Hi! I am not sure I understand well your issue. I think because you are going to clone your gene into an E.coli expression plasmid, all features you will need such as T7 promoter, T7 gene 10 leader, RBS, flag/tag, terminator are already there. You only have to make sure that the MCS on your gene and the expression vector doesn’t result in frameshift. I don’t understand why you need to incorporate a MCS on your gene. Normally people only incorporate one restriction site at each end. Some vectors do not need MCS such as Topo-based vector or gateway vectors.

Hi Abdelhalim,

of course, all that stuff is already in the vector. I just thought I'd ask what could be nice to have in the polylinker, as I have the option to design one myself. Of course, I have my favorite list of RE sites that will be included but I hope that this discussion might lead to better multiple cloning sites in future vectors, with the goal to make the vector pretty universal for cloning and expression.

Example: One feature that should be present is a restriction site which generates a T/A overhang, to enable direct Taq-PCR cloning.

Unless you have an N-terminal tag, would it make sense to include a ribosomal binding site, at actually it should be 9 bases before the ATG?

Hi Wolfgang

I've made many MCSs and each time I ask myself this exact question, so I will be watching this thread with interest.

There clearly must be constraints on MCS design (eg on the number and types of RE sites that can be included, as a large gathering of pallindromic sequences must eventually be problematic to the vector (or the cloning of the MCS)). Unfortunately I haven't come up with any magic formulae yet. Generally I include a T/A overhang, and I also add in pairs of enzymes with compatible ends (eg XbaI/NheI or BamHI/BglII). But apart from that I am generally conserving space.

Sorry I can't add anything more adventurous or more useful, but as I said, I'll watch this thread with interest.

Al

Hi Wolfgang

Yes, I think making a vector with T/A overhang would be great. I only don’t see what improvement does it bring over existing vectors? Is it for the pleasure to construct one or is it for a commercial purpose? I once did something but only slightly similar by reconstructing the original MCS of a mammalian expression plasmid to use it as an empty control vector but I didn’t think in adding more restriction sites.

I think it would be nice to engineer expression vectors with new tags/flags which may offer some advantages over existing ones. Because it has been quite a bit I didn’t express a protein in E.coli I have had to read at a glance about the RBS stuffs and even learned new things I didn’t know about at the time. I think you should keep the RBS in its place otherwise it should be included with the construct. Now I will have to screen for cryptic RBS sites or anti RBS sites inside one of my proteins because we thought it was degraded inside E.coli. It could be just alternative initiation sites. So, thanks for posting this question. I will be also following the thread.

Yea, there is some "fun project" involved, but also the fact that for commercial entities, sometimes it is quite expensive to obtain vectors. That's why I plan to get as much out of an ongoing gene synthesis as possible.

Probably this is often neglected: For a small company, there is also the pressure to keep costs low (instead of writing grants, you "just" need to keep the investors satisfied and somehow earn the money for the monthly paychecks for those who are doing the work here :).

I see! I think it is a great idea. Anyway, even if you synthesize the whole plasmid with the aim to use it commercially you will still have to pay royalties for some of the “features” which are patented. Maybe their patents have expired.