How to remove antibiotic resistance from bacteria

Dr Jamey Kain, from the Glowing Plant Project, explains an ingenious process he developed for removing antibiotic resistance genes from agrobacterium.

[@ChristieNel]

Sometimes I'm tempted to switch from engineering to microbiology. This stuff is so fascinating.

[@raulmacias6018]

Has anyone been shipped out the beta version of the maker’s kit?

[@darkquaesar2460]

If only you didn't use antibiotic resistance as genetic markers for colony purification.

[@notyourluckyday]

Not that I know anything on the subject, but why not use restriction enzymes to cut and switch the DNA segments manually? Or is that what you are doing when you talk about the "rare recombination event"?

[@TAXABiotechnologies]

notyourluckyday Restriction enzymes require a unique insertion site, and because our plasmid is over 200kb there aren't too many unique sites we can use.

[@cosmicaug]

notyourluckyday, the problem is not them being able to do stuff with restriction enzymes (or by other means). The problem is them being able to find transformants which have picked up the correct construct. When they are done attempting to do a transformation, they may have a microcentrifuge tube with many millions of viable bacteria in it. When you spread those on a plate you have those millions of bacteria with a few dozen bacteria that have picked up the correct construct. If you let that grow, you just have a bacterial lawn and no way to know where the bacteria you actually want are (it's all a uniform layer of bacteria). The question is how you pick out the bacteria that you actually want? The answer is that, under these conditions, you cannot. That's why you need a selection marker. Normally you use positive selection markers which are genes that favor survival (antibiotic resistance markers would be of this type when used in conjunction with antibiotics). In contrast, a negative selection marker is a gene that is selected against under some conditions (examples would be the sacB mentioned in the video or a "suicide gene" such as ccdB). Either way, when you grow such under the appropriate conditions, most of the millions of bacteria in that tube will not be able to survive and the ones you actually want will survive. When you let that grow you will get a few dozen discrete colonies composed of the genotypes you actually want (if nothing has gone wrong). The clever thing they have done is swap in a construct with a positive selection marker with one that has both a positive and a negative selection marker.

[@notyourluckyday]

August Pamplona Well yes I realize the necessity for the correct construct selection, what I was curious about is what exactly they did for the swapping process you mentioned, as they said it was a rare occurrence.

[@cosmicaug]

Ah, never mind! All of these techniques rely on the selection of rare events even when using restriction enzymes. As for recombination, there are protocols out there for this but I am not specifically familiar with them (I need to do some reading). In fact, an extremely popular method for cloning genes called the Gateway system uses recombination instead of restriction enzymes (I am pretty familiar with that one) but that is not what they have done for the first step (it relies on and is optimized for a very specific set of sequences for the recombination sites which they do not have). I suspect you could contrive a sequence for the insert from the first step that would work with the gateway system enzymes to make it excise itself with relatively high efficiency but I would have to work it out on paper or with something like ApE to be sure (see biologylabs.utah.edu/jorgensen/wayned/ape/ ).

[@notyourluckyday]

August Pamplona Well thanks for the info and link! Can't wait to learn more about it!

[@nitroagent6494]

So is this the last big issue you have with any of your products?

[@TAXABiotechnologies]

NitroAgent 64 There is one more possible catch/problem which concerns the removal of herbicide markers from the plants. This should be easy to cross out using mendelian genetic's techniques, but if the herbicide insert gets close to the glowing genes we could possibly have an issue.

[@nitroagent6494]

Thank you for the update.

[@GregDowning]

How does this technique compare to using something like CRISPR to modify the dna? There was a really good radiolab podcast on CRISPR a few weeks ago that explained it well, www.radiolab.org/story/antibodies-part-1-crispr/