5
$\begingroup$

I'm currently working on a synthetic biology project which involves working with lots of different parts. I would ultimately like to integrate these genes by transforming a single plasmid. I've heard (casually) that 9-10 foreign genes on a transformed plasmid is often the practical upper limit for these types of experiments, without a real explanation other than "beyond that, it just doesn't work".

Is there a practical upper limit to size of amount of genes in a transformed plasmid, assuming all need to be expressible? What causes this upper limit?

Improve this question
$\endgroup$
4
  • $\begingroup$ I think the upper limit is more about the size of the plasmid and the efficiency of getting very large constructs into cells, at least in my experience working with mammalian systems. Beyond that you would need to start thinking about other vectors besides plasmids, such as viral delivery systems, Vaccinia vectors, artificial chromosomes, and the like. $\endgroup$
    – MattDMo
    Commented May 31, 2013 at 1:25
  • $\begingroup$ I'll most likely be working with E.coli. Putting aside differences in transformation, would there be any reason to suspect differences in functional expression of each gene? (If there is, I've never heard of it. It would seem like genes should get expressed equally in no matter what lengths of nucleotide). $\endgroup$
    – LanceLafontaine
    Commented May 31, 2013 at 1:29
  • 1
    $\begingroup$ As long as you can get the construct into the cells in one piece without some kind of degradation or shearing, I don't see why they shouldn't get expressed. Most of my experience with E. coli is just using them as a tool to generate plasmids or proteins, and I know that some of their characteristics are quite different than eukaryotes. I was just thinking, though, would a BAC or something similar serve your purpose? 150-350 kb is a heck of a lot bigger than your standard pUC19... $\endgroup$
    – MattDMo
    Commented May 31, 2013 at 1:37
  • $\begingroup$ A BAC might work. Thank you for the advice. You should turn your comments into an answer. $\endgroup$
    – LanceLafontaine
    Commented May 31, 2013 at 2:05

1 Answer 1

Reset to default
4
$\begingroup$

From my experience in the mammalian world (and this may apply to bacterial systems as well), it's not so much the number of genes in the plasmid as its actual size. The larger the construct is, the more difficult it will be to get it into your target cells in one piece, without degradation or shearing. Since the transformation efficiency is lower, you are getting fewer whole constructs per cell, so depending on how you've set up your promoters, the overall expression level can be significantly lower. The trouble with splitting your genes amongst two or more plasmids is that each will have different transformation efficiencies, and there will be a certain (perhaps large) number of cells that don't get the full complement of genes, interfering with phenotype analysis. And again, you'll also have to consider differential expression rates.

However, once you get your vector into the cells in one piece, theoretically they should all get expressed at approximately equal levels (assuming identical promoters). It's possible that steric hindrance among multiple transcription complexes may occur - I just don't know enough about bacterial transcription and the effects of circular plasmids to say.

One way to get around many of these factors would be to use a bacterial artificial chromosome or BAC. BACs are 7-10 kb vectors that can have inserts of up to 1 million bp cloned into them and are then electroporated into cells. One of (the many) cool things about them is they control their own duplication and partitioning at cell division, so succeeding generations should have essentially the same copy number as the original. They were heavily used during the Human Genome Project to amplify large sections of DNA for sequencing, but they are also used in many other studies, including synthetic biology. OpenWetWare has a list of some common ones, and NEB sells pBeloBAC11 systems.

Improve this answer
$\endgroup$
2
  • $\begingroup$ Also bear in mind that being based on the F plasmid, BACs are low copy-number plasmids. This means you may have to take care with antibiotic concentrations, and if levels of gene expression are important in your work you shouldn't expect as much as you would get from a standard high copy number plasmid. $\endgroup$
    – Alan Boyd
    Commented May 31, 2013 at 16:25
  • 1
    $\begingroup$ @AlanBoyd thanks for the edits - sorry about mitosis, like I said I'm a mammalian guy :) $\endgroup$
    – MattDMo
    Commented May 31, 2013 at 16:27

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .