Gluten Screen Test.

From 2012.igem.org

Revision as of 22:56, 27 June 2012

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.

After conducting some initial research on invertase, it appears that most experimentation has been done using two strains of Saccharomyces called 303-67 and FH4C (mutant strain). The strains act differently according to the amount of glucose present in the media, as well as other fundamental differences.

I received a quote from GenScript for a purified gluten peptide sequence, but one of our advisors is concerned that in a gel run the fragments may be too small to distinguish from individual amino acids. The only solution appears to be to order the PQPQLP sequence with a quencher and fluorophore attached. As of 6/15/12 I am still waiting for a response from both Biomatik and Genscript on the differences between the recommended quencher and fluorophore combinations (other than price). Both companies claimed I would be hearing back today at some time. After doing more research on the DABCYL/Glu(EDANS)-NH2 quencher and fluorophore, I found many papers documenting the combination for various protease assays. From my understanding it would be a viable combination for use in our project. Here is a link about the combination's use in an experiment for various assays http://www.jimmunol.org/content/183/10/6708.full.pdf

Here is a link to various protocols needed to run the enzyme activity assay from the University of Washington. https://2011.igem.org/Team:Washington/Protocols

Over the weekend I researched the possibility of using mass spec as an assay to measure enzymatic activity. After speaking with an expert at CSU, Don Dick, I was referred to Jessica Prenni who has experience in mass spec with beer. I emailed Dr. Prenni and am currently awaiting her response.

I also received a quote back from Genscript and Biomatik, but it appears we are foregoing the route of the straight PQPQLP sequence for the quencher and fluorophore option. I suspect we may order a sample of gluten online if we decide to use mass spec as an option for monitoring enzymatic activity.

Here is a very informative link on the quantification of wheat gluten using mass spec. The publication also mentions that one of the best ways to characterize gluten proteins in wheat is using 2D gel electrophoresis. http://www.springerlink.com/content/11u4752uu71510v1/fulltext.pdf

I have emailed two different companies about the possibility of a gluten ELISA assay sponsorship. On the quote I received back from one company, the difference in quencher and fluorophore combinations is the wavelength they emit. This however does not explain why some combinations would cost more than 3x as much others. I emailed GenScript asking what the differences were as well as asking them for the discounted rates.

I called both Genscript and Biomatik today. With Biomatik I was only able to leave a message, so I will be waiting for them to call me back. I spoke with customer service at GenScript and was told I would receive a quote with the 15% discount applied, and that they would forward me the response on the differences between the quencher and fluorophores as described by their specialists. If the quote and description are adequate, we should be able to order the sequence today (6/19/12).

We met with Chris Strickland yesterday (6/19/12) and he tasked us with finding an appropriate set of equations to model collision rates within the reactions. Specifically, I am researching the gas collision theory to see if it can be applied to fluids. From what I understand it should be a valid set of equations to use on a liquid reactions, assuming some modifications are made. The following are links on collision theory: This link is the collision theory based primarily around gas reactions. http://en.wikipedia.org/wiki/Collision_theory This slide show reviews some of the derivation of the collision theory. http://www.powershow.com/view/51cd1-YTM4Z/Collision_Theory_flash_ppt_presentation This link shows some different variations of the collision theory equations. http://www.life.illinois.edu/crofts/bioph354/diffusion1.html

I did some various derivations and modifications of collision theory equations to find a viable modeling system. If we start by assuming there is no activation energy (i.e. a reaction occurs with every collision) then my equations should prove sufficient. This would provide a starting ground to build an initial model of the reaction between the K (Kumamolisin) enzyme and 33-Mer sequence of Gluten. After having an initial model, we could then add more advanced components depending on the information available about our two components. We met with Dr. Prenni today (6/20/12) about the possibility of using mass spec as an enzymatic assay. She said she thought there would be too many proteins to identify the one we wanted, and even more difficult to see if this specific sequence had been broken down. She instead recommended a Western Blot test. In order to perform this test we need an antibody that targets a specific binding site on the gliadin sequence.

Today (6/21/12) we have a meeting with Chris Strickland to further discuss the modeling component of our project. We are first trying to model the collision rate between the enzyme Kumamolisin and the 33-mer. David is currently working on a Thermodynamic based approach to the prolem, while I am researching a collision theory approach. We may more forward with both theories in parallel in order to have both options to compare our experimental results with.

We met with Chris today (6/21/12) about modeling, and he thinks starting from scratch will be too difficult and greatly inaccurate. So we are instead going to use an existing modeling software called Smoldyn, which specializes in micro-scale chemical reactions. Chris has tasked those interested in the modeling component of the project with reading the user manual for Smoldyn and to find the parameters of our reaction necessary to run an initial model of our system. http://www.smoldyn.org/index.html Here is the link for the Smoldyn user manual. http://www.smoldyn.org/Smoldyn_doc1.pdf

(6/26/12) Today I spoke with Dr. Brian Geiss about the protocol for preparing the enzyme and protein with the quencher and fluorophore. He said he wanted to set up a meeting with us sometime this week to discuss the assay as well as how to prepare for it. I sent him an email telling him we are available all week in the afternoon and am currently awaiting his response. We also have a meeting with Chris Strickland today about furthering the modeling component of our project. So far I have read over the more relevant pieces of the user manual for Smoldyn and it appears to be a very in-depth detailed software. I am trying to understand the various components we will need in order to successfully run an initial attempt. The manual is clear on setting up parameters within Smoldyn, but not necessarily the features we will need for our molecules.