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 and much needed chemical detector as one possible method of testing the idea of "DIY Bio". This theoretical "Melaminometer" detector registers presence of both melamine and/or cyanuric acid; original proposal here:

Large portions of the technical content of this article are based on wikipedia.org entries.

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 cause renal failure, illness and/or death. Industrial quality assurance measures and/or oversight have 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.

Technical Details

Melamine is an organic base and a trimer of cyanamide, with a 1,3,5-triazine skeleton. (See Triazine) Melamine combines with cyanuric acid to form melamine cyanurate. Melting point 350 °C.

http://upload.wikimedia.org/wikipedia/commons/thumb/0/05/Melamine.svg/150px-Melamine.svg.png Melamine C3H6N6

A 1,3,5-triazine, also called s-triazine, is an organic chemical compound whose chemical structure has a six-membered heterocyclic aromatic ring consisting of three carbon atoms and three nitrogen atoms. triazine is one of three organic chemicals, isomeric with each other, whose empirical formula is C3H3N3. The atoms in triazine rings are analogous to those in benzene rings, which makes triazines aromatic compounds like benzene. The most common derivative of 1,3,5-triazine is 2,4,6-triamino-1,3,5-triazine, commonly known as melamine or cyanuramide. 2,4,6-Trichloro-1,3,5-triazine is easily hydrolyzed to cyanuric acid by heating with water at elevated temperatures. Trichloro-1,3,5-triazine is the starting point for the manufacture of many herbicides such as simazin. Another important derivative is 2,4,6-trihydroxy-1,4,5-triazine better known as cyanuric acid.

http://upload.wikimedia.org/wikipedia/commons/thumb/c/c1/135TriazineStructure.png/100px-135TriazineStructure.png Traizine

http://upload.wikimedia.org/wikipedia/commons/e/e2/Triazine_isomers.PNG Triazine Rings

Cyanuric acid or 1,3,5-triazine-2,4,6-triol is a chemical compound.

http://upload.wikimedia.org/wikipedia/commons/a/a1/S-triazine-2%2C4%2C6-triol_and_s-triazine-2%2C4%2C6-trione.PNG Cyanuric acid C3H3N3O3

Melamine cyanurate, also known as melamine-cyanuric acid adduct or melamine-cyanuric acid complex, is a crystalline complex formed from a 1:1 mixure of melamine and cyanuric acid. The substance is not a salt despite its non-systematic name melamine cyanurate. The complex is held together by an extensive two-dimensional network of hydrogen bonds.

http://upload.wikimedia.org/wikipedia/commons/thumb/4/4d/Melamine-cyanuric_acid_chemical_structure_color.png/200px-Melamine-cyanuric_acid_chemical_structure_color.png Melamine-cyanuric acid complex C6H9N9O3 (C3H6N6·C3H3N3O3)

<iframe src=http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-5170 width=640 height=480></iframe>

MetaCyc Pathway: melamine degradation

Standard Methods of Detection

1. High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid. Food Addit Contam. 2007 Dec;24(12):1319-25.

Melamine has been used for the adulteration of cereal flours in order to increase their apparent protein content. Crude melamine may contain several by-products, i.e. ammeline, ammelide, and cyanuric acid. The simultaneous analysis of all four chemicals is difficult because of the formation of an insoluble salt between melamine and cyanuric acid. A simple and convenient high-performance liquid chromatography (HPLC) method for the detection of the adulteration of cereal flours with all four chemicals is proposed herein. The precipitate formation between melamine and cyanuric acid was prevented by using alkaline conditions (pH 11-12) for both standards preparation and sample extraction. The method uses matrix-matching, which involves the construction of a calibration curve on a blank (negative control) matrix, which is then used for the quantitation of melamine and by-products in adulterated (positive) samples. Matrix-matching compensates for analyte losses during sample preparation, and for matrix effects. The method was successfully applied to wheat, corn, and rice flours, and is expected to be applicable (with some modifications) to soy flour as well. The method allows for the detection of melamine, ammeline, and ammelide at approximately 5 µg g-1, and cyanuric acid at approximately 90 µg g-1 in wheat flour.

1. [Full Text] GC-MS Screen for the Presence of Melamine, Ammeline, Ammelide and Cyanuric Acid (Version 2.1). U.S. FDA May 22, 2007 10:36 AM ET

This procedure was developed by PRLNW and FCC to screen various matrices for the presence of melamine and some related compounds at the established minimum reporting level (MRL) of 10 µg/g and above using gas chromatography/mass spectrometry. Samples are extracted using a mixture of acetonitrile/water/diethylamine and the analytes are subsequently converted to trimethylsilyl derivatives for analysis.

1. [Full Text] Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC-MS/MS. U.S. FDA Laboratory Information Bulletin LIB No. 4421 Volume 24, October 2008

This method consists of an initial extraction with 2.5% aqueous formic acid, followed by a series of filtration, centrifugation, and dilution steps. Both compounds are analyzed in the same chromatographic program using a zwitterionic HILIC LC column. Electrospray ionization is used in both the negative ion (CYA) and positive ion (MEL) modes. Two selected reaction monitoring (SRM) transitions are monitored for both compounds. The amount of compounds present is determined with a calibration curve consisting of sample extracts from infant formula fortified from 0.25 to 5 µg/g that have been taken through the extraction procedure. The range of recovery from fortified infant samples (n =38) was 70-114 % (RSDs 4.5- 22.7 %), and 72-110% (RSDs 5.7-24.9%) for cyanuric acid and melamine, respectively. The limits of quantification and confirmation are 0.25 µg/g for both analytes in dry infant formula.

Primary Metabolic Pathways

http://jb.asm.org/content/vol183/issue8/images/medium/jb0811395001.gif

Comparison of the reactions catalyzed by melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 (A) and AtzA from Pseudomonas sp. strain ADP (B).

http://jb.asm.org/content/vol183/issue8/images/medium/jb0811395004.gif

Time course of melamine and ammeline deamination by cell extracts prepared from the TriA clone E. coli(pJS3). Error bars represent the standard error of the mean; n = 2.

http://88proof.com/synthetic_biology/img/Figure1_Metabolism_of_Melamine_by_Klebsiella_terragena.png

Pathway of melamine metabolism by Pseudomonas strain A and presumptive pathway of melamine metabolism by K. terragena.

http://88proof.com/synthetic_biology/img/Figure2_Metabolism_of_Melamine_by_Klebsiella_terragena.png

Growth of K. terragena with melamine as the sole N source.

http://88proof.com/synthetic_biology/img/Figure_MetaCyc_Pathway_melamine_degradation

[MetaCyc Pathway: melamine degradation]

1. [Full Text] The degradative pathway of the s-triazine melamine. The steps to ring cleavage. Biochem J. 1982 Dec 15;208(3):679-84.
2. [Full Text] Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227. J Bacteriol. 1991 Feb;173(3):1215-22.
3. [Full Text] Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains. J Bacteriol. 1991 Feb;173(3):1363-6.

The enzymes are encoded by the genes trzB, trzC, trzD for metabolizing ammeline to biuret. Enzymes: B, ammeline aminohydrolase; C, ammelide aminohydrolase; D,and cyanuric acid amidohydrolase.

1. [Full Text] Metabolism of Melamine by Klebsiella terragena. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, p. 2832–2835 Vol. 63, No. 7
2. [Full Text] Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227. Appl Environ Microbiol. 1999 Aug;65(8):3512-7.
3. [Full Text] Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different. J Bacteriol. 2001 Apr;183(8):2405-10. PMID: 11274097

The gene encoding melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. [...] Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp. strain ADP. [...] Melamine (2,4,6-triamino-1,3,5-triazine), a related s-triazine that predates the use of atrazine (29), is also metabolized by soil bacteria. [...] The triA gene was cloned into the EcoRI and HindIII sites of pUC18, and the resulting plasmid, pJS3, was transformed into Maximum Efficient E. coli DH5alpha (Gibco BRL, Gaithersburg, Md.). The melamine degradation phenotype of transformed cells was confirmed by incubating cell extracts with melamine, followed by analysis using high-pressure liquid chromatography (HPLC) [...] The triA sequence has been entered into GenBank under accession number AF312304. [...] Sequence analysis indicated a single open reading frame 1,425 nucleotides in length.

1. Ultrasonic extraction and determination of cyanuric acid in pet food. doi:10.1016/j.foodcont.2008.04.004

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] 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.

1. [Full Text] The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway. Appl Environ Microbiol. 1997 Mar;63(3):916-23.
2. [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. Microbial aspects of atrazine degradation in natural environments. Biodegradation Volume 13, Number 1 / January, 2002 doi:10.1023/A:1016329628618
2. Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies. Appl Microbiol Biotechnol. 2002 Jan;58(1):39-45.

pADP-1 from Pseudomonas sp. ADP, has been completely sequenced and contains the genes for atrazine catabolism. Insertion sequence elements play a role in constructing different atrazine catabolic plasmids in different bacteria. Atrazine chlorohydrolase has been purified to homogeneity from two sources. Recombinant Escherichia coli strains expressing atrazine chlorohydrolase have been constructed and chemically cross-linked to generate catalytic particles used for atrazine remediation in soil. The method was used for cleaning up a spill of 1,000 pounds of atrazine to attain a level of herbicide acceptable to regulatory agencies.

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. 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
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

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.

Popular Media Articles

1. Killer pet food ingredients identified. The New Scientist Volume 196, Issue 2631, 24 November 2007, Page 4

Historical Perspective

late 1970s: Partial, tentative, data into melamine degradation is published.

mid 1980s: HPLC analysis allows quantitative measurement of melamine pathway; prior to this, identification of intermediates is difficult.

early 1990s: Melamine catabolism studied as means to degrade atrazine herbicide in the U.S. Pseudomonas used and first plasmids created.

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: Metabolism of melamine and/or related compounds in bacteria metabolism studied in further detail using genetic analysis.

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

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

Dry Lab Design Method

1. Quickly (re-)define project
2. Verify theoretical feasibility
3. Repeat from above

1. Research existing metabolism/catabolism/enzymatic activity
2. Research existing microbe structure
3. Verify theoretical feasibility of inserting metabolic path into desired microbe
4. Verify theoretical compatibility of biological components
5. Repeat from above

1. Research method for testing microbe
2. Repeat from above for design of test method, as necessary

1. Model theoretical operation of microbe
2. Model theoretical compatibility of components
3. Model testing method
4. Obtain expert assistance/advice on design & models
5. Repeat from above

1. Scope building organism (cost, labor, designer)
2. Funding source
3. Proceed to wet lab phase