User:Jonathan Cline/Notebook/Melaminometer/Brainstorming

Dry Lab Design Method

1. Project definition
   1. Iteratively (re-)define project
   2. Verify theoretical feasibility
   3. Repeat from above
2. Project Feasibility
   1. Research existing metabolism/catabolism/enzymatic activity
   2. Research existing microbe structure
   3. Verify theoretical feasibility of inserting metabolic path into desired microbe
      1. Need a suitable vector -- not all plasmids are appropriate for all gut bacteria
      2. Must be stable, i.e., reproduce from generation to generation
   4. Verify theoretical compatibility of biological components
      1. Find possible promoters, repressors = regulators for gene expression
      2. Design decision: in vivo microbial system, or in vitro cell free system?
   5. Repeat from above
3. Project Testability
   1. Research method for testing microbe
   2. Research required controls, blanks, positives, samples
   3. Repeat from above for design of test method, as necessary
4. Project Modeling
   1. Model theoretical operation of microbe
      1. Pathways + membrane + multi-cell
      2. Gene regulation
   2. Model theoretical compatibility of components
   3. Model testing method
   4. Obtain expert assistance/advice on design & models
   5. Repeat from above
5. Project Implementation Scoping
   1. Scope building organism (cost, labor, designer)
   2. Funding source
   3. Materials source
   4. Proceed to wet lab phase

Wet Lab Implementation Method

1. Verify Prior Research
   1. Obtain close analogues to original microbes & plasmids
      1. Culture control & target
   2. Modify strain
   3. Verify strain
      1. Compare growth & products
      2. Measure against control
      3. Compare to model
      4. Improve model
   4. Repeat as necessary for validation of original & current protocols
2. Optimize for Desired Function
   1. TBD
   2. Isolate to enzymes
   3. Switch strains or microbes

Designing the plasmid

Strategies

• indirect method
  • detect the byproduct(s) after metabolizing melamine
• direct method
  • detect the melamine by specific antibody (inspired by ELISA method)

Designs

• Idea #1: Re-construction of Melamine Degradation Pathway + pH sensor

Idea is OK, however need to address cyanuric acid as well. it is not necessary to distinguish between the two, only to detect both. --JC

• Idea #2: Re-writing Signal Transduction (Melamine Sensor)

• Idea #3: Re-design Melamine Sensory Protein

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 (yellow+blue), there's both!

Cons:

• Two strains of bacteria = more complicated process (blent says: we can have one plasmid with both melamine and cyanuric acid detection and put it in one single host/chassis)
• 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"

The sensitivity of melamine detection methods

Method	Sensitivity (in ppm)
LC/MS/MS	0.05 ppm (current Taiwan standard)
GC/MS	0.5 ppm
HPLC	2.0 ppm - 2.5 ppm (previous Taiwan standard)
ELISA	Romer Labs: AgraQuant® Melamine Sensitive ELISA Test Kit Quantitation Range: 0.1 - 5.0 ppm in milk 0.5 - 25 ppm for milk powder Limit of Detection: 8 ppb in milk 150 ppb for milk powder Abraxis, Warminster, Pa. LDD (lease detectable dose) 10 ppb in their manual 9 ppb in PBS (FDA report) 1 ppm in dog food (FDA report) different dilution methods vary the performance of detection it is difficult to detect melamine in mixed sample (real sample)

• Limitation of Melamine Detection Methods (in Chinese)

25 Labs in Taiwan provide melamine detection service

Organization and Lab	Equipment and Service
SGS TAIWAN LIMITED: Ultra Trace Industrial Safety Hygiene Lab	LC/MS/MS
National Animal Industrial Foundation	LC/MS/MS
TÜV Rheinland Aimex Ltd (Taiwan)	LC/MS/MS
Intertek Taiwan	LC/MS/MS, HPLC
Wei-Chuan Corp.	GC/MS, HPLC
FSI (Food Safety Institute) International - Asia Pacific Office	LC/MS/MS, GC/MS, HPLC
FIRDI (Food Industry Research and Development Institute)	LC/MS/MS, HPLC
GSL (General Standard Laboratory)	LC/MS/MS
Sun Dream Environmental Technology Corp.	LC/MS/MS
Cheng-Shiu University: Super Micro Mass Research and Technology Center	LC/MS/MS
Sinphar Group	LC/MS/MS, LC/MS
Meiho Institute of Technology: Biotechnology Service Center	LC/MS/MS
Hwayo Tech & Lab Co., LTD.	GC/MS, HPLC
SL (Super Laboratory) in Taiwan	HPLC
Lin Family Biotech Co.	HPLC
National PingTung University of Science and Technology: CAAPIC (Center for Agriculture and Aquaculture Product Inspection and Certification)	LC/MS/MS
VCRO (Virginia Contract Research Organization) Co., Ltd.	LC/MS/MS
Taiwan Eel Farming Industry Development Foundation	LC/MS/MS
BSMI (Bureau of Standards, Metrology and Inspection), R.O.C.	LC/MS/MS
Department of Health, Taipei	LC/MS/MS
Department of Health, Kaohsiung	LC/MS/MS
BFDA (Bureau of Food and Drug Analysis): North, Central, South and Eastern Departments]	LC/MS/MS

• list from the Department of Health in Taiwan
• Food Safety Web in Taiwan

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.
    • as an uneducated opinion, I'm partial to cyanobacteria since they seem friendly (and easy to differentiate our GMO from wild type by adding salt). Dont know enough about compatibility or growing though. -JC
• How will CO2 production affect microbe? -JC
• How will diffusion into microbe affect metabolism? -JC
• How will products build up in microbe & diffuse out? -JC

Environmental

• Metabolizing melamine/etc throws off NH4+ ammonium cation with each degradation step.
  • Are you sure about that? MetaCyc says it's NH3.
    • I'm confused on this point. Some pathway diagrams show NH3. MetaCyc shows ammonium. Result graph from K. terragena shows concentration of NH4. Likely both will exist and swap back & forth to reach equilibrium depending on pH of the environment. -JC
  • 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.
      • more free H+ (or + in general) means more acidic means much lower pH. NH3 ammonia is high pH since it is more "-" thus basic. -JC
  • 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!
        • Good point about the vapor. Maybe could add heat. or if chassis already requires 30C temperature, maybe vapor is already produced. -JC
    • use pH strip or drop kit, scale of red indicates melamine metabolized. What is blank pH of typical sample; infant baby formula likely very neutral; milk is very neutral. -JC
    • Depending on concentration of NH3/NH4, could measure conductivity as a measure of melamine reduction products. This would be very easy to threshold/calibrate. Maybe much easier than using gene expression for visual indicator. This could really be a key point. -JC