Talk:CH391L/S13/GeneticMarkers

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*'''[[User:Gabriel Wu|Gabriel Wu]] 17:30, 4 March 2013 (EST)''': On a practical note, ampicillin (amp), kanamycin (kan), and chloramphenicol (cam) are the most used antibiotic selection markers in synthetic biology.  Amp in my experience is convenient, but somewhat "fragile" (relative to kan and cam).  Convenient: typically "rescuing" your cells post-transformation is unnecessary when using amp. "Rescuing" is necessary for kan and cam.  Cam and kan are useful for long time course experiments (longer than 24 hours) because amp generally breaks down (or is broken down) over time.
*'''[[User:Gabriel Wu|Gabriel Wu]] 17:30, 4 March 2013 (EST)''': On a practical note, ampicillin (amp), kanamycin (kan), and chloramphenicol (cam) are the most used antibiotic selection markers in synthetic biology.  Amp in my experience is convenient, but somewhat "fragile" (relative to kan and cam).  Convenient: typically "rescuing" your cells post-transformation is unnecessary when using amp. "Rescuing" is necessary for kan and cam.  Cam and kan are useful for long time course experiments (longer than 24 hours) because amp generally breaks down (or is broken down) over time.
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**'''[[User:Benjamin Gilman|Benjamin Gilman]] 17:53, 5 March 2013 (EST)''': Gabe brings up a good point, amp is an easy antibiotic to use because it doesn't kill the cells quickly, so you don't have to wait for beta-lactamase to build up before you plate them on antibiotic.  Ampicillin is somewhat unstable, so many labs use it's relative cerbenicillin, which survives longer and breaks down into less toxic products.  Ampicillin is cheaper though, and works pretty well most of the time, so it remains the popular choice.
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**'''[[User:Benjamin Gilman|Benjamin Gilman]] 17:53, 5 March 2013 (EST)''': Gabe brings up a good point, amp is an easy antibiotic to use because it doesn't kill the cells quickly, so you don't have to wait for beta-lactamase to build up before you plate them on antibiotic.  Ampicillin is somewhat unstable, so many labs use it's relative carbenicillin, which survives longer and breaks down into less toxic products.  Ampicillin is cheaper though, and works pretty well most of the time, so it remains the popular choice.  
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****'''[[User:Alvaro E. Rodriguez M.|Alvaro E. Rodriguez M.]] 23:39, 14 March 2013 (EDT)''': Lol, Ben where do you buy cerbenicillin?, you mean carbenicillin hahaha, but I agree with you you can get Amp for 19 bucks vs Crb that is $39, still relatively cheap and yes more stable.
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*****'''[[User:Benjamin Gilman|Benjamin Gilman]] 01:46, 18 March 2013 (EDT)''': I've often seen carb go for about 10x the price of amp, so I'm not sure where you got those numbers from.  That's definitely the reason that amp is still more popular.  Regardless, I added a few things to the page about why carb is used.
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***'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 18:41, 7 March 2013 (EST)''': Are the products from Amp breakdown toxic to humans? Poisoning bacteria with something that turns into stronger poison doesn't sound all that bad otherwise.
***'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 18:41, 7 March 2013 (EST)''': Are the products from Amp breakdown toxic to humans? Poisoning bacteria with something that turns into stronger poison doesn't sound all that bad otherwise.
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****'''[[User:Benjamin Gilman|Benjamin Gilman]] 23:16, 7 March 2013 (EST)''': As far as I know, the cleaved beta-lactams aren't particularly toxic to humans, but they might cause a problem when amp is used for selection.  If amp resistant cells are poisoned by the byproducts of beta-lactamase cleavage, the overall number of surviving colonies will be low relative to the number of successful transformants.
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**'''[[User:Catherine I. Mortensen|Catherine I. Mortensen]] 11:39, 7 March 2013 (EST)''':I've heard of some other antibiotics such as tetracycline, streptomycin, and chloramphenicol. When are these particularly useful?
**'''[[User:Catherine I. Mortensen|Catherine I. Mortensen]] 11:39, 7 March 2013 (EST)''':I've heard of some other antibiotics such as tetracycline, streptomycin, and chloramphenicol. When are these particularly useful?
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***'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 18:38, 7 March 2013 (EST)''': The wikipedia page for [http://en.wikipedia.org/wiki/Streptomycin#Mechanism_of_action Strep] says that at low concentrations, it only inhibits growth instead of outright killing the cells. It cites a Biochemistry textbook for this. For Tet, the fact that it's counterselectable, as Larry mentioned, is extremely useful. According to [http://pharmaxchange.info/press/2011/05/mechanism-of-action-of-tetracyclines/ this], tetracycline binding to the 30S ribosomal subunit is reversible, which sounds useful, althoughI don't know how you'd actually reverse it.
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***'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 18:38, 7 March 2013 (EST)''': The wikipedia page for [http://en.wikipedia.org/wiki/Streptomycin#Mechanism_of_action Strep] says that at low concentrations, it only inhibits growth instead of outright killing the cells. It cites a Biochemistry textbook for this. For Tet, the fact that it's counterselectable, as Ben mentioned, is extremely useful. According to [http://pharmaxchange.info/press/2011/05/mechanism-of-action-of-tetracyclines/ this], tetracycline binding to the 30S ribosomal subunit is reversible, which sounds useful, although I don't know how you'd actually reverse it.
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****'''[[User:Benjamin Gilman|Benjamin Gilman]] 23:16, 7 March 2013 (EST)''': In my experience, streptomycin (strep) isn't frequently used for selection with E. coli, this may be because two genes are required for strong resistance to it.  I've heard of it being used with other bacteria, possibly because they aren't as sensitive to antibiotics like amp, cap, or kan.  Strep is often added to the media that human tissue culture cells are grown in, along with penicillin, to provide some broad-spectrum protection against bacterial contamination.
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***'''[[User:Benjamin Gilman|Benjamin Gilman]] 23:16, 7 March 2013 (EST)''': One of the main reasons for the popularity of several antibiotics is that often you need to select for two or more markers simultaneously, like when you need cells transformed with multiple plasmids.  The cells I use for expressing proteins for instance are grown on amp/cap plates because they have a plasmid with the protein-coding gene and an ampR marker and a plasmid that codes for additional tRNAs with a capR marker.
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*'''[[User:Gabriel Wu|Gabriel Wu]] 17:33, 4 March 2013 (EST)''': For those who work outside of E. coli, there's a class of selection markers used more commonly in yeast known as auxotrophic markers. Maybe not this time, but this topic should be addressed on this page at some point.
*'''[[User:Gabriel Wu|Gabriel Wu]] 17:33, 4 March 2013 (EST)''': For those who work outside of E. coli, there's a class of selection markers used more commonly in yeast known as auxotrophic markers. Maybe not this time, but this topic should be addressed on this page at some point.
**'''[[User:Benjamin Gilman|Benjamin Gilman]] 17:39, 5 March 2013 (EST)''': The [http://openwetware.org/wiki/CH391L/S12/Selectablegeneticmarkers selectable genetic markers] page from last year's class gives a brief overview of different categories of markers, including auxotrophic markers.  Briefly, auxotrophs are organisms that are incapable of synthesizing a particular molecule required for growth, in this case because they've had a necessary gene knocked out.  The mutant auxotrophs grow when supplemented with the required molecule but require complementation with the deleted gene to survive without it .  Auxotrophic markers are more often associated with yeast than bacteria mostly because the range of antibiotic resistance markers in yeast is much smaller.  The GFAT-expressing gene, ''glmS'', I discussed in class is an example of an auxotrophic marker which works in ''E. coli'', and a related GFAT gene, ''gfa1'', works similarly as a marker in fission yeast (''S. pombe'').
**'''[[User:Benjamin Gilman|Benjamin Gilman]] 17:39, 5 March 2013 (EST)''': The [http://openwetware.org/wiki/CH391L/S12/Selectablegeneticmarkers selectable genetic markers] page from last year's class gives a brief overview of different categories of markers, including auxotrophic markers.  Briefly, auxotrophs are organisms that are incapable of synthesizing a particular molecule required for growth, in this case because they've had a necessary gene knocked out.  The mutant auxotrophs grow when supplemented with the required molecule but require complementation with the deleted gene to survive without it .  Auxotrophic markers are more often associated with yeast than bacteria mostly because the range of antibiotic resistance markers in yeast is much smaller.  The GFAT-expressing gene, ''glmS'', I discussed in class is an example of an auxotrophic marker which works in ''E. coli'', and a related GFAT gene, ''gfa1'', works similarly as a marker in fission yeast (''S. pombe'').
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*'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 19:22, 7 March 2013 (EST)''': I'm guessing satellite colonies are only problem for antibiotics that are pumped into the surroundings? A transporter like TetA should maintain the Tet concentration around it, preventing any cheaters from getting through.
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**'''[[User:Benjamin Gilman|Benjamin Gilman]] 23:37, 7 March 2013 (EST)''': I think you meant to say it the other way around, satellite colonies aren't a problem with antibiotics that are just pumped out of the cell.  Amp selection is the most prone to satellite colonies, because the beta-lactamase degrades enough amp around a resistant colony that additional cells without the ampR marker can grow.  I'm not sure why some of the other antibiotic-degrading enzymes don't cause the same problem, but I'd guess that the cells are less permeable to kan and cap, so they are degraded less quickly and higher concentrations of antibiotic can be used in the media.
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*'''[[User:Alvaro E. Rodriguez M.|Alvaro E. Rodriguez M.]] 23:51, 14 March 2013 (EDT)''':Also the explanation of screening vs. selection could be much simpler.
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**'''[[User:Benjamin Gilman|Benjamin Gilman]] 02:46, 18 March 2013 (EDT)''': I reworked it, but I wanted to avoid just saying something like "screening makes you check every cell while selection just gives you the right ones" without some explanation of how either might conceptually work in bacteria.

Current revision

  • Gabriel Wu 17:30, 4 March 2013 (EST): On a practical note, ampicillin (amp), kanamycin (kan), and chloramphenicol (cam) are the most used antibiotic selection markers in synthetic biology. Amp in my experience is convenient, but somewhat "fragile" (relative to kan and cam). Convenient: typically "rescuing" your cells post-transformation is unnecessary when using amp. "Rescuing" is necessary for kan and cam. Cam and kan are useful for long time course experiments (longer than 24 hours) because amp generally breaks down (or is broken down) over time.
    • Benjamin Gilman 17:53, 5 March 2013 (EST): Gabe brings up a good point, amp is an easy antibiotic to use because it doesn't kill the cells quickly, so you don't have to wait for beta-lactamase to build up before you plate them on antibiotic. Ampicillin is somewhat unstable, so many labs use it's relative carbenicillin, which survives longer and breaks down into less toxic products. Ampicillin is cheaper though, and works pretty well most of the time, so it remains the popular choice.
        • Alvaro E. Rodriguez M. 23:39, 14 March 2013 (EDT): Lol, Ben where do you buy cerbenicillin?, you mean carbenicillin hahaha, but I agree with you you can get Amp for 19 bucks vs Crb that is $39, still relatively cheap and yes more stable.
          • Benjamin Gilman 01:46, 18 March 2013 (EDT): I've often seen carb go for about 10x the price of amp, so I'm not sure where you got those numbers from. That's definitely the reason that amp is still more popular. Regardless, I added a few things to the page about why carb is used.
      • Aurko Dasgupta 18:41, 7 March 2013 (EST): Are the products from Amp breakdown toxic to humans? Poisoning bacteria with something that turns into stronger poison doesn't sound all that bad otherwise.
        • Benjamin Gilman 23:16, 7 March 2013 (EST): As far as I know, the cleaved beta-lactams aren't particularly toxic to humans, but they might cause a problem when amp is used for selection. If amp resistant cells are poisoned by the byproducts of beta-lactamase cleavage, the overall number of surviving colonies will be low relative to the number of successful transformants.
    • Catherine I. Mortensen 11:39, 7 March 2013 (EST):I've heard of some other antibiotics such as tetracycline, streptomycin, and chloramphenicol. When are these particularly useful?
      • Aurko Dasgupta 18:38, 7 March 2013 (EST): The wikipedia page for Strep says that at low concentrations, it only inhibits growth instead of outright killing the cells. It cites a Biochemistry textbook for this. For Tet, the fact that it's counterselectable, as Ben mentioned, is extremely useful. According to this, tetracycline binding to the 30S ribosomal subunit is reversible, which sounds useful, although I don't know how you'd actually reverse it.
        • Benjamin Gilman 23:16, 7 March 2013 (EST): In my experience, streptomycin (strep) isn't frequently used for selection with E. coli, this may be because two genes are required for strong resistance to it. I've heard of it being used with other bacteria, possibly because they aren't as sensitive to antibiotics like amp, cap, or kan. Strep is often added to the media that human tissue culture cells are grown in, along with penicillin, to provide some broad-spectrum protection against bacterial contamination.
      • Benjamin Gilman 23:16, 7 March 2013 (EST): One of the main reasons for the popularity of several antibiotics is that often you need to select for two or more markers simultaneously, like when you need cells transformed with multiple plasmids. The cells I use for expressing proteins for instance are grown on amp/cap plates because they have a plasmid with the protein-coding gene and an ampR marker and a plasmid that codes for additional tRNAs with a capR marker.
  • Gabriel Wu 17:33, 4 March 2013 (EST): For those who work outside of E. coli, there's a class of selection markers used more commonly in yeast known as auxotrophic markers. Maybe not this time, but this topic should be addressed on this page at some point.
    • Benjamin Gilman 17:39, 5 March 2013 (EST): The selectable genetic markers page from last year's class gives a brief overview of different categories of markers, including auxotrophic markers. Briefly, auxotrophs are organisms that are incapable of synthesizing a particular molecule required for growth, in this case because they've had a necessary gene knocked out. The mutant auxotrophs grow when supplemented with the required molecule but require complementation with the deleted gene to survive without it . Auxotrophic markers are more often associated with yeast than bacteria mostly because the range of antibiotic resistance markers in yeast is much smaller. The GFAT-expressing gene, glmS, I discussed in class is an example of an auxotrophic marker which works in E. coli, and a related GFAT gene, gfa1, works similarly as a marker in fission yeast (S. pombe).
  • Aurko Dasgupta 19:22, 7 March 2013 (EST): I'm guessing satellite colonies are only problem for antibiotics that are pumped into the surroundings? A transporter like TetA should maintain the Tet concentration around it, preventing any cheaters from getting through.
    • Benjamin Gilman 23:37, 7 March 2013 (EST): I think you meant to say it the other way around, satellite colonies aren't a problem with antibiotics that are just pumped out of the cell. Amp selection is the most prone to satellite colonies, because the beta-lactamase degrades enough amp around a resistant colony that additional cells without the ampR marker can grow. I'm not sure why some of the other antibiotic-degrading enzymes don't cause the same problem, but I'd guess that the cells are less permeable to kan and cap, so they are degraded less quickly and higher concentrations of antibiotic can be used in the media.
  • Alvaro E. Rodriguez M. 23:51, 14 March 2013 (EDT):Also the explanation of screening vs. selection could be much simpler.
    • Benjamin Gilman 02:46, 18 March 2013 (EDT): I reworked it, but I wanted to avoid just saying something like "screening makes you check every cell while selection just gives you the right ones" without some explanation of how either might conceptually work in bacteria.
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