HELP: From Plasmid Vector to Bacterial Chromosome - (Mar/16/2015 )

Dear All,

Although I'm a microbiologist my experience is more in classical microbiology and I have little experience with cloning. So, as I start a new project that has a small but important cloning set in the beginning, I've come to this forum in search of advice and to steepen my learning curve

In my new project, I will track up to four different E. coli genotypes (different strains) simultaneously in a single culture over an extended period of time. In order to achieve this aim, I intent to insert four different colors of fluorescent proteins onto the chromosome of each strains so that I can measure the abundance of each using a flow cytometer. I choose to insert the fluorescent proteins into the chromosome instead of keeping them on the plasmid to ensure stability. 

I've read about "BD colors fluorescent proteins" and using a set up consisting of fluorescent proteins DsRed2, ZsYellow1, HcRed1, or AmCyan1 my experiment is possible with the right flow cytometer, which is available to me at my university. 

These fluorescent proteins are available from the company (BD Biosciences Clontech) as vectors containing multiple cloning sites and an antibiotic resistance marker.

My basic questions I how do I insert the genes for these fluorescent proteins onto the chromosomes of each strain?

From what I know is that this type of allelic exchange can be accomplished by using a suicide plasmid and two homologous regions between the plasmid vector and host chromosome--but that's pretty much where my knowledge (at least for the moment!) ends. For instance, I take it that I have to clone the fluorescent protein's gene into a suicide plasmid first? And if so, what suicide plasmid do I need? Also, might there be other methods to do this, like using a transposon system?

I would appreciate any guidelines--like a basic to complex workflow--that you're able to suggest. Or even if this is too basic, some elementary references that I could look up for such a problem.

Thank you so much for your help

I hope I can return the favor in the future

Cheers,

CAM

Like yourself, I haven't actually done this myself, and I'm not a bacteriologist (I'm a virologist), but your scheme is correct. You are looking for homologous recombination between the plasmid and the chromosome. This link should help (pdf). Have a further look on current protocols, they typically have detailed and very informative protocols available (if you have access).

As to systems, the one that is all the rage at the moment is CRISPR/Cas, which I understand is quick and very very accurate.

Indeed, thanks Bob (sorry for the late reply). I'm still looking into the specific cloning system I will use your pdf was indeed a good intro.