Team:Danzi Kesh 8/Description
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Description
Theoretical background
We decided to focus on the allergen gluten, which causes the celiac disease. Celiac disease is a genetic autoimmune disease that damages the villi of the small intestine and interferes with absorption of nutrients from food. Celiac disease can affect men and women in all ages and races. It can lead to a number of other disorders including infertility, reduced bone density, neurological disorders, some cancers, and other autoimmune diseases.
The disease is due to sensitivity to the gliadin protein, components, with another protein named glutamine, which also contains components that are harmful for celiac sufferers. The gliadin protein is a proteoglycan; this is actually a protein that has sugar. The glutamine protein has a molecular structure of a three-dimensional network.
The gluten mainly found in cereals such as wheat, barley, and rye. In wheat for example, the gluten constitutes to 80 percent of the protein in the nucleus.
The celiac disease is an autoimmune disease. The immune system cells accidentally identify the gliadin protein as a strange object which absorbs in the inner wall cells of the small intestine. The sufferer's immune system attacks the cells in the intestine and causes problems with the absorbing of food. Food hypersensitivity disorder caused by an. inflammatory response to wheat gluten and similar proteins of barley and rye. The resulting intestinal inflammation often causes symptoms related to malabsorption, but in many patients extra-intestinal symptoms dominate, and in others the disease is clinically silent.
There are no pharmaceutical cures for the celiac disease, 100% gluten-free diets are the only existing treatment for celiac today.
Biopsy of small bowel showing coeliac disease manifested by blunting of villi, crypt hyperplasia, and lymphocyte infiltration of crypts.
The world without Alergln
Currently celiac patients do not take the risk. Since there is no cure for the disease, they can just avoid eating foods containing gluten. They are forbidden to eat the most common and favorite foods: pasta, pizza, bread, cakes and biscuits, sweets and more....
Many times you can find substitutes for the forbidden foods, but in some cases the gluten-free food will have an aftertaste and often very expensive.
Most of the Israeli food products labeled as "may contain gluten ", not necessarily the food products contain gluten.
What does Alergln Provide?
The Alergln kit allows for quick testing of food products. This quick test is very reliable and allows a professional analysis on the amount of gluten that a particular food product has. We assume that some foods labeled that "may contain remnants of gluten", remnants of gluten will not be found, which would enable many buyers to eat food which should be forbidden, as well enjoy a lower product prices.
The aim of our project is to create a personal kit, one that identifies gluten in food, reliably and in reasonable time. This kit will allow to ease the celiac patient's life and reduce their expenses to a great extent.
The "Alergln" Kit is based on a fascinating innovative technology in the diagnostic world, just as the kit to identify pregnancy brought with it a series of scientific technology, equally, this kit brings innovation and a breakthrough in a way of thinking and designing the kit. In the future, we believe that the idea can be implemented and utilized as well in areas to identify other substances, not necessarily food materials.
The "Alergln Project" was created after the realization that the number of people diagnosed in having gluten sensitivity (celiac disease) increases from year to year. The number of diagnosed celiac patients increases annually worldwide mostly due to awareness of this phenomenon. This awareness eases and enables many people real quality of life without the horrendous stomach pains. Celiac disease patients are not treated with drugs because the drug is not available, these patients simply avoid eating gluten. Therefore, they must verify whether the food they intend eating has gluten or not. The scales to check are often complicated and not necessarily accurate.
The "Alergln" kit will serve the above population, prevent them from concern for the presence of gluten in food and can eat with peace of mind and without worrying about their lives.
The success of the project will allow patients to eat foods that "may contain gluten," but there is no gluten, the difference in prices is very high and hence the kit cancels the presence of gluten in regular foods and allows the patient to eat without any concern, and at a lower cost.
The feasibility of the product
In recent years, the awareness of the of celiac disease has increased, and perhaps that is why the number of people diagnosed is increasing constantly. Allegedly we are talking about unexplained "abdominal pains", however, many times it turns out that the pain stems from sensitivity to gluten and can be avoided by refraining from eating foods that contain gluten. After the survey we conducted interviews with family physicians and pediatricians, we decided that this kit is extremely necessary for celiac patients and therefore has economical potential.
Why is it worthwhile to buy the product ?
- Quality of life without pain: We are talking about abdominal pain, in reality, it disrupts the normal way of life, use of the product could prevent unnecessary pain due to a lack of knowledge about the contents of the product.
- A quick, accurate and inexpensive test: restaurant businesses are not obliged to marking gluten content in the food, sometimes the situation may be critical.
- There is no embarrassment: a school student would not have to stand embarrassed, wondering what to eat. This kit can help him choose the right food that has no gluten or avoid eating foods containing gluten.
- Cut expenses: Americans spend 5 billion dollars on gluten-free food every year, about $ 4,000 more than an ordinary person for the unmet medical need for celiac patients and therefore it is necessary to have a unique diet which is very expensive. Sometimes the nutritional information marking "may contain gluten" is not accurate, and in this case you can use our kit. The celiac disease is a hereditary disease, meaning that every family has a reasonable possibility of purchasing "Alergln" through other family members.
References
protocols
Project Achievenments
In the course of the work and preparations towards the big iGEM project, we feel that we have gone through a significant process as a group.
The intensive and long work enriched our knowledge in scientific topics where we worked. Without a doubt we feel
that we received a one-time opportunity to work in real scientific research where we would never have been able to acquire as students in our regular, standard curriculum in biotechnology. We enriched our knowledge in every aspect of biotechnology and around it, and even experimented on designing and implementing a big project, and work as part of a team where we learnt tocooperate and give space to each member of the team. All these things together led to creating new social relationships.
In the course of the work we met businessman, parliament members, doctors in different fields of science, and we created connections and relationships with people who promoted us and can promote us in the future.
In addition to all of that, we feel that the work we did in our free time strengthened the work ethic of every one of us, the capability to plan time in the right way and to stick to the target.
However, we encountered a number of difficulties where at the end of the day we overcame them.
Each member of the team contributed to the team with his or her skills towards the project, meaning that we gave up our summer vacation, gave up family trips and most of the time we also gave up hanging out with friends.
This and more, at the beginning of the school year we did not stop working on the project and this meant missing lessons, and of course catching up on all the material in our free time
Another challenge we had, not all the students were fully committed to the hard work the project needed.
At times there were a few students who were ready to perform any task, and even when there was a problem performing
the task, there were not always students who came forward to give a hand in solving the problem.
Towards the end of this intensifying process, we can say that we went through a unique and one-time experience, where during the time we came across quite a few challenges, however, we learnt to jump over the hurdles and overcome every challenge that came our way, there is no doubt we received many values due to our being a team and we will reach our goal … Boston we are coming!
system design
Project Goal: Design a kit to identify gluten in food
The kit will contain a biosensor that identifies gluten and will confirm the presence by creating a change in color. Designing the kit is on the use the Gln-H protein. This periplasmatic protein is made up from two lobes, one big and one small. The protein identifies the gluten through a connection to amino acid glutamine found in a relatively high concentrate. The connection to glutamine is done in the molecular center of the protein.
In order to implement the process we were required to divide it into a number of stages as described below:
Designing 6 DNA segments encoded to Gln-H protein
Three bacterial sources served us as a source to express the Gln-H protein: TM1, GBS, E.coli.
To each one of the three a new gene encoded to the protein was reassembled. To each one of the genes we added a reporter. Because we used two types of reporters (GFP, HAD) on each the DNA sources two copies of the gene to the Gln-H protein, so in the end we created 6 biosensors. For example, from the DNA segment encoded to the Gln-H protein and its source in the GBS bacteria will be comprised once with the HAD reporter and once again through GFP.
Work design for the six mutant genes was done using APE software; the six mutants were created to increase the chances of success of the planned system.
Below are details of the structure of the 6 mutants:
In order to engineer the protein in which the ends will close when we link to gluten, we changed the protein's terminal ends position. This is by binding linkers to the edges of the N Terminal, the original C terminals of the small sub-unit and the big one respectively and opening the ends (C, N terminals) in other places. Figure 2 describes the process of transferring C ends, N terminals; figure 2 shows the end position and the position of the linker in Gln at the end of the process. It is important to note that the linker includes 3 amino acids – serine, glycine that influence the amount of flexibility of the molecules at the time linking to glutamine. (figure 1, figure 2).
In the figure the linkers added to the ends of the C terminal and N terminal are highlighted in red
This mutation cycle allows one of the new Terminal ends of the protein to unite (when on each end is half of another reporter gene), in actual fact the two parts of the gene come together and form comfortable conditions for creating color. The change in color will occur only when the connection is through gluten.
In fact we have created three mutant cycles in three genes encoded to the Gln-H protein. Each of the genes (when translated to protein) will act as follows: Without gluten - the protein will be in the open position and therefore the half enzyme will remain inactive and there will not be a color reaction.
The presence of gluten - the protein closes, following; the half enzyme will close and become an active enzyme with the result of a color reaction.
The reporter genes – HAD, GFP
- Enzyme – HAD
HAD enzyme removes a phosphate group from the molecule substrate paranitrophenyl - a common phosphate powder, this substrate binds to the enzyme that removes from itself a phosphate group, removal of the phosphate causes a change in color to bright yellow. Of course the HAD molecules are active only in the presence of glutamine (it is closed due to the closure of the lobes (gln_h) and a yellow color appears indicating the presence of gluten in the platform. - GFP
The protein absorbs the blue light and emits back luminescence, the luminescence is seen under a U.V. light. in both cases the reporter molecules will see a color change when the gluten connects to the protein, a conformational structural change of the protein will occur which will allow the two reporter gene parts to unite and activate a color change.
Product Design
The final stage of the process and it is not part of the laboratory process is – product design.
Assuming that the laboratory idea described will be a success we will move onto designing the product, Implementation of the business portfolio on the Website and distribution of the kit for use by celiac patients.
Appendix
Sources of the genes
Thermatoga Maritima bacteria
Bacteria which originate from hot springs, hyperthermophilic bacteria, its enzymes are very stable and have a special structure. Using the enzymes from these bacteria we will manipulate the protein.
GBS Bacteria
Geobacillus stearothermophilus (formally Bacillus stearothermophilus) is a rod-shaped, Gram-positive bacterium and a member of the division Firmicutes. The bacteria is a thermophile and is widely distributed in soil, hot springs, ocean sediment, and is a cause of spoilage in food products. It will grow within a temperature range of 30-75 degrees Celsius. Some strains are capable of oxidizing carbon monoxide aerobically. It is commonly used as a challenge organism for sterilization validation studies and periodic check of sterilization cycles.
It was first described in 1920 as Bacillus stearothermophilus, but, together with Bacillus thermoglucosidasius, it was reclassified as a member of the genus Geobacillus in 2001. Recently, a DNA polymerase derived from these bacteria, Bst polymerase, has become important in molecular biology applications.
Bst polymerase has a helicase-like activity, making it able to unwind DNA strands. Its optimum functional temperature is between 60 and 65°C and it is denatured at temperatures above 70°C.
E.coli
E. coli bacteria were discovered in the human colon in 1885 by German bacteriologist Theodor Escherich. E. coli is often referred to as the best or most-studied free-living organism. More than 700 serotypes of E. coli have been identified. The E. coli that are responsible for the numerous reports of contaminated foods and beverages are those that produce Shiga toxin, so called because the toxin is virtually identical to that produced by Shigella dysenteria type 1.
The bacterium can be grown and cultured easily and inexpensively in a laboratory setting, and has been intensively investigated for over 60 years. E. coli is the most widely studied prokaryotic model organism, and an important species in the fields of biotechnology and microbiology, where it has served as the host organism for the majority of work with recombinant DNA E. coli is a Gram-negative (bacteria which do not retain crystal violet dye), facultative anaerobic (that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation or anaerobic respiration if oxygen is absent) and nonsporulating bacteria.
RESULTS
Expression
The aim of our project was to express one of our six Gln-H proteins.
The six mutant genes will be expressed in the bacterial system; the degree of expression will be tested for each of the
genes. The protein that will be expressed with the correct amount should connect to the gluten and create a color reaction that proves the connection. The color reaction will only be created if the structure of the protein was done correct and accurate in designing the protein.
For this purpose We created 6 mutant proteins from 3 different bacterial sources. The six mutants are different from each other in the way they report on the presence of gluten – half of them them includes the GFP protein and half of them the HAD enzyme.
Stage 1: Cloning the six mutant genes of the Gln-H protein
The cloning is done into the pET-R 3 plasmid. These six parts will be found in the Igem library according to the following
details. The name of the gene includes its source (the name of the bacteria we took the DNA sequence of the wanted protein) and the name of the reporter gene.
TM- HAD
BBa_k1790000- http://parts.igem.org/Part:BBa_K1790000:Design
TM-GFP
BBa_k1790001- http://parts.igem.org/Part:BBa_K1790001:Design
GBS- HAD
BBa_k1790002- http://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_K1790002
GBS- GFP
BBa_k1790003- http://parts.igem.org/Part:BBa_K1790003
EC- GFP
BBa_k1790004- http://parts.igem.org/Part:BBa_K1790004
EC- HAD
BBa_k1790005- http://parts.igem.org/Part:BBa_K1790005
The genes were cloned to the Pet-R 3 plasmid and after that a transformation was implemented to the component bacteria – type: DH5 –alpha E.Coli.
For every ligation product (each one of the mutants) 2 types of transformation were performed: heat shock and electroporation. This action was performed in order to increase the chance of transformation feasibility.
At the end of the transformation the cells were seeded on a platform including –LB+amp+1%glu.
Stage 2- Expressing the genes after cloning
After purification of the proteins in the cells (using lysozyme and sonication), we carried out a specific separation of Gln-H using Ni-NTA Colonna Agarose (QIAGEN).
The Purification System is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins which contain an affinity tag of six or more histadine residues. This technology allows one-step purification of almost any His-tagged protein from any expression system under native or denaturing conditions.
We ran the collection of fractions of the entire extraction process of the protein Gln-H, on SDS PAGE. This stage finds the rotein that expresses the best way possible the system we created.
Size of expected protein – 55KDa
Following are the results for each of the mutants.
Picture 1
gene No. 1 EC_HAD is expressed in the system, probably was split into two sub units, we presume the reason is in the separation process, a change in the electrical charge due to a change in PH may cause protein catabolism to sub units. Another possibility is the existence of other proteins that were separated in the elution process to release the Gln-H protein. Nevertheless, one can see that the protein is expressed well in the system.
Picture 2
gene No. 2 EC-GFP we can see the expression of the Gln-H protein in channels 7-14. Expression of the protein in the gene is lower; the intensity of the mark is very low compared to the protein extracted from gene No. 1.
Picture 3
gene No. 15 GBS-GFP is expressed in channels 8-14, expression of the Gln-H protein is lower than the protein expression from gene No.1 however greater than the protein extracted from gene No.2.
Picture 4
gene No.3 EC-HAD is expressed in channels 5-14, expression of the Gln-H protein in the gene in considerably lower than protein expression from gene No. 5, howeve,r higher than expression of the protein extracted from gene No.6
Picture 5
gene No.5 TM-HAD is expressed from channel 5 in all channels, 5-14 expression of the Gln-H protein in the gene is the highest observed.
Picture 6
gene No. 6 TM-GFP is expressed in channels 5-15, expression of Gln-H protein in the gene is lower than the protein in other genes.
Throughout the process we made sure of the existence of control
- Transformation stage, the control plate had bacterial cells without the recombinant plasmid and their seeding on a selective platform containing Amp, and of course the control plate was clean from bacteria.
- Protein cleaning stage, the control plate was a selective platform containing Amp but without IPTG molecule and therefore, we did not see protein, lactose-free that activates the T7- insert encoded to the Gln_H protein will not be expressed.
Discussion
- We have designed and constructed 6 synthetic gluten biosensors based on GlnH from different sources and combined with 2 reporter genes (GFP or HAD).
- 3 of the constructs were expressed and purified to near homogeneity from e. coli cells.
- The activity and the sensitivity of the purified biosensors should be evaluated in various conditions to validate the design.
- Different new combinations of GlnH and reporter genes should be constructed to improve the function of the biosensors.
In the Future
Our vision is that Alergln will be accessible and found in all pockets of celiac disease patients.
Alergln will be sold at affordable prices for every pocket and distributed at food chains, drugstores, and even public services and schools.
Commercial competition that the - Alergln will cause the gluten-free food manufacturers to lower their prices, alongside social pressure, and the drive to change legislation and provide equal rights for celiac sufferers.
We hope that medical clinics will offer potential patients celiac diagnosis tests.
Schools will serve gluten-free food and school cafeterias will sell gluten-free food.
At the scientific level, we hope hacking will result in the manufacture of diagnostic kits based on the identification of additional materials using transmembrane proteins.
Next steps
- Completion of finishing operations and product design on a scientific level.
- Writing of rights for Alergln out of concern for spiritual buyer rights.
- Aggressive marketing activities among celiac patients associations worldwide.
- Completion of the legislative phase in the Israeli parliamentcolony and continued the process as we did with genes 1,2,4.
Appendix 1
Difficulties on the way
Throughout the process we encountered difficulties with genes 3,5,6. First, the ligation process did not succeed- the genes did not enter into plasmid expression, the transformation process was done in a different way in order to increase the chances of success. In consultation with the laboratory team leader, Dr. Itamar Yadid, it was decided that we will perform a different process with these genes. First we cut them, then we increased them by phusion PCR reaction, then we performed ligation and sedimentation of ethanol and performed a transformation using electroporation method for bacteria bl21 (de3), the next
step we performed PCR.