I am doing research on screening of metagenomic library for enzymes and bioactive molecules. I want to know about the expression vectors for preparing functional metagenomic library.
You can use any type of vector, depending on how involved you want to get. When you're looking for expression of xeno-genes in your chosen host organism, you're going to be limited by two main factors.
(1) Promoter incompatibility. This is usually solvable by adding your own inducible promoters. For example, I once did a screening with in pBR322 in E. coli BL21. I had two promoters, T7 and lac facing from either side of the plasmid into my cloned inserts. I got 100s of clones that did what I wanted in my screening. 80% were oriented along the T7 direction, 20% in the lac direction. 90% of all clones, independent of promoter, required promoter induction by IPTG. So that's one easy thing you can do to *greatly* increase the types of genes you can see. And it's really easy to do using a standard E. coli plasmid with minimal gene synthesis to place the convergent promoters.
(2) Ribosome binding site incompatibility. You can't place "your own" RBS, because the distance to the start codon needs to be just right (~5-10 bp in E. coli). This isn't practical with random metagenomic fragments. (unless you settle for fusion proteins.... I haven't seen that, but think it might work. It could also help solubilize proteins from other species. Try it :). The other way to solve it is to go with broad-host range plasmids, and use non-e.coli hosts that can deal with different sets of RBS from the environment. For example incP, incQ cosmids work in a lot of species, and can be transferred at high efficiency by conjugation from E. coli. I think you should get into this only if the standard E. coli plasmid approach doesn't give you what you want.
Hi,
In my PhD thesis I used PCC2Fos of Copy Control kit. It works very well and I got an average fragments of 30 kbp. As John said, in this case the expression will be dependent of the original promoter, which can limite the range of species that you will reach.
Given the wide choice of host-vector systems (plasmids, fosmids, cosmids, BACs etc), the normal approach is to select the vector on insert size criteria. For single enzyme screening, plasmids are fine (although we and others have had issues with preferential selection of very small inserts), but many labs use fosmids (simple to use, good insert size etc). Issues of host and promoter compatibility etc have been widely reviewed.
For bioactive molecules such as secondary metabolites, where expression of an entire biosynthetic pathway is required, cosmids and BACs are the norm. Technically much more difficult, but refer to papers by Sean Brady and Mark Liles, both of whom have done this very successfully (with inserts of up to 100kb).
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