Melaminometer

Project Proposal

Use of synthetic biology has been proposed by OpenWetWare and related individuals/labs/groups as a means for non-scientists to use biotechnology. I propose creation of a simple chemical detector as one possible method of testing the idea of "DIY Bio". This theoretical "Melaminometer" detector registers presence of melamine; original proposal here:

Original Proposal

I would like to propose a real example regarding diy bio.

Currently in asia, as reported on HK news, there are 10,000 babies in the hospital after being poisoned with melamine from purposely-tainted milk products. Note that melamine has now been found in M&M's, in oreo cookies, in cereal.. I dunno about you guys, I like M&Ms.

http://en.wikipedia.org/wiki/Melamine#2008_Chinese_milk_scandal

http://en.wikipedia.org/wiki/2008_Chinese_milk_scandal

It occurred to me this week as I was attempting to buy some cereal over here in asia.. and looking at the palettes of quaker oats "on discount", I'm not too encouraged that all the contaminated food is being, or will be, actually disposed of.

I would like a melamine detector so I could test the food in my fridge and the food from the store. Some of the contaminated food will get "curiously" re-directed to resellers as discount stock and may be re-sold for years through local channels - the locals need this detector too.

So sure would be nice to have a cheap melamine detector. I'm not talking about something fancy, like taking a sample of food to send to a lab which reports the result in 1 week or 1 day. I'm talking about something I can stir up in the kitchen for years to come that turns red within an hour for "don't eat this."

This should be a simple DIYBIO project, right?

I am encouraged by open source biology because open source has been shown to have the quickest response time for problems found in the field. Simple example: When someone found a major flaw in Intel Pentium chips, the Illegal Instruction errata, and Intel admitted it was a valid problem, it took the Linux community something less than a week (if memory serves -- something like 3 days?) to come up with a runtime patch which scanned all applications at runtime for the security risk. This was a very high tech solution to a very threatening computer security problem which "endangered" everyone who had an Intel computer. Whereas, Microsoft took months to release a patch, and SUN microsystems I believe took even longer to patch their version of unix. Open source took days, and commercial entities took months (not even willing to admit there was a problem).

Keeping all this in mind, how would I build melamine detecting "yogurt" in my garage right now? I mean a solution which is cheap (less than $0.50 per use), stand- alone, and usable by an 8 year old, so that the non-bio savvy masses can test their own oreo cookies before dunking them into soy milk, Shouldn't open source bio heads be able to get a working device validated in less than 3 months? (BTW, if precursors etc to melamine needs detecting, then the device should do that as well.)

A simple problem. At least from the applications angle.

Background

Synthetic biology has provided an alternative method to chemical analysis and synthesis by providing biological methods of processing substances. Synthetic biology uses and modifies common biological organisms such as bacteria or yeast to perform new biological functions. The modification to the organism is done at a genetic level to allow for modified gene expressions which create new protein structures and enzymatic interactions. In a controlled manner, the organism can be used to process and/or create new chemical structures via the enhanced biological function.

The compounds melamine and cyanuric acid have recently been found to be present and harmful in commercial food products. Melamine apparently combines with cyanuric acid to form a crystaline structure Melamine cyanurate. The crystals collect to form kidney stones, causing illness and/or death. Industrial quality assurance measures and/or oversight hae not removed the risk of contamination and may not do so in the near future.

A method for detecting these harmful chemicals would allow consumers to avoid ingesting harmful foods. The method for detection should operate at the point of care:

• Consumer usable
  • Simple to use
  • Relatively reliable (does not need to be as rigorous or precise as industrial detection methods)
• Consumer affordable
• Consumer available "off the shelf"

The above is theoretical.

Scientific American Magazine (2007)

Protein Pretense; August 2007; Scientific American Magazine; by Alison Snyder; 2 Page(s)

Traditionally, food protein is measured by a method developed by Danish brewer Johann Kjeldahl in the late 1800s. In this analytical technique, a strong acid digests a sample, breaking down the organic matter and releasing nitrogen, which is then converted to ammonia. The amount of ammonia indicates how much nitrogen was in the original sample and, hence, the amount of protein. This "proved to be a robust, precise method," says Julian McClements, a food scientist at the University of Massachusetts Amherst. It is attractive because it can be used for a variety of products and protein types. Another, similar nitrogen-based technique, called the Dumas test, is also popular with industry. It relies on burning the sample to release nitrogen. The Association of Analytical Communities (AOAC) International, a scientific association that sets standards for analytical methods, lists the Kjeldahl and Dumas techniques as the standard methods for measuring protein in food.

After hundreds of dogs and cats fell ill this past spring [Spring 2007], government officials traced the source to melamine, a nitrogen-rich compound found in plastics and fertilizer that, when ingested by the animals, crystallized in their kidneys and caused renal failure. The U.S. Food and Drug Administration later announced that producers may have deliberately added the compound to wheat gluten and rice protein concentrates to inflate the measured amount of protein. The greater the protein level in the concentrates, the higher the market price the products fetch. Regardless of whether its addition was deliberate or accidental, melamine snuck past standard industry protein analysis, suggesting that the century-old test methods should be reevaluated. Several alternatives exist, but the food industry has yet to make a switch.

Washington Post (2007)

Science and Medicine: Melamine David Brown and Robert Poppenga Washington Post Staff Writer and Veterinary Toxicologist Tuesday, May 8, 2007; 11:00 AM

Dr. Poppenga is a board-certified veterinary toxicologist and is currently a professor of clinical veterinary and diagnostic toxicology at the University of California at Davis. He is also the section head of toxicology at the California Animal Health and Food Safety Laboratory and has been actively involved in investigating pet and livestock exposure to melamine and other contaminants found in wheat gluten and rice protein concentrate imported from China.

[...]

Robert Poppenga: It is unlikely that melamine itself is causing the pet illnesses. The current thinking is that melamine in combination with cyanuric acid (another contaminant in the wheat gluten and rice protein concentrate used in the pet foods) may be responsible, although this has not been proven. There is no reason to add melamine to pet food - melamine is believed to have been added to the wheat gluten and rice protein concentrate to artificially increase their content of nitrogen (and as a result their apparent protein concentration). The more protein in the material, the higher the selling price.

Product Need

• Commercial food testing is unlikely to change in the short term
• Commercial testing has failed to meet safety standards demanded by consumers
• A method for establishing harmful content in food is needed at "Point-of-care"
  • Harmful product may especially exist in resource-constrained communities where commercial testing is lax
  • Consumers should have a means of testing food themselves.

Primary Metabolic Pathways (Synthetic Biology approach)

TBD

Slightly Similar Metabolic Pathways

Melamine Formaldehyde

1. Biodegradation of melamine formaldehyde by Micrococcus sp. strain MF-1 isolated from aminoplastic wastewater effluent. International Biodeterioration & Biodegradation Volume 57, Issue 2, March 2006, Pages 75-81 doi:10.1016/j.ibiod.2005.11.006

Atrazine

• Herbicide
  • Partially studied pathway
  • Enzymes partially known

1. [Full Text] Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria. Appl Environ Microbiol. 2000 October; 66(10): 4247–4252. PMCID: PMC92292

For instance, in 1937, melamine (2,4,6-triamino-1,3,5-s-triazine) was reported to be nonbiodegradable. In 1964, however, bacteria capable of slow degradation were isolated. More recently, melamine was reported to be readily biodegraded[8]. [8] = Experientia. 1983 Nov 15;39(11):1191-8, Isolation and cultivation of microbes with biodegradative potential, Cook AM, Grossenbacher H, Hütter R.

1. [Full Text] Isolation and characterization of an atrazine-degrading bacterium from industrial wastewater in China. Letters in Applied Microbiology Volume 36 Issue 5, Pages 272 - 276. Published Online: 8 Apr 2003 PMID:12680937 doi:10.1046/j.1472-765X.2003.01307.x
2. Microbial aspects of atrazine degradation in natural environments. Biodegradation Volume 13, Number 1 / January, 2002 doi:10.1023/A:1016329628618
3. Combined metabolic activity within an atrazine-mineralizing community enriched from agrochemical factory soil. International Biodeterioration & Biodegradation Volume 60, Issue 4, 2007, Pages 299-307 doi:10.1016/j.ibiod.2007.05.004
4. Atrazine degradation in anaerobic environment by a mixed microbial consortium. Water Research Volume 38, Issue 9, May 2004, Pages 2277-2284 doi:10.1016/j.watres.2003.10.059
5. [Full Text] Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. J Bacteriol. 1996 Aug;178(16):4894-900. Erratum in: J Bacteriol 1999 Jan;181(2):695.

The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R. TrzA catalyzes the deamination of melamine and the dechlorination of deethylatrazine and desisopropylatrazine but is not active with atrazine. AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine.

Atrazine biodegradation can occur by two known pathways:

1) Atrazine can be dechlorinated followed by removal the other ring substituents via amidohydrolases by the enzymes AtzA, AtzB, and AtzC. The end product is cyanuric acid. The best characterized organism that performs this pathway is Pseudomonas sp. strain ADP.

2) The other pathway involves dealkylation of the amino groups. Subsequent dechlorination yields cyanuric acid. The end result is 2-chloro-4-hydroxy-6-amino-1,3,5-triazine, which currently has no known path to further degradation. This path occurs in Pseudomonas species and a number of bacteria.

[...] The genes for enzymes AtzA-C have been found to be highly conserved in atrazine-degrading organisms worldwide. The prevalence of these genes could be due to the mass transfer of AtzA-C on a global scale. In Pseudomonas sp. ADP, the Atz genes are located non-contiguously on a plasmid with the genes for mercury catabolism. This plasmid is conjugatable to Gram negative bacteria in the laboratory and could lead to the worldwide distribution, in view of the extensive release of of atrazine and mercury. AtzA-C have also been found in a Gram positive bacterium but are chromosomally located.[7] The insertion elements flanking each gene suggests that they are involved in the assembly of this specialized catabolic pathway.

[...] A stable four-member bacterial community, characterized by colony morphology and 16S rDNA sequencing, was rapidly able to mineralize atrazine to CO2 and NH3. Two primary organisms were identified as Arthrobacter species (ATZ1 and ATZ2) and two secondary organisms (CA1 and CA2) belonged to the genera Ochrobactrum and Pseudomonas, respectively. PCR assessment of atrazine-degrading genetic potential of the community, revealed the presence of trzN, trzD, atzB and atzC genes. Isolates ATZ1 and ATZ2 were capable of dechlorinating atrazine to hydroxyatrazine and contained the trzN gene. ATZ2 further degraded hydroxyatrazine to cyanuric acid and contained atzB and atzC genes whereas ATZ1 contained atzC but not atzB. Isolates CA1 and CA2 grew on cyanuric acid and contained the trzD gene. Complete atrazine degradation was a result of the combined metabolic attack on the atrazine molecule, and complex interactions may exist between the community members sharing carbon and nitrogen from atrazine mineralization.

Related Articles

1. Metabolism of Melamine by Klebsiella terragena. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, p. 2832–2835 Vol. 63, No. 7
2. Biodegradation of melamine formaldehyde by Micrococcus sp. strain MF-1 isolated from aminoplastic wastewater effluent. International Biodeterioration & Biodegradation Volume 57, Issue 2, March 2006, Pages 75-81
3. Killer pet food ingredients identified. The New Scientist Volume 196, Issue 2631, 24 November 2007, Page 4
4. Ultrasonic extraction and determination of cyanuric acid in pet food. doi:10.1016/j.foodcont.2008.04.004

Historical Perspective

mid 1990s: biotech interest in melamine metabolism due to possibility of enhancing plant nitrogen takeup in optimizing fertilization. Not much known about metabolic pathway. Nothing known about related gene expression. Bacterial strains known; Pseudomonas isolates, two Klebsiella isolates, and a Rhodococcus isolate).

mid 2000s: Conversion of melamine and/or related compounds in bacteria metabolism studied in further detail.

2007: Pet food contamination triggers more intensive research into chemically or physically determining presence of melamine.

2008: Large-scale contamination found in milk products exported from China. Hospitalized population reported between 10,000 and 50,000 people in popular media.