Gel quantitation with Lambda/HindIII for starters, spectrophotometer later. I've got specs for a DIY spectrophotometer which is super simple to build.
Where will you get antibiotic for your plates?
The pGREEN plasmids confer ampicillin resistance, and I got my ampicillin in powder form from an aquarium supplier. I also have fish tetracycline, in case we decide to go with tetracycline resistance.
The questions about FRET, promoters, conformational changes &c are also further ahead than I am in my work -- we're still trying to figure out whether simply using GFP as a reporter (i.e., getting it to express when melamine deaminase is expressed) will do what we want, since I'm still working on figuring out whether melamine deaminase will be expressed all the time or if it will only be expressed in the presence of melamine. If it's the former, we happen to be working with a team at National Yang-Ming University in Taiwan which built a bacterial pH detector for this year's iGEM. If we go that route, we'll use the pH detector to detect the breakdown product of melamine deaminase + melamine (ammonia). For that matter, ammonia is very easy for the human nose to detect, so simply having the breakdown product might be enough. I don't mind trying an idea and finding out that it doesn't work; I learn as much from failure as I do from success.
If so, will you also be building your own SDS-PAGE setup and western blot apparatus?
Most likely, yes. I've held off on that because I haven't figured out how I want to handle polyacrylamide safely.
How will you be validating your results?
That's kind of a broad question, but of course repeatability, comparison to LC/MS and ELISA results, &c are all important. The NYMU guys are a big part of that stage of things.
Where will you get melamine to test with?
Aldrich sells it, 5g for $15.
Are you planning to make yogurt with the resultant bacteria?
Only insofar as it's a workable way to store cultures long-term when one doesn't have a proper freezer. I don't think it will be a very good delivery medium. Tablet form might actually be easiest, like the pills you can buy at the pharmacy.
Will you add antibiotic to the yogurt to maintain the plasmid?
Only if I'm using yogurt for storage purposes, and I sure as hell won't be eating it. I'm violently allergic to ampicillin.
Is the idea to ultimately mix food into the yogurt and look for fluorescence with the naked eye?
Or to culture bacteria on/in the food, yes. Being able to look for fluorescence with the naked eye (well, and a blue light) would certainly be optimal. Will we achieve that? Probably not without additional tweaking, since the concentration of melamine that is deemed dangerous is actually quite low. That said, there are some neat tricks in BioBricks for upregulating reporter genes.
no subject
How will you quantitate your DNA?
Gel quantitation with Lambda/HindIII for starters, spectrophotometer later. I've got specs for a DIY spectrophotometer which is super simple to build.
Where will you get antibiotic for your plates?
The pGREEN plasmids confer ampicillin resistance, and I got my ampicillin in powder form from an aquarium supplier. I also have fish tetracycline, in case we decide to go with tetracycline resistance.
The questions about FRET, promoters, conformational changes &c are also further ahead than I am in my work -- we're still trying to figure out whether simply using GFP as a reporter (i.e., getting it to express when melamine deaminase is expressed) will do what we want, since I'm still working on figuring out whether melamine deaminase will be expressed all the time or if it will only be expressed in the presence of melamine. If it's the former, we happen to be working with a team at National Yang-Ming University in Taiwan which built a bacterial pH detector for this year's iGEM. If we go that route, we'll use the pH detector to detect the breakdown product of melamine deaminase + melamine (ammonia). For that matter, ammonia is very easy for the human nose to detect, so simply having the breakdown product might be enough. I don't mind trying an idea and finding out that it doesn't work; I learn as much from failure as I do from success.
If so, will you also be building your own SDS-PAGE setup and western blot apparatus?
Most likely, yes. I've held off on that because I haven't figured out how I want to handle polyacrylamide safely.
How will you be validating your results?
That's kind of a broad question, but of course repeatability, comparison to LC/MS and ELISA results, &c are all important. The NYMU guys are a big part of that stage of things.
Where will you get melamine to test with?
Aldrich sells it, 5g for $15.
Are you planning to make yogurt with the resultant bacteria?
Only insofar as it's a workable way to store cultures long-term when one doesn't have a proper freezer. I don't think it will be a very good delivery medium. Tablet form might actually be easiest, like the pills you can buy at the pharmacy.
Will you add antibiotic to the yogurt to maintain the plasmid?
Only if I'm using yogurt for storage purposes, and I sure as hell won't be eating it. I'm violently allergic to ampicillin.
Is the idea to ultimately mix food into the yogurt and look for fluorescence with the naked eye?
Or to culture bacteria on/in the food, yes. Being able to look for fluorescence with the naked eye (well, and a blue light) would certainly be optimal. Will we achieve that? Probably not without additional tweaking, since the concentration of melamine that is deemed dangerous is actually quite low. That said, there are some neat tricks in BioBricks for upregulating reporter genes.