4 replies to this topic

[Tichduong]

Hi there,
I'm really in need of help! i'm trying to express the E7 gen of HPV16 in BL21 (DE3) E. coli, using plasmid pGAT. Unfortunately, the expression is not stable.

I expressed it three times in 5ml LB culture by picking recombinant clone on petri dish. At first, the construction worked very well and there was a thick band at the position of E7 fused with GST. One month later, I transform the plasmid pGAT E7 again to BL21 DE3 and expressed it, but only at the third time, that bands appeared while in the second time, only the bands of E. coli showed up in the gel.

Then, I used the same clone that expressed at the third time and cultured it, expressed it in 500 ml LB culture but the gene again did not express!

Have any of you faced this problem? I would really appreciate any idea from you guy!

Thanks so much!

Huong Ha

[perneseblue]

In the three times that the gene was expressed, what where the culturing conditions? The volume of the culture, the temperature, the type of flask used, agitation rate, media used?

Given that 5ml cultures expressed okay, but not 500ml, it seems that expression of your protein is sensitive to culturing condition (most likely oxygen availability).

I would find the culturing conditions that works, and scale up the number of cultures, rather than the size of the culture.

Dropping the culturing temperature sometimes helps, as it reduces growth rate and thus oxygen demand.

[protolder]

Hola, sometimes it happens, so trasform each time that you want to express, extend any colonie in a new plate and use the suspension as preinoculum, or look for a lacIq bl21 strain, in order to control expression before induction, because sometimes there is a quasi constitutive induction, and if the protein is toxic, plasmid which have loose insert are selected, I think. Buena suerte

[DaRQsiDe]

Try the flasks with slits on the sides, they help with the aeration.

[pDNA]

what antibiotic resistance does your pGAT vector uses?

Regards,
p