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Research and Design Project: Biopharmaceuticals and Insulin-generating Enteric Bacteria Research: The Use of Synthetic Biology in the Detection of Tuberculosis Biopharmaceuticals are medical drugs such as interferons, vaccines, and hormones produced using biotechnology. The first example of a biopharmaceutical was insulin produced biosynthetically using recombinant human DNA. Humulin, as it is marketed, is the product of genetic engineering. Synthetic biology aims to create entirely new biopharmaceuticals by using engineering processes to synthesize new biological molecules. This presents opportunities to learn more about disease mechanisms and processes while also synthesizing new drugs to overcome drug-resistance, a growing problem in medicine.
Currently one of the main diseases being combated using biopharmaceuticals developed using synthetic biology is extremely drug resistant and multidrug resistant tuberculosis. Tuberculosis is a highly infectious disease caused by Mycobacterium tuberculosis. Tuberculosis most commonly affects the pulmonary system, spread by the transmission of respiratory fluids from one person to another, usually by sneezing or coughing. In healthy individuals, the immune system is usually able to prevent the bacteria from causing an active infection, but they still carry the disease and can spread it to others. This latent form of tuberculosis is present in about one- third of the world’s population. Tuberculosis is still a devastating disease in the developing world and causes the second most deaths due to an infectious agent, after HIV/AIDS. An especially dangerous strain of tuberculosis is multidrug resistant tuberculosis (MDR-TB), which does not respond to isoniazid and rifampicin, the two most effective and standard tuberculosis antibiotic treatments. Extensively drug resistant tuberculosis (XDR-TB) responds to even fewer drugs than MDR-TB. In 2011, there were 630,000 cases of MDR-TB, about 9% of which were Researchers are using synthetic biology to create new drugs to combat MDR-TB and XDR-TB. By synthesizing new drugs, it is hoped to discover an antibiotic that MDR-TB and XDR-TB will respond to and even be more effective than existing treatments. One problem being solved in drug discovery using synthetic biology is screening potential drugs for effectiveness. One of the most powerful tuberculosis drugs is ethionamide, a last line of defense against tuberculosis. Ethionamide is converted by EthA into a form that kills the bacterium. However, some tuberculosis bacteria produce EthR, which inhibits EthA, rendering ethionamide ineffective as an antibiotic. Wilfred Weber and his team developed a synthetic gene circuit to determine drugs that could inhibit the protein EthR (thereby rendering ethionamide ineffective).
In the presence of a suitable inhibitor, a color change would occur. Using this screening process developed by synthetic biology, a nontoxic, cell permeable was identified: 2-phenylethyl- butyrate. When tested, 2-phenylethyl-butyrate effectively shut off resistance to ethionamide in In addition to developing a drug that helps solve the problem of drug-resistant tuberculosis, this process of using synthetic gene circuits to screen chemicals for effectiveness and nontoxicity as drugs could be applied to other diseases. For example, this system could be applied to find drugs to inhibit the resistance of other pathogens such as MRSA to certain antibiotics. This system of screening for drugs could also be utilized to develop more effective Synthetic biology provides many opportunities for advancement in biopharmaceuticals, ranging from the development of new drugs to methods of overcoming drug-resistance. As the field of synthetic biology continues to develop, it will certainly play an important role in the development of more effective medicines and treatments.
"Expanding Nature’s Toolkit: How Synthetic Biology Is Changing the Face of Medicine." Sciencebuz. N.p., 2012. Web. 8 July 2013. <http://sciencebuz.com/articles/expanding-nature’s-toolkit-how-synthetic-biology-is- Tomilson, Catherine. "Ethionamide." TB Online. Global Tuberculosis Community Advisory <http://www.tbonline.info/posts/2011/8/24/ethionamide/>.
Weber, Wilfried, Ronald Schoenmakers, Bettina Keller, Marc Gitzinger, Thomas Grau, Marie Daoud-El Baba, Peter Sander, and Martin Fussenegger. "A Synthetic Mammalian Gene Circuit Reveals Antituberculosis Compounds." Proceedings of the National Academy of Sciences of the United States of America 105.29 (2008): 9994-998. Web. 6 July 2013. <http://www.pnas.org/content/105/29/9994>.
Weber, Wilfried. "Synthetic Biology in Drug Discovery and Combating Drug Resistance." Lecture. Synthetic Biology Workshop - From Science to Governance. Sofitel Hotel, Brussels. 18 Mar. 2010. Public Health. European Commision. Web. 6 July 2013. <http://ec.europa.eu/health/dialogue_collaboration/docs/ev_20100318_co10.pdf>.
World Health Organization. "Tuberculosis (TB)." WHO. United Nations, 2013. Web. 7 July 2013. <http://www.who.int/topics/tuberculosis/en/>.
Design: Insulin-generating Enteric Bacteria All persons affected by type 1 diabetes mellitus must receive injections of insulin or wear an insulin pump in order to survive, as their bodies do not produce insulin due to the destruction of beta cells in the islets of Langerhans. About 40% of those affected by type 2 diabetes (caused by insulin resistance) are treated with insulin injections. This treatment will focus mainly on type 1 diabetes, but could easily be used for the treatment of type 2 diabetes as well. The body regulates blood sugar primarily through a feedback cycle using insulin and glycogen. The release of insulin allows cells to take in sugar, lowering blood sugar. When blood sugar is too low, glycogen is released, prompting the liver to release sugars into the bloodstream. Type 1 diabetic patients do not produce insulin due to the destruction of beta-cells in the pancreas while type 2 diabetic patients are insulin resistant. The possibility of synthesizing bacteria that could exist within the body to produce insulin (or insulin substitutes) could be effectively used in order to treat diabetes, removing needle sticks, blood sugar highs and lows, and provide a more flexible As previously stated, the current options for the treatment of type 1 diabetes mellitus are insulin injections and insulin pumps. Both provide problems and inconveniences for diabetic patients. Insulin injections require daily injections at various sites on the body, which are effective at delivering insulin but cause discomfort and be inconvenient for diabetics. Additionally, blood glucose highs and lows may occur as a result of insulin injections. However, they are more inexpensive and easier to use than insulin pumps. Insulin pumps deliver more precise amounts of insulin and can be adjusted to suit lifestyles and are thus more flexible. However, insulin pumps require extensive training and they must be attached to the body at all times, causing inconvenience and a constant reminder of diabetes. As methods of treating diabetes, both insulin injections and pumps are effective but cause daily inconveniences for This design project focuses mainly on the problem of having enteric bacteria (in this case, E. coli) produce insulin at the proper times, i.e. in response to the consumption of glucose and other carbohydrates by the diabetic patient. In this design, the absence of glucose will inhibit the production of insulin, since there is already a built in detector for the presence or absence of glucose in bacterial cells. In the absence of glucose, bacteria such as E. coli undergo the glyoxylate cycle to synthesize carbohydrates. Succinate, one of the intermediates in the glyoxylate cycle, is the inducer for insulin production in this system. Because the presence of succinate is an internal stimulus in response to a lack of glucose in the environment, it is useful as an inducer for a system. The absence of succinate within the cell (indicating the presence of glucose in the environment, i.e. the gastrointestinal tract) would allow for the expression of the LuxS gene, producing the LuxS enzyme, which produces autoinducer-2 (AI-2). AI-2 is a signaling molecule utilized in a process known as quorum sensing. Normally, quorum sensing allows bacteria to “sense” the presence of other bacteria and work in unison when a certain number of bacteria or present. In this system, a modified version of quorum sensing is used to “signal” other bacteria when a sufficient number of other bacteria are not producing succinate, indicating that there is a large amount of glucose present. When AI-2 reaches a certain concentration, the bacteria will release insulin into the small intestine. This is accomplished using a two-component signaling system. AI-2 binds to the lsr transporter and is taken into the cell where it is phosphorylated, becoming phospho-AI-2. Phospho-AI-2 binds to a repressor known as LsrR. This turns on the lsr promoter, allowing the open reading frame coding for insulin to be expressed. The open reading frame contains both the human insulin gene (INS) and a TAT peptide export signal gene. The TAT peptide export signal allows insulin to easily leave E. coli without having to use complex biochemical processes, making it optimal for this system. For this system, if it were working “perfectly”, in the absence of glucose no insulin would be produced and in the presence of glucose insulin would be produced, as seen in the truth However, in actuality, it is likely that a small amount of insulin would be produced even in the absence of glucose. In reality, however, this is not necessarily a negative. Insulin pumps, the technology on which this design is some what based produce a low level of basal insulin throughout the day to prevent highs and lows in blood glucose levels. Therefore, a small amount of insulin being produced continuously despite the absence of glucose (which is likely to occur IGEBs present many advantages over current technology in insulin therapy. The main disadvantages of insulin injections and insulin pumps are the inconveniences they provide for diabetics. Insulin pumps resolve many of the problems present with insulin injections, while providing disadvantages as well. IGEBs aim to resolve problems presented by insulin pumps. One of the main issues of insulin pumps is the physical pump that is constantly attached to the body, providing discomfort. IGEBs are a completely internally contained system, requiring no external devices. IGEBs also are completely self-adjusting, requiring no external output to adjust production of insulin levels. Insulin pumps require the patient to adjust insulin levels when eating or exercising. However, IGEBs will adjust without requiring human input, using the system described in the previous section. Additionally, IGEBs retain many of the advantages of insulin pumps, including a more flexible lifestyle and reducing blood sugar highs and lows. However, IGEBs also face many potential problems. One of the largest issues is how the bacteria will survive the gastrointestinal tract. If the bacteria are ingested (a similar idea to probiotics), the bacteria (or the capsule surrounding them) must be able to withstand the acidic conditions of the stomach. The next potential problem is the bacteria adhering to the villi in the small intestine without being flushed out by the body, which could require displacing the existing gut flora. Another issue is ensuring that enough insulin is absorbed into the bloodstream by the small intestine. Studies have shown that it is possible for insulin to be absorbed through the small intestine, but it is still unclear how much can be absorbed. Another issue is preventing the bacteria from mutating into a less than desirable form that could harm the patient, or transferring its genes through conjugation to other gut flora, which could result in the patient The testing of this system would involve observing the production of insulin by these bacteria in the absence and presence of glucose. The bacterial cells would be exposed to cycles of absence and presence of glucose of varying lengths of time and amounts of glucose. Insulin production would be tracked through these cycles to determine how much insulin is being produced and when. This testing would help adjust the bacterial insulin production to ideal levels for human insulin therapy. Further testing would also allow the development of technology to allow IGEBs to survive in the small intestine, providing an effective means for the treatment of Bowen, R. "Absorption of Amino Acids and Peptides." Digestion. Colorado State University, 8 <http://www.vivo.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_aacids.html> Crane, C.W., B.Sc., M.B., M.C. Path., F.R.I.C., and George R. W. N. Luntz, M.R.C.P. "Absorption of Insulin from the Human Small Intestine." Diabetes 17 (1968): 625-27. "Human Insulin Gene, Complete Cds." National Center for Biotechnology Information. U.S. National Library of Medicine, 12 Feb. 2001. Web. 10 July 2013. <http://www.ncbi.nlm.nih.gov/nuccore/J00265.1>.
"MetaCyc Pathway: Glyxoylate Cycle." MetaCyc. BioCyc Database, 04 Dec. 2007. Web. 10 July 2013. <http://www.biocyc.org/META/NEW- IMAGE?type=PATHWAY&object=GLYOXYLATE-BYPASS>.
Miller, MB, and BL Bassler. "Quorum Sensing in Bacteria." Annual Review of Microbiology 55 (2001): 165-99. PubMed.gov. Web. 9 July 2013. <http://www.ncbi.nlm.nih.gov/pubmed/11544353>.
O'Donnell, Stacy, RN, BS, CDE, and Andrea Penney, RN, CDE. "Insulin Injections vs. Insulin Pump." Diabetes Research, Care, Education & Resources. Joslin Diabetes Center, 11 <http://www.joslin.org/info/insulin_injections_vs_insulin_pump.html>.
"Part:BBa I761002 TAT Signal+INS_A." Registry of Standard Biological Parts. IGEM, 19 Oct. 2007. Web. 9 July 2013. <http://parts.igem.org/Part:BBa_I761002>.
Shichiri, Motoaki, M.D., Nobuaki Etani, M.D., Ryuzo Kawamori, M.D., Kenkichi Karasaki, M.D., Akira Okada, M.D., Yukio Shigeta, M.D., and Hiroshi Abe, M.D. "Absorption of Insulin from Perfused Rabbit Small Intestine in Vitro." Diabetes 22.6 (1973): 459- 65. Diabetes. American Diabetes Association. Web. 2 July 2013. <http://diabetes.diabetesjournals.org/content/22/6/459>.
Taqa, ME, JL Semmelhack, and BL Bassler. "The LuxS-dependent Autoinducer AI-2 Controls the Expression of an ABC Transporter That Functions in AI-2 Uptake in Salmonella Typhimurium." Molecular Microbiology 42.3 (2001): 777-93. PubMed.gov. Web. 9 July 2013. <http://www.ncbi.nlm.nih.gov/pubmed/11722742>

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