If you only have one loxP site per plasmid, then you will generate a dimer plasmid that goes loxP - gene 1 - plasmid 1 - loxP - gene 2 - plasmid 2 - (back to loxP and gene1).

Nebojša Bogdanović The yellow bar in the figure should be 'gene 2' right?

Should be 5'-G1-G2 after recombination of two plasmids.

Hello Yuan-Yeu Yau,

Yes, you're right, in the yellow figure, it should be gene 2.

If you only have one loxP site per plasmid, then you will generate a dimer plasmid that goes loxP - gene 1 - plasmid 1 - loxP - gene 2 - plasmid 2 - (back to loxP and gene1).

Thank you very much Michael. It is the case that i have one loxP site per plasmid. Is there any paper You recommend that would bring this topic little closer to me?

There are undoubtedly papers out there but I don't have a reference at hand. But it is a general principle that a single recombination event between two circular molecules will yield a single molecule comprised of both the original ones. That is true regardless of mechanism, CRE, FlP, Int, homologous recombination, etc.

Nebojša Bogdanović

See the picture I drew. The loxP sites in the hybrid plasmid are in the same orientation. If I can find a paper I will send it to you.

Your drawing looks correct.

Thank you Michael and Yuan-Yeu for clearing this out. I really appreciate your help. Yuan-Yeu, figure is very helpful! Next step for me is to use two identical vectors with two different genes i need to co-express since pUCDM+ several tested genes used for single expression doesn't yield any protein.

Hello everyone, I have made these plasmids and now according to the recombination scheme seen here, it is expected that the new (recombined) plasmid confers the resistance to the antibiotic genes present in both origin plasmids. One origin plasmid has an ampicillin resistance gene and another a chloramphenicol resistance gene, and no loxP site breaks any of them. However, when I cultivate the DH5-alpha to amplify the new recombined plasmid, I can see an overnight growth of around 0.8 OD600 when I add the following amount of these two antibiotics (50 µg/ml and 20 µg/ml, amp and ch, respectively). Has anyone tried to use only one antibiotic without detrimental consequences for plasmid integrity? I have no other problems with the same strain (same batch) when I use it for my other transformations and plasmid amplification. Thank you everyone for the valuable (future) suggestions.

It isn't clear whether the problem is the double antibiotics, or the new plasmid. You might inoculate separate tubes/flasks and grow one with amp only, one with ch only, and one with both to see if there is something to be learned. If the plasmid is stable it should not matter whether you use either one or both, but if the plasmid is not stable then it might.

Nebojša Bogdanović I try to understand....if you use only 1 antibiotic, how can you get the correct hybrid one? You could have 3 species exist--plasmid A (ampr), plasmid B (chr) and hybrid plasmid A - plasmid B (ampr+chr). Besides, in the chart, the Cre reaction is 'bi-directional'. That means under the present of Cre protein, the hybrid plasmid can also be excised into 2 original plasmids (see attachment, right panel). So, you might have 3 plasmids co-exist. And the reaction favors 'excision', not 'integration' of plasmids.

@Yuan-Yeu Yau. I didn't make the hybrid plasmid by recombination in E. coli. I used the commercial overexpressed Cre recombinase, mixed it with two original plasmids in vitro and i got a new bigger plasmid that should look like what both of you have described earlier. The reaction mixture is then "pcr purified" and recombinant pDNA is isolated. That pDNA is transformed into E.coli Dh5a to amplify it. Those cells do not grow so well and thus the plasmid yield is low. Is it maybe clearer now Yao? Thank you both very much. I appreciate your answers. As this isn't my project, but a tool I'm using, i don't have the full time to read and deconvolute the whole process so any discussion we have here can be really helpful.

Nebojša Bogdanović Thanks for explain. Yes, I am clearer about your experiment procedure now.

Nebojša Bogdanović < The reaction mixture is then "pcr purified" and recombinant pDNA is isolated. >

Can you tell me how exact this was carried out? or do you have a protocol explaining that. I tried to figure it out.

Yeah, i can of course. Protocol is very simple. Mix the two desired plasmids 250 ng each, add cre recombinase, add cre buffer, incubate 30 min at 37C, 10 min at 70C, pause a +4C. From this point, you can run the gel and /or purify the mixture and transform into E coli. I hope this helps.