RecA is a recombinase involved in DNA repair that's responsible for recombination between long (hundreds of base-pairs) homologous stretches of DNA. If the DNA gets say, damaged during replication, RecA can repair it using a (theoretically) undamaged copy.

However, it's usually knocked out in cloning strains of E. coli because it can either cause plamids with long repeat regions, like lentiviral vectors for instance to become unstable due to RecA sometimes "resolving" the long repeats into a single copy which causes a deletion in the plasmid. Another problem is that if you're cloning a sequence that's homologous to the E. coli genome, recA will sometimes recombine the plasmid into the chromosome which can be a problem for a lot of reasons.

It also has a tendency to sometimes recombine a multiple copies of a plasmid into a single "multimer" which is often unstable. Large natural plasmids often have their own resolvase gene to turn these multimers back into individual plasmids, but most cloning plasmids have had these cut out long ago during the construction process.

RecA is not the only homologous recombinase in the average E. coli genome that can affect the stability of a cloning plasmid, but the others tend to work on shorter regions of homology and are not usually as obviously problematic for cloning.

Hi there,

To answer I just copy and paste text from RecA wikipedia page:

" E. coli strains deficient in RecA are useful for cloning procedures in molecular biology laboratories. E. coli strains are often genetically modified to contain a mutant recA allele and thereby ensure the stability of extrachromosomal segments of DNA, known as plasmids... Transformants retain the plasmid throughout cell divisions such that it can be recovered and used in other applications. Without functional RecA protein, the exogenous plasmid DNA is left unaltered by the bacteria. Purification of this plasmid from bacterial cultures can then allow high-fidelity PCR amplification of the original plasmid sequence"

RecA is a recombinase involved in DNA repair that's responsible for recombination between long (hundreds of base-pairs) homologous stretches of DNA. If the DNA gets say, damaged during replication, RecA can repair it using a (theoretically) undamaged copy.

However, it's usually knocked out in cloning strains of E. coli because it can either cause plamids with long repeat regions, like lentiviral vectors for instance to become unstable due to RecA sometimes "resolving" the long repeats into a single copy which causes a deletion in the plasmid. Another problem is that if you're cloning a sequence that's homologous to the E. coli genome, recA will sometimes recombine the plasmid into the chromosome which can be a problem for a lot of reasons.

It also has a tendency to sometimes recombine a multiple copies of a plasmid into a single "multimer" which is often unstable. Large natural plasmids often have their own resolvase gene to turn these multimers back into individual plasmids, but most cloning plasmids have had these cut out long ago during the construction process.

RecA is not the only homologous recombinase in the average E. coli genome that can affect the stability of a cloning plasmid, but the others tend to work on shorter regions of homology and are not usually as obviously problematic for cloning.

Both Dominique and Alexandra are on point. Just to relate it to your question...

Your CRISPR plasmid is foreign, heterologous and extrachromosomal DNA that needed to be stably integrated or transformed to your E.coli strain like Stbl3. It is not required for growth or anything, but to ensure your DNA will be introduced to your cells flawlessly.

@Dominique Liger @ Alexandra Johnson @ Edward Alain Pajarillo

Excellent answer!

Before I never distinguish TOP10 and Stbl3, so I used TOP10 to amplify all my vectors. So if I want to amplify any vectors, especially lenti vectors with long repeats, it's better to use Stbl3.

Then, what is the advantage of TOP10 compared to Stbl3??

The main advantage I could think of is that Top10 is endA-, which means you don't have to worry much about endonuclease contamination of minipreps. Sbtl3 is endA+ so minipreps with it require additional wash steps to remove it. I made the mistake of trying to clone with a plasmid from an EndA+ strain without the extra purification steps first - the plasmid was fine after extraction but once I put it into a restriction enzyme buffer EndA became active and chopped my plasmid down to nothing. Top10/DH10B tend to have a higher electroporation efficiency than most strains for reasons that aren't totally understood. Both Top10 and Stbl3 have a recA mutation, and to be honest I don't know if there's a good reason people use Stbl3 over other recA- strains or if it's just an artifact of tradition. I don't know enough about the history of the strain and haven't worked much with it to give a definite answer.

This is a great blog post from AddGene that lists many lab strains, their genotypes, and a short description of what each genotype means:

http://blog.addgene.org/plasmids-101-common-lab-e-coli-strains

Becoming familiar with strains and their various advantages/problems/similarities has been a huge boon to my ability to clone successfully.

@Alexandra Johnson

Thanks! I like the table in the link!

In this case, both TOP10 and Stbl3 are RecA negative. In addition, TOP10 dose not have endA, which will improve the yield; also, TOP10 has better electroperation efficiency. So in general, TOP10 can be used widely.