I'm going to over express several genes in a single strain simultaneously. (It would be convenient if I clone all of them into a single plasmid, so that I don't have to make many rounds of integration onto the chromosome, and don't have to worry about plasmid compatibility.)
Suppose the ligation and plasmid construction is not a problem, what are the limiting factors of the plasmid size? Note, the plasmid is not derived from phage.
Some say transformation efficiency would be affected, but isn't it true that one successful colony of transformation would probably be enough?
Some people mentioned the stability of the plasmid. Is it a big issue? What if the selection pressure is always there?
Any other factors?
The strain at hand is a yeast. But I'm also interested in the cases of bacteria.
Thanks!
I agree with Sam. In addition the longer the plasmid size the more problems it poses both for its manipulation and for E.coli.
I did some work with several species of yeast sometimes ago. First of all, you don’t need a plasmid to integrate whatever cassette you want into genomic DNA. However, you should bear in mind that S. cerevisiae has only homologous recombination as E.coli, which make it relatively easy to carry it out. S.pombe and others species may sometimes prove quite difficult because your cassette can integrate through homologous recombination and non-homologous end joining. In this case it is better to have longer flanking homology ends to the site of targeted integration.
Transformation efficiency can be attributed in part to the size of your insert, so, this may be an issue if the two genes you plan to add into your vector are significantly large.
Personally I would do a co-transformation, using two vectors with different selective markers, but that is just because I feel more comfortable doing that rather than making a construct that may, in the long run, end up taking more time to get it to properly work, transform and express.
Hi Jinbei,
(The information below applies to bacteria)
> what are the limiting factors of the plasmid size?
Plasmid stability/ segregation and ease of work. It simply gets more difficult to handle when they are large (> 50 kb).
>Some say transformation efficiency would be affected
Yes, the bigger the plasmid the lower the transformation efficiency. However, plasmids up to 200 kb can be transferred by electroporation (hand-on experience). For even bigger plasmids conjugation would be my choice of transfer method.
Working with large plasmids (> 20-30 kb) I would look for plasmids that are not high copy number and have partitioning systems (par) eliminating any potential segregation issue.
Best,
Rahmi
It really depends on the size range you are considering. If you are taking a plasmid from 4kb to 6kb then I doubt you will see much difference. If you are taking a plasmid from 6kb to 12 kb, then the effect might be more dramatic. So it really depends on the size range.
plasmid size can accomplished a maximum of 52kb in size (that are cosmids) which come under non chromosomal vectors.
if u want to increase ur size or u want to increase the gene of instertion try chromosomal vectors which can even accomblished a size of 1Mbp.
Respected Scientists, I am trying to insert a gene of almost 10 Kb into pCAMBIA 1305.1 plasmid (12.8 Kb) digested with respected enzymes. I have divided the gene into two fragments (S1 and S2) of 5 kb each. I have successfully inserted S1 and now the size becomes almost 17 kb. Now I am trying to introduce the remaining S2 into partially made vector but couldn't succeed after many attempts. I have tried different vector to insert ratios but couldn't get any colonies. I need your precious suggestions for the task and will be very thankful if you can please share your knowledge and experience. Thank you very much.
Try a different E. coli strain for large size plasmids. I've had the same problem. Usually Neb10 competent cells will do the trick.
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