Question on recombination in E.coli and in baculovirus

[Aawara Chowdhury]

In <%xGyj.88$8m2.34 from newsfe05.lga>,
 DK <dk from no.email.thankstospam.net> wrote:

> I am considering co-expressing three proteins from the same 
> baculovirus. For that, a transfer plasmid from Novagen is 
> available which has two polh promoters and two p10 promoters. 
> Each of the same promoters is found on different strand
> (i.e., one strand has one polh and one p10 and another strand
> has one polh and one p10) "to minimize recombination" according 
> to the company. Promoters are ~ 70 and 90 bp.  
>
> More than that, I might need to express two of the proteins 
> as fusions with the same small protein (162 bp). There is no 
> problem placing those on different strand. 
>
> Because I don't really know much about homologous 
> recombination in either system, I worry if there are caveats 
> related to recombination and resulting instability of the 
> 1) transfer vector in E.coli  2) resulting baculoviral DNA
> in the insect cells. 

I wouldn't worry about insect cells - given the time length of expression,
after infection with baculovirus (or you concerned about recombination
when you make the virus?).

In E. coli, if I understand you correctly, the repeats will be inverted
(the polh and p10 promoters are on opposite strands, so they must be
inverted with respect to each other, correct?)

I don't think you'll have major issues with stability, but if you do,
propagate the plasmid in an E. coli strain like STBL2.  We have had
very good luck propagating retroviral vectors, and retrovirus clones 
in this strain, and both of these types of clones have two copies of 
direct repeat, the LTR (U3RU5), and also four copies of inverted 
repeats - at the U3 and U5 ends.  The direct repeats LTRs range in 
size from about 300 bp to 1 kb, and the inverted repeats are 15 - 30 bp 
in length.

Anyway, these constructions are completely stable in STBL2.  I think
we originally obtained this from Invitrogen.

AC
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