User:Jonathan Cline/Notebook/Melaminometer/Brainstorming

Designing the plasmid

Which enzymes should we produce?

Per the second bullet point at User:Jonathan_Cline/Notebook/Melaminometer/Toxicology_Details, it sounds like melamine and cyanuric acid aren't that harmful in isolation, but when combined they produce renal toxicity by forming the melamine cyanurate salt. Should we think about developing multiple strains of test bacteria, one which reports the presence of melamine and one which reports the presence of cyanuric acid? Or should we develop a plasmid which carries reporters for both melamine and cyanuric acid (with different colours, e.g. yellow and blue?)

Pros:

• More realistic determination of risk
• Single strain has a simple detection protocol -- if the sample turns yellow there's melamine, if it turns blue there's cyanuric acid, if it turns green, there's both!

Cons:

• Two strains of bacteria = more complicated process
• Cyanuric acid is a product of melamine degradation, so if the test organism contains the full melamine degradation pathway, the whole sample will start out yellow, shift to green, and finally turn blue as melamine is deaminated into cyanuric acid.
  • Could solve this problem by only including melamine deaminase and cyanurate amidohydrolase, i.e., the first step of each pathway. Melamine deaminase will deaminate the melamine twice, but the third deamination never takes place if there's no ammelide aminohydrolase. Thus, the sample will only turn green if both melamine and cyanuric acid are present.
    • Another benefit of including just the first step of each pathway: the first step for both pathways is present in A. acidovorax subsp. citrulli. Should it turn out to be more cost-effective, practical, whatever, to clone our genes of interest from a living organism rather than synthesizing them de novo, we'll only have to work with one organism -- and it's an organism that isn't pathogenic to humans, unlike Pseudomonas.

Possible solution:

• Agree that 1-step degradation of melamine is best, to eliminate crosstalk between melamine->product and cyanuric acid.
• Ratio of melamine-to-cyanuric acid may or may not be important? 10-to-1 same as 1-to-1 for crystal formation.
  • Reference 2006 igem arsenic detector for 'amplifer' circuit used.
    • http://parts.mit.edu/wiki/images/5/5d/IGEM2006-Edinburgh-Powerpoint.pdf

Sensitivity

• What's the threshold for detection? How can this threshold be modified?
  • "Melamine levels in imported Chinese candies recalled last week in California were as high as 520 parts per million, about 200 times greater than the level set Friday by the FDA for "tolerable" risk."
  • Must detect close to "2.5 ppm of melamine and its analogues"

Chassis

• Desired to be non-pathogenic, wide temperature stability, non-smelly
  • "Pseudomonas sp. strain ADP is not amenable to genetic studies. [...]"
  • I'm generally in favour of Lactobacillus sp., although Gram-positive bacteria are more difficult to transform than E. coli are. There are good electroporation protocols for lactobacilli though.

Environmental

• Metabolizing melamine/etc throws off NH4+ ammonium cation with each degradation step.
  • Are you sure about that? MetaCyc says it's NH3.
  • Will the microbe be affected with change in pH?
    • I'm bad at chemistry: does that mean the pH goes up or down? Lactobacilli tend to like acidic environments.
  • Can ammonium be used as a super-easy detection mechanism?
    • if user smells ammonium, then melamine is present.
      • "The average human nose can detect ammonia concentrations on the order of 50 ppm or less." [1]
      • Need to test whether it's released as a gas. If so, then it should be quite easy to detect -- though note that concentrations of 100ppm are irritating to the nasal passages!
    • use pH strip or drop kit, scale of red indicates melamine metabolized. What is blank pH of typical sample; infant baby formula likely basic (?)