Quick note from the Bibel lab 4 - our selection strategy has hope

Usually, we're sad when we don't see any colonies (clumps of bacteria) on our plates. But today we were super stoked by the absence of growth because it meant that the wild-type (unaltered) bacteria we want to genetically manipulate are susceptible to the antibiotic kanamycin (which was present in the plates). This was exciting because it means that we can use kanamycin as a "selection marker." Basically, we want to remove ("knock out") a gene from these bacteria. But we know that 1) not all the bacterial cells will cooperate with this plan and 2) it will make that do cooperate (have the gene knocked out) unhappy (less fit and thus less able to compete against those bacterial cells that did not cooperate (the unaltered ones)). Therefore, we need a way to give the altered ones an advantage that's so strong it will allow us to kill off the non-cooperators and thus "select for" those that cooperated. The way we're planning to do this is by using homologous recombination to trick the bacteria into swapping in a kanamycin resistance gene in place of the gene we're removing. This way, in the cells where the swappage works, the bacteria will have kanamycin resistance and thus be able to grow in the presence of kanamycin. But, the bacterial cells that didn't cooperate with us will pay the ultimate price... This strategy only works, however, if the bacteria aren't naturally resistant. So were were super happy to see they weren't!
We're basically following the strategy and protocol from this paper where they did this for every gene in Bacillus subtilis!
Koo, B. C., Kritikos, G., Farelli, J. D., Todor, H., Tong, K., Kimsey, H. H., … & Gross, C. A. (2017). Construction and analysis of two genome-scale deletion libraries for bacillus subtilis. Cell Systems, 4(3), 291-305.e7. doi.org/10.1016/j.cels.2016.1...
And the authors made them publicly accessible through the Bacillus Genetic Stock Center (BGSC). bgsc.org
That's where we got that 168Δmdh strain. 168 is the classic laboratory strain of Bacillus subtilis, and the Δ "delta" notation means knock out so this is 168 with the mdh gene (instructions for making malate dehydrogenase) knocked out (removed). We're working to copy the kanamycin cassette (the kanamycin resistance gene and surrounding regions) from that strain and stick it in our Bacillus safensis strains in place of the mdh gene. Fingers crossed! There's still lots of hurdles to overcome, but at least we know that it theoretically should work!
More on antibiotic selection: bit.ly/antibioticselections & • Using antibiotics as s...
More updates from the Bibel lab: • Quick "lab notes" from...
more about bacterial media & agar plates: bit.ly/bacterialmedia
more about genetic knockout: bit.ly/knockdownvsknockout & • Genetic Knock-Down (e....
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@suryadarshan2381]

Yo this is soooo cool I literally did this today in the lab (Just started my summer internship) 🤩

[@thebumblingbiochemist]

Awesome! Congrats!!!