As to where those satellite cells come from, they were likely there all along! A big area of recent research is studying so-called “persister cells” which kind of “hibernate” while the bla-containing cells do all the work. Then they “wake up” when things have cleared up. more on this here: Medaney, F., Dimitriu, T., Ellis, R. J., & Raymond, B. (2016). Live to cheat another day: bacterial dormancy facilitates the social exploitation of β-lactamases. The ISME journal, 10(3), 778-787. doi.org/10.1038/ismej.2015.154 It ties in to some of what I’ve talked about in the past regarding the growing problem of antimicrobial resistance (AMR): bit.ly/antibiotic_resistance_mech ; UA-cam: ua-cam.com/video/ueP_9Vj-Q0k/v-deo.html for more on how we take advantage of antibiotic resistance mechanisms in the lab to select for bacteria containing plasmids of interest: bit.ly/antibioticselections & ua-cam.com/video/YXg56OH3N3A/v-deo.html and here’s more about ampicillin instability (and how it’s worse that way than other antibiotics): Ryan, K. J., Needham, G. M., Dunsmoor, C. L., & Sherris, J. C. (1970). Stability of antibiotics and chemotherapeutics in agar plates. Applied microbiology, 20(3), 447-451. doi.org/10.1128/am.20.3.447-451.1970
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
I remember I had to subclone my gene into a pet20 protein expression vector and I would always get very little colonies after ligation. Is it common to only get a few colonies after sub cloning? They were extremely small sometimes. I ran an agarose gel with appropriate restriction enzymes and luckily saw my gene of interest, but I always thought that was odd. Maybe cells like DH5 alpha or XL1 blue make better colonies than BL21 gold? Cells? Hahah anyways I always learn so much from your videos😊 I think it would be awesome if you can give a few pointers on how to write a manuscript/make nice looking figures ?
It definitely isn't uncommon to get very few colonies. If you aren't doing so already, I recommend plating your entire transformation - spin down the cells after the outgrowth, remove the media, and resuspend in ~100 μL LB, then plate that all. Typically I do my cloning/subcloning in DH5 cells (higher copy number, no T7) and then transform them into BL21(DE3) for expression. More on why here: bit.ly/dh5alpha ; UA-cam: ua-cam.com/video/MwMTFQira4A/v-deo.html As for manuscripts, I've only worked on one so don't have enough experience to feel qualified to make a video on that - sorry!
As to where those satellite cells come from, they were likely there all along! A big area of recent research is studying so-called “persister cells” which kind of “hibernate” while the bla-containing cells do all the work. Then they “wake up” when things have cleared up.
more on this here: Medaney, F., Dimitriu, T., Ellis, R. J., & Raymond, B. (2016). Live to cheat another day: bacterial dormancy facilitates the social exploitation of β-lactamases. The ISME journal, 10(3), 778-787. doi.org/10.1038/ismej.2015.154
It ties in to some of what I’ve talked about in the past regarding the growing problem of antimicrobial resistance (AMR): bit.ly/antibiotic_resistance_mech ; UA-cam: ua-cam.com/video/ueP_9Vj-Q0k/v-deo.html
for more on how we take advantage of antibiotic resistance mechanisms in the lab to select for bacteria containing plasmids of interest: bit.ly/antibioticselections & ua-cam.com/video/YXg56OH3N3A/v-deo.html
and here’s more about ampicillin instability (and how it’s worse that way than other antibiotics): Ryan, K. J., Needham, G. M., Dunsmoor, C. L., & Sherris, J. C. (1970). Stability of antibiotics and chemotherapeutics in agar plates. Applied microbiology, 20(3), 447-451. doi.org/10.1128/am.20.3.447-451.1970
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
I remember I had to subclone my gene into a pet20 protein expression vector and I would always get very little colonies after ligation. Is it common to only get a few colonies after sub cloning? They were extremely small sometimes. I ran an agarose gel with appropriate restriction enzymes and luckily saw my gene of interest, but I always thought that was odd. Maybe cells like DH5 alpha or XL1 blue make better colonies than BL21 gold? Cells? Hahah anyways I always learn so much from your videos😊 I think it would be awesome if you can give a few pointers on how to write a manuscript/make nice looking figures ?
It definitely isn't uncommon to get very few colonies. If you aren't doing so already, I recommend plating your entire transformation - spin down the cells after the outgrowth, remove the media, and resuspend in ~100 μL LB, then plate that all. Typically I do my cloning/subcloning in DH5 cells (higher copy number, no T7) and then transform them into BL21(DE3) for expression. More on why here: bit.ly/dh5alpha ; UA-cam: ua-cam.com/video/MwMTFQira4A/v-deo.html
As for manuscripts, I've only worked on one so don't have enough experience to feel qualified to make a video on that - sorry!