4 replies to this topic

[scwvin]

Hey everyone, I am seeking for a suitable vector to clone multiple genes into it and express them synergistically in E.Coli. Any recommendation? Thanks.

[pDNA]

You should definitly go for a low copy or single copy vector ...if you want to co-express several genes or a whole pathway.

Look for a vector with a SC101 origin of replication or a so-called mini-F plasmid ...there is one good available from Keasling and Kristala Jones Prather called pKLJ12.
You can get it easily from the Japan Vector Collection.

Regards,
p

[Qundo12]

scwvin, on 25 October 2010 - 08:19 PM, said:

Hey everyone, I am seeking for a suitable vector to clone multiple genes into it and express them synergistically in E.Coli. Any recommendation? Thanks.

You can use the pETDuet-1 (medium copy number) or pACYCDuet-1 (low copy number) vectors of Novagen, they have 2 T7 promoters and two MCSs with many RE sites. Normally I express 5 to 6 genes on the same plasmid.
There are some disadvantages, these vectors must be expressed in hosts containing a source of the T7 polymerase such as (DE3) lysogenic strains, and the T7 promoter is quite leaky with background expression (not good for producing toxic products). However, the expression level of this promoter is very strong.
In my lab, we replaced these T7 promoters with tac promoter, so we can express genes in many E. coli strains.

Edited by Quasimondo, 28 October 2010 - 04:09 PM.

[scwvin]

Thanks pDNA. I am planning to have about 5 proteins synergistically expressed together. The problem I am facing is the designing of the whole system and the stability of the protein produced. Thanks again for the wonderful information that you provided.  

pDNA, on 26 October 2010 - 01:19 AM, said:

You should definitly go for a low copy or single copy vector ...if you want to co-express several genes or a whole pathway.

Look for a vector with a SC101 origin of replication or a so-called mini-F plasmid ...there is one good available from Keasling and Kristala Jones Prather called pKLJ12.
You can get it easily from the Japan Vector Collection.

Regards,
p

[scwvin]

Hi Quasimondo, you successfully expressed all the genes? Are the genes involving in the same function? Do you use any bioinformatic tools or software? Thanks for the information. Have a nice day...

Quasimondo, on 28 October 2010 - 04:09 PM, said:

scwvin, on 25 October 2010 - 08:19 PM, said:

Hey everyone, I am seeking for a suitable vector to clone multiple genes into it and express them synergistically in E.Coli. Any recommendation? Thanks.

You can use the pETDuet-1 (medium copy number) or pACYCDuet-1 (low copy number) vectors of Novagen, they have 2 T7 promoters and two MCSs with many RE sites. Normally I express 5 to 6 genes on the same plasmid.
There are some disadvantages, these vectors must be expressed in hosts containing a source of the T7 polymerase such as (DE3) lysogenic strains, and the T7 promoter is quite leaky with background expression (not good for producing toxic products). However, the expression level of this promoter is very strong.
In my lab, we replaced these T7 promoters with tac promoter, so we can express genes in many E. coli strains.