We know that putting repetitive sequences in a single plasmid may initiate recombination in those plasmids. If recombination happens, the sequences in between those repetitive sequences may get excised out.
Can this be prevented by having the repetitive sequences in two separate plasmids (with two different backbones)? Would recombination still occur in between the separate plasmid systems?
P.s. I am working with a bacteria which is almost genotypically identical to its wild type variant. It is also not a recA- strain.
Are you talking about in bacteria and do you mean separate plasmid systems in the same cell (one cell with two separate plasmids) or in different cells?
If you are using a RecA- strain of E. coli, you should be ok if you clone a repeated sequence. It is true than in a RecA+ strain you might get plasmid deletions.
Regarding the specific question you ask, it very much depends upon what is surrounding the repeat elements as to what sort of recombination events and outcomes you would get if you clone two homologous sequences in two different plasmids.
But if you think about it, when you clone ANY fragment into a plasmid, and have a 100 copies of that plasmid in the cell, you have substrate for recombination in a RecA+ strain, hence you frequently get dimers, trimers, tetramers etc
Prof. Michael J. Benedik
Thank you for your answer in this question and in my previous question regarding recombination.
Since I am encountering recombination events in my single plasmid system in Lactococcus, I have thought about two ways to deal with it -
1. Knock-out/Fully repress recA gene in Lactococcus genome
2. Two plasmid system, each with separate Ori.
I will first try a dual plasmid system (strategy2) where the repetitive sequences are split in two different plasmids. I am working on Toehold Switches - the switch and trigger sequences have reverse complementarity of about 65bp.
Strategy 1 may have pleiotropic effects if recA is knocked out, like you had mentioned. So I have built a sgRNA/dcas9 plasmid for full repression of recA gene.
I hope the first strategy works, so that I don't have to go into the second one. What do you think?
Regards and Thanks.
@Alexandra Johnson
Yes, a two plasmid system with different Ori, for expression in Lactococcus.
You can certainly try the two plasmid system to see if it works for you. You might also consider having the two elements on the same plasmid, but one inverted relative to the other. That way any recombination events don't lead to deletion.
What is the length of the repeat elements and how similar are the sequences?
Prof. Michael J. Benedik
The Toehold switch-trigger pairs are designed to make strong secondary stem loops among themselves and also have strong affinity to each other.
They have complementarity of 65bp only. But I guess their strong affinity/complementarity makes things worse in my case.
have you tried separating them from each other on the same plasmid. You might significantly reduce the recombination if there was 500-1000 bp between them.
Prof. Michael J. Benedik
I had taken that into account during the initial designing itself since recombination frequency increases proportionally to greater genetic distance.
The Toehold Switch and Trigger sequences only have 380bp in between then.
These RNAs are also expressed by their promoters in opposite direction in the plasmid so as to reduce any chance of RNA pol walkthrough.
"I had taken that into account during the initial designing itself since recombination frequency increases proportionally to greater genetic distance."
This is not relevant in your case, the greater the distance between the repeats, the less the recombination will occur. The above sentence is talking about INTER-molecular recombination such as two chromosomes. Your situation is presumably an INTRA-molecular recombination event and will be dictated by the length of the homologous regions (increases) and the distance between them (decreases). But 380 is already somewhat substantial so I don't know whether you would get a benefit from extending that to 1-2 kb or not.
What are the products you get from the recombination event? Have you sequenced them? And what percent of plasmids have such events?
What origin of replication are you using? Rolling circle replication plasmids (especially with only a single stranded ori) like pWV01 tend to be much more unstable to secondary structure and short repeats than theta replicating origins such as pAMB1.
If you're using a RCR plasmid I would try swapping the origin for a theta replicating one first before modifying the strain.
Alexandra Johnson
Yes the plasmid I am facing issues with, uses the rolling circle replication.
Thank you for the suggestion. The second plasmid that I am using, employs theta replication mechanism.
Michael J. Benedik
Yes I have sequenced them and found out that that plasmid DNA sequences in between the switch and trigger pairs get excised out.
I may try keeping larger spacer region in between like you suggested if the current strategies don't work out.
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