Well, according to my status bar, I am 40% of the way to my projected goal for InaDWriMo. However, progress is temporarily halted since my boss wants me to stop writing. I told her OK, but I am writing in my free time so I think I'll stop at the end of the month.
Now for the other (not so) fun stuff.
I am actually in a shitty mood because things aren't working. There is a month remaining in gradstudent lab and I need to finish more than a months worth of stuff. When I defended my dissertation in September, I thought that there was ample time, but that was before the triple mutant from hell.
I need to make a triple mutant lacking genes A, B and C. Right now, I have double mutant AB and single mutant C. I've tried introducing the knock-out construct for deleting genes AB into mutant C, but it is toxic and the cells get really pissed and lose the KO construct before homologous recombination can take place. I tried to get around this by growing the cells in the presence of antibiotic since the vector contains resistance to one antibiotic and the genes are being replaced with a cassette that imparts resistance to another antibiotic. I obtained many colonies with the correct antibiotic resistance phenotype, but all of them still contain the genes I am trying to delete. The number of colonies and the rate of growth of said colonies indicate that they are not spontaneous, antibiotic-resistant mutants, indicating that the resistance gene is somewhere in the chromosome. Where? I don't know. I only know where it is NOT.
Obviously that wasn't working out, so I tried the opposite approach. Delete C in the AB double mutant background. Fine. I introduce the KO construct and the cell grow normally (yay) Unfortunately, that is the end to the good news. The KO vector for C is a temperature-sensitive vector and gene C is only deleted, not replaced with an antibiotic-resistance gene or cassette. So, it is screening hell. This approach works in the following way: The vector is integrated after growth at a non-permissive temperature. Cells are returned to the permissive temperature and and are passaged several times. At some point dilutions are plated on media containing no antibiotic. These colonies are replica plated onto additional plates that do contain antibiotic. Those that are sensitive, are screened for the mutation using PCR...colony PCR.
Unfortunately, the locus I am deleting doesn't seem to amplify using colony PCR, so I ended up trouble-shooting PCR. PC fucking R people. This is like trouble-shooting shampooing my hair. Finally, I found primers that work, except they don't yield a product if the gene is deleted. Awesome. A fucking negative result. This means that I have to recheck any colonies that don't yield a product and so far, all of those potential candidates have yielded a product the second time through. Still, it is better than growing up 5 zillion cultures and extracting DNA for PCR. Now, I am adding an antibiotic-resistance gene to this construct, so that I will have a selection. Good thing I am a rock star at cloning.
I am also considering that this triple mutant is lethal, although it really shouldn't be. We'll see. I am going to give it the ol' college try one last time with the new vector and see how it goes. If I can't get this mutant, then I will just have to drop the strain from the manuscript. :(
So, yeah. I am irritated to say the least.
Now for the other (not so) fun stuff.
I am actually in a shitty mood because things aren't working. There is a month remaining in gradstudent lab and I need to finish more than a months worth of stuff. When I defended my dissertation in September, I thought that there was ample time, but that was before the triple mutant from hell.
I need to make a triple mutant lacking genes A, B and C. Right now, I have double mutant AB and single mutant C. I've tried introducing the knock-out construct for deleting genes AB into mutant C, but it is toxic and the cells get really pissed and lose the KO construct before homologous recombination can take place. I tried to get around this by growing the cells in the presence of antibiotic since the vector contains resistance to one antibiotic and the genes are being replaced with a cassette that imparts resistance to another antibiotic. I obtained many colonies with the correct antibiotic resistance phenotype, but all of them still contain the genes I am trying to delete. The number of colonies and the rate of growth of said colonies indicate that they are not spontaneous, antibiotic-resistant mutants, indicating that the resistance gene is somewhere in the chromosome. Where? I don't know. I only know where it is NOT.
Obviously that wasn't working out, so I tried the opposite approach. Delete C in the AB double mutant background. Fine. I introduce the KO construct and the cell grow normally (yay) Unfortunately, that is the end to the good news. The KO vector for C is a temperature-sensitive vector and gene C is only deleted, not replaced with an antibiotic-resistance gene or cassette. So, it is screening hell. This approach works in the following way: The vector is integrated after growth at a non-permissive temperature. Cells are returned to the permissive temperature and and are passaged several times. At some point dilutions are plated on media containing no antibiotic. These colonies are replica plated onto additional plates that do contain antibiotic. Those that are sensitive, are screened for the mutation using PCR...colony PCR.
Unfortunately, the locus I am deleting doesn't seem to amplify using colony PCR, so I ended up trouble-shooting PCR. PC fucking R people. This is like trouble-shooting shampooing my hair. Finally, I found primers that work, except they don't yield a product if the gene is deleted. Awesome. A fucking negative result. This means that I have to recheck any colonies that don't yield a product and so far, all of those potential candidates have yielded a product the second time through. Still, it is better than growing up 5 zillion cultures and extracting DNA for PCR. Now, I am adding an antibiotic-resistance gene to this construct, so that I will have a selection. Good thing I am a rock star at cloning.
I am also considering that this triple mutant is lethal, although it really shouldn't be. We'll see. I am going to give it the ol' college try one last time with the new vector and see how it goes. If I can't get this mutant, then I will just have to drop the strain from the manuscript. :(
So, yeah. I am irritated to say the least.
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3 comments:
Your triple mutant makes me want to puke!
At least you're doing it in bugs instead of say, mice, where you'd have to wait 6 months to find out the damn thing didn't work...still that sounds frustrating as hell though. Hope it works eventually.
OMG! This stupid mutant is making me homicidal. I will not let the bacteria win. I don't care how many years of evolution they have on me.
I don't know how people make KO mice. They must be really patient. I would spontaneously combust if I had to wait six months to see if my mutation was present only to find that it wasn't.
Triplemutants are a pain. Never really understood why one works, double can work but three - there is a limit kind of...
so, back to my questions/trouble shooting help.
have you tried the triple antibiotic resistence? I saw that you had it on temp permissive... have to tried some different temps? Sometimes the triple/double mutant behaves even stranger with temp sensitivity so maybe you can lower/higher the temp and see if there is a difference.
And if it helps, would it be easier to insert a disruption rather than a complete knock out? you know, sometimes that makes a small gene product but then you'd know if the triplemutant (with complete knock out) would be lethal.
I wish you the best of luck!! I'm going to try and fix my two single mutants and my recombinant protein next week. It's been acting up a looooong time (read months and months). I've redone the construct like three times and now the sequencing doesn't work. I'm leaning towards more suicidal than homocidal but sure, I can adapt ;)
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