Gluten Screen Test.

From 2012.igem.org

I am currently researching how to create a screen test for gluten in a beer sample. More specifically Hordein, the gluten protein found in barley which is a key ingredient in beer.

According to various group research we suspect there is a repeating sequence in both wheat and barley gluten proteins that will allow Hordein, the Gluten protein of barley, to be broken down in a similar manner to gliadins, the Gluten protein of wheat. This could potentially be conducted using an enzyme to break down the gluten protein, in a method similar to that used by the University of Washington in 2011's iGEM competition.

here is a link on beer and hordein quantification http://pubs.acs.org/doi/pdf/10.1021/pr2008434 https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay

I am currently searching for a purified polypeptide sequence of gluten that we can use as a control for experimental basis. The University of Washington used a PQPQLP sequence with a fluorophore and quencher attached using a custom sequencing option from a company called Anaspec (Anaspec provided a 20% discount for educational research). According to Anaspec's website the average delivery time for a custome peptide strand is between 2 and 3 weeks. This could be problematic and another solution may need to be found.

I found today that CSU has a Lab dealing with DNA sequencing, I sent an email asking if they also did custom peptide sequencing. Their product turnaround is faster than many commercial biotechnology companies. Also, I found several other companies that have a quicker turnaround than Anaspec for custom sequencing. It turns out that CSU's department does not do custom peptide sequencing, but a company called biomatik is looking promising.

Here is a patent for a gluten assay that goes incredibly in-depth into the foudation of Gluten, its components and sequencing, its relation to celiac patients, etc. Very informing. http://www.patentstorm.us/patents/7534426/description.html

Over the weekend I read the reading for the journal club, requested quotes from two more companies on the custom peptide sequence and updated the goodle document. The reading was very informative and confirmed my other research that the sequence PQPQLP was primarily responsible for the digestive difficulties of Celiac patients. I received a repsonse from Biomatik stating that my quote could not be completed because of the quencher modification on the C-terminus. I am still awaiting a reply from three companies as of 6/11/12. I updated some gluten information on the google doc as well as the temperatures for which ale and lager are brewed. This allows us to be certain that our concept would work under actual brewing temperatures.

Here is a link with great information on yeast expression and secretion vectors. http://www.patentstorm.us/patents/5024941/fulltext.html

I have done a lot of research on the definition of "gluten-free" according to various organizations and standards and it appears as though <20 ppm seems to be classified as "glute-free." As something like our idea has never been done before, from my understanding we would qualify as "gluten-free" assuming we could meet the <20 ppm requirement.

Here is a patent for a genetically engineered strain of yeast that produces a gluten-free wheat protein that is indistinguishable from the actual protein. http://www.freepatentsonline.com/4826765.html

Today I received my quote back from Anaspec with a time frame of 4-5 weeks for delivery. I returned their email with a response explaining the iGEM competition through CSU and our educational research purpose. I mentioned that UW's iGEM team from 2011 received a 20% discount and I asked if we coul receive a similar deal. I also mentioned our time constraint and asked if there was anyway to receive the sample in a more timely fashion. I also emailed New Belgium to potentially setup a meeting to discuss the brewing process as well as our project idea.

Today I received the quote back from Anaspec and received a 20% discount on the previous quote, but the delivery time frame was still 4-5 weeks. This time frame is not acceptable given the limited window of the project. Therefore I am researching other fluorophore and quencher combinations offered by different biosynthetic companies. Here is a helpful link on how to choose quenchers and fluorophores. http://www.biosyn.com/faq.aspx?qid=303

Two companies, Biomatik and Genscript, got back to me today with other options for quencher and fluorophore combinations. Each company provided two combinations, with one of the combinations being the same from each company. Biomatik recommended 5-TARMA/5-FAM and DABCYL/Glu(EDANS)-NH2, while Genscript recommended Abz,Tyr(3-NO2) and DABCYL/Glu(EDANS)-NH2. The DABCYL/Glu(EDANS)-NH2 combination requires the addition of a Glucose chain attachment, but that does not appear to be a problem. I asked what the key differences in those combinations were from each company and am currently waiting a repsonse. We are also considering using a different assay while waiting for our custom peptide sequence. This assay is a 2D Silverstain gel and would potentially allow us to see is our enzyme is properly cleaving the PQPQLP sequence. I sent two quotes for purified PQPQLP sequences to Biomatik and Genscript to see if we could get a quicker and less expensive solution to our problem. I am currently waiting for a repsonse on those also.

I am going to start researching the qualifications for meeting "gluten-free" standards according to U.S. requirements. I am also going to do research on invertase as a yeast secretion solution.

There are no gluten-free regulatory definitions in place according to the FDA, but actions are under way to better define gluten-free. http://www.fda.gov/Food/LabelingNutrition/FoodAllergensLabeling/GuidanceComplianceRegulatoryInformation/ucm111487.htm#q8

I contacted the advisor and main contact (Dr. Mills) for the 2011 University of Washington iGEM team to see if they had any leftovers from last year's gluten project. We are piggybacking off many of their concepts and experiments for original purpose and it would likely save time and money if they were able to offer assistance in any form.