Hi everyone,
I need to make a single base change (no scars or markers can be left) in the E. coli chromosome and would like to avoid using the traditional method (clone, insert in genome, recombine again to lose plasmid). Are there new popular PCR based methods for doing this? Or a CRISPR product?
Thank you all.
Teca
you best bet is CRISPR combined with lamda red system. multiple papers and plasmids are available on how to do this.
Hi Teca.
Check this efficient and economic PCR-based site-directed mutagenesis method:
https://experiments.springernature.com/articles/10.1385/0-89603-332-5:157?gclid=CjwKCAjwzJjrBRBvEiwA867bytDKO_chSQj36kmbD7mfRCV_4RpJKhiwHHz68CuB6plf4sNu5OYpWxoCN04QAvD_BwE
Nevertheless, I also think that if you already have standardized Crispr , just go for it
Recombineering is still the fastest method if you are looking to make a single new strain of bacteria. All you need to do is transform your E. coli with a temperature unstable Lambda Red plasmid (e.g. pSim5), and then perform recombination with an oligo (but I'd recommend a short PCR product) that has your bp change. You will have to screen a lot of colonies to find your insert, which is the time consuming part. Also, single base pair changes are difficult but not impossible to screen by PCR on a large scale. Once a positive is found, grow your E. coli at a higher temp, and you will cure your bacteria of the recombineering plasmid quickly.
If you plan to make a variety of changes at a single site, then you should invest the time needed to get a CRISPR system working. This will increase the efficiency greatly. There is no way around the time it will take to design, make, and test your sgRNA, and you will still need to perform a similar recombineering protocol to get that to work.
it depend on how much bases you want to insert,if 80bp or less you can use this annealing oligo primers method and you can find it here https://eu.idtdna.com/pages/education/decoded/article/annealing-oligonucleotides
Thank you all for your answers.
George, I need a single mutant and so looks like trying the recombineering and sequencing is the best way forward. It raises some additional questions - the lambda proteins need single stranded DNA, so would it not be more efficient to use oligos? Also, would making several rounds of transformation with the same oligo/PCR increase the frequency of mutants? And is there a magic number for the initial number of colonies that will need checking before a mutant appears? (In other words, what is the estimated frequency of mutation using a system such as the one you mention?)
Best
Teca
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