normally use. The host should have an IPTG-inducible T7 polymerase gene.
2. LB will do because there's no lactose or glucose.
3. antibiotic is Amp. Ap normally means amp.
4.please ensure the your protein sequence is in frame when designing the primer to do PCR>
thanks, I see, so the pET-16b vector with the gene of interest was sent in DH5alpha cells to amplify the plasmid but not to express the protein....
I'm confused as to why you need a dam- dcm- strain for protein expression. It seems unlikely.
unlikely yes, but I've seen it happen before... evil protein expression.
Dear Bearer,
1. In pET16b your gene is cloned downstream of a T7 promoter. For Driving protein expression you must transform your construct into BL21 DE3 or JM109 De3 E coli. These strains have the DE3 mutation, a λ prophage carrying the T7 RNA polymerase gene which is induced by IPTG. When u induce with IPTG, it drives the expression of T7 RNA polymerase, which in turn transribes your gene of interest cloned under the T7 promoter in pET16b vector.
2. LB medium should be fine for growth and induction
3. Ap stands for Ampicillin resistance.
4. You have to induce the culture with IPTG for recombinant protein expression. Basically you grow your culture to log phase, and then induce the culture with IPTG. The IPTG concentration can vary from 0.2mM to 1 mM.
All the best
aviprem
http://aviprem.gq.nu
I think pET-16b is good to express a protein of interest in larger amounts in E.coli.
My questions:
which medium should I use to culture? will LB do?
Has pET-16b an antibiotica resistance gene? There is a region in the vector map designated "Ap (4646-5503)"; what does it mean? Is it ampicillin resistance gene?
Do I have to add an inductor to start expression of my protein from pET-16b?
I will be pleased for any hint...