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Ryan's Research: Gluten Screen Test

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.

Two links on beer and hordein quantification:

1. Link 1
2. Link 2

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.

(6/11/12) 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.

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 "gluten-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.

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 UWs iGEM team from 2011 received a 20% discount and I asked if we could 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.

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 (source).

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.

(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.

Here is a link to various protocols needed to run the enzyme activity assay from the University of Washington.

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.

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.

(6/19/12) 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.

(6/20/12) We met with Chris Strickland yesterday 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.
• This slide show reviews some of the derivation of the collision theory.
• This link shows some different variations of the collision theory equations.

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.

(6/21/12) We have a meeting with Chris Strickland today 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 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. Here is the link for the Smoldyn user manual.

(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.

This link gives measurement of gluten in various types of beer.

Here is a link about EnvZ and ompR use from the Missouri Miners 2011 iGEM project.

(7/2/12) Today I am going to be working on further determining the relative amounts of Kumamolisin and gluten necessary to run an accurate simulation in Smoldyn. I will also be periodically learning some Python coding for Smoldyn programming. I am still researching the possibility of the CXCR3 receptor gluten assay. I will be bouncing ideas off of Steven who is also researching the topic. It appears as though many schools in the past have used EnvZ and ompR in their projects as a receptor/signaling pathway.

Here is the link for the full publication about the levels of gluten in various beers.

In order to calculate the number of molecules of gluten in a given sample of beer, I used the molecular weight of the 33-mer repeating gluten sequence as a base model. In one publication I found that the average concentration of gluten for a lager beer was roughly 4.175 mg/L. I then used the molecular weight of the 33-mer (g/mol) to calculate the mol/L of the gluten. Then, using a 5-gallon (18.927 L) carboy as my volume, I calculated the number of moles of gluten. Finally, I multiplied this result by Avagodro's Number to find the number of molecules of gluten. I repeated the same process for wheat beer (28.6 mg/L) to obtain the number of molecules of gluten in a 5-gallon carboy. I don't know if I mentioned this, but on Friday 6/22/12 we ordered our custom peptide sequence for the enzymatic assay. After much research, debate, and frustration, we ended up going with the 5-FAM/TAMRA combination, as recommended by Dr. Brian Geiss. We ordered from Biomatik and received an educational research discount. The delivery time frame is estimated to be 3-4 weeks. We can expect delivery between 7/13 - 7/20. I will be checking up on our order's progress in the coming weeks.

(7/3/12) Today we had our meeting with Dr. Brian Geiss and spoke about several different topics. The first topic was about our desire to test enzymatic activity of crude Kumamolisin produced from bacteria using constituative promoters (they are always on, i.e. not inducible) in the DH5-alphas. Dr. Geiss said we could run an SDS-Page Gel to verify that our bacteria system was producing our enzyme. The next topic we discussed was using inducible promoters in BL-21 E. coli to produce enzyme K. Dr. Geiss is providing the bacterial cells and we are in the process of designing and ordering the promoters. Once the promoters arrive we will be able to test this as an alternative system to the constituative promoter system (inducibles will also be run on a Page gel). Dr. Geiss recommended we test our quencher and fluorophore system once we received it using Protinase K, or something that will cleave within that sequence. This would allow us to see that our custom peptide system was working correctly, so we could proceed with our assay.

We also discussed our end goal of having our yeast secrete enzyme K. He said we needed to purify enzyme K in order to cleanly test it against our custom peptide sequence. Once purified, we will be able to run in on his platereader to obtain our data. We then moved to discussion of our novel idea to use the GPCR CXCR3 to detect gluten in samples. He said that our main concern would be the successful fusion of CXCR3 and signal transduction of the binding of gliadin. We told him that we were speaking with Dr. Kevin Morey and that we would further pursue the concept with him. The final topic we covered was our modeling component. We mentioned that we were having trouble finding data needed to accurately model enzyme kinetics. He mentioned the idea of using a light polarization assay to calculate values related to enzyme K binding to the custom peptide sequence.

(7/5/12) Today I learned several different procedures for bench work we are doing on a regular basis. These procedures include making/pouring agarose gel, purifying gel bands, and running a nanodrop device. I also emailed Biomatik asking for an update on our custom peptide order. I am working on finding diffusion coefficients for Kumamolisin and Gluten 33-mer for the modeling component of our project. Tomorrow we have the New Belgium microbiology tour where we should learn more about the brewing process as well as discuss our project with them.

(7/9/12) Today I am further researching the idea of creating a Gluten-detecting assay. Our method would utilize a gliadin-binding receptor with a two stage signal transduction system. Several other iGEM teams as well as researchers have published work on the fusion of receptors to create a transmembrane receptor for various purposes. One specific iGEM team used this concept to create a vanillin receptor that produced a fluorescent protein that varied in color based the concentration of ompR produced. Although similar constructs (similar to what we are trying to develop) have been created, none of them used G-protein coupled receptors. This is where the potential problem arises. Fusion of the CXCR3 GPCR may be possible, but may not necessarily transduce the signal properly. Since I cannot find published work on this topic, we may have to take a shot in the dark and just make an attempt. I will discuss several ideas and topics with fellow group members and advisers to see what they think. New Belgium brewery also discussed the possibility of having us design primers for them. They need primers to help detect the presence of certain contaminants in the brewing tanks.

Here is a paper that better characterizes the GPCR CXCR3.

(7/11/12) Today I am trying to learn about some of the basic functionality of python. I have limited previous experience with coding, but python appears to be quite user-friendly. I also have been doing bench work with Guy to further expand my practical lab knowledge. The first thing we did was a gel extraction, which I did once before. This procedure takes about 20 mins and follows a very intuitive protocol. After completing the gel extraction, we went to Lucas' lab to use the fluorometer to measure the concentration of our 189 and Kan DNA. The protocol for this procedure is slightly more involved, but wasn't overly complicated. Our results however proved disappointing, with very low concentrations of 189 and Kan. We are going to have to redo one of the original procedures and maybe use some combination of the previous measures or modify them in some manner.

(7/12/12) Today we met with Chris Strickland to discuss our modeling progress as well as information about using the Cray Supercomputer. Dave showed Chris the rate limiting equation he had derived for the breakdown of gluten by Kumamolisin. The equation appears to be solid and should prove useful in determining the rates at which gluten breaks down. Chris told us our accounts had been added to the Cray and that we could now log-in from anywhere on campus. After Chris left, Dave tasked Ethan and I with writing Python code that would put multiple output files into one unified file with labels that described which file corresponded to which output.

(7/13/12) Today we have a meeting with Dr. Brian Geiss to discuss operation of the platereader for when our custom peptide arrives. We also have questions about the experiment involving the polarization of light to measure various qualities of our enzyme and gluten substrate. After meeting with Dr. Geiss we now know how to use the platereader for several tests. These tests include fluorescent, kinetics, and ELISA. Brian Searcy brought in both a barley and sorghum beers to test on the Gluten ELISA. Since sorghum is gluten-free, it shouldn't show up on the ELISA, whereas barley should. We are trying to work in with another lab member who is using the platereader, so I am not sure when we will actually test our gluten ELISA.

Here is a paper on two commercial types of gluten ELISA assays.

Here is a link for an enzyme assay protocol.

(7/23/12) Today I worked on preparing the Proteinase K for a reaction with our peptide sequence. This took several hours as the pipetting was ample and tedious. The finished 384-well plate was run in the platereader, but the result was very poor. This was to be expected as the methods used to pipette and monitor the setup of the experiment were primitive compared to the methods used in Dr. Geiss' lab.

(7/24/12) Today I will attempt to redo the experiment on the platereader using more accurate and monitored methods. I will actively keep a notebook of the progress during the experiment to minimize human error.