Team:Mingdao/Demonstrate
INTRODUCTION
INTRODUCTION
Obesity and type 2 diabetes represent a serious health threat to the population all over the world. Glucose overdose has the major impact on this problem. However, sugar does make you feel happy. Some people may choose to limit, or even quit, sugar intake. Others may do more exercise to stay fit and healthy. In plain words, you have to either decline sweet happiness or pay for it with gaining fat or spending hard time burning calories.
In Mingdao’s project this year, we will help you “crush” the sugar by doing nothing except taking one pill full of our engineered probiotics. Really, just sit and read our iGEM story as well as enjoy your sweets and sugary drinks.
In the research, we addressed the health problems caused by excess glucose, providing information of controlling blood glucose by medicine, briefing previous iGEM team projects dealing with the issue of obesity and diabetes, and detail the mechanism of glucose metabolism and pleasure.
In the design and experiment, we will show you how we created probiotic-based glucose retrieval system with (1) glucose transporter device, which facilitates the cell glucose uptake with high efficiency, (2) glucose responsive suicide circuit, which produces lysis protein and nuclease to destroy bacteria when running out of glucose, and (3) probiotic transgenesis system, which be utilized to stably transform Lactobacillus acidophilus by chromosomal homologous recombination.
In the end, we introduced the modeling of the glucose uptake efficiency of our genetically engineered bacteria based on our experimental results. In addition, we showed the design of our acid-resistant pill and the prototype to mimic the stomach-gut environment and simulate the pill passing through gastric acid and releasing probiotics in the gut.
RESEARCH
What does glucose do to your body?
SUGAR exists in the types of glucose, fructose, galactose, sucrose, lactose, maltose and starch, as well as in wide varieties of drinks and food such as juice, honey, fruit, yogurt, candies and desserts. Simply speaking, sugar is everywhere, anyone can’t avoid it. Glucose is the smallest form of sugar and used as energy source for the body. And further, glucose would make you feel pleasure when hitting your tongue letting you crave more sugar like addicted.
OBESITY raises increasing concerns worldwide. Too much sugar in the body will be converted to fat and stored in the liver and cells. Excess sugar will make you overweight and obese. Meanwhile, fat is easily gained but hard to break down.
TYPE 2 DIABETES is another health problem because of excess sugar in the blood for a long term period. Insulin produced by the pancreas stimulates cells to absorb extracellular glucose. But in patients with type 2 diabetes, cells somehow become insulin resistant and need more amounts of insulin to maintain normal function. So the patients are advised to adjust their diet and control the blood sugar level.
The fat converted from excess glucose may contribute to atherosclerosis and increase the risk of cardiovascular problems. In addition, a study provided evidence that high blood glucose could lead to ALZHEIMER'S DISEASE and may damage your brain to cause dementia (Scientific Reports 2016;6:25589).
PROBLEM - Excess Sugar Consumption
How to deal with the excess glucose problems
To stay fit and keep healthy, you can choose to control desire and avoid sugary food or drinks. Or what else you can do to avoid the excess glucose in the blood?
DOING EXERCISE is good to your health and burning glucose you gained. Accordingly, you have to running (at the speed of 5 mph) for 13 min to burn off calories from 330ml of sugary soft drink, 28 min for a medium cup of mocha coffee, 43 min for a sandwich with chicken & bacon or a ¼ pizza (published by Royal Society of Public Health, 2016). When you think about it, I guess you probably already give up.
ARTIFICIAL SWEETENERS in diet drinks are common synthetic substitutes for glucose such as aspartame and saccharine. One survey by Boston University School of Medicine found that diet drinks are associated with higher risk of dementia caused by strokes (published on Stroke, 2017). Another study published on Canadian Medical Association Journal (CMAJ., 2017) reported that artificial sweeteners linked to risk of weight gain and heart disease. The sweeteners may have negative effects on metabolism, gut bacteria and appetite.
TAKING MEDICINE to reduce blood glucose is one of the treatments for diabetes. Canagliflozin is a drug of an inhibitor of subtype 2 sodium-glucose transport proteins (SGLT2) (Can J Diabetes., 2017). SGLT-2 play a major role in renal glucose reabsorption. When blocking SGLT-2 by canagliflozin, the blood glucose could be eliminated through the urine. However, the drug has adverse side effects such as fungal infection, thirst, increased urination and low blood pressure. DPP-4 inhibitor is a relatively new antidiabetic drug (Postgrad Med., 2017). DPP-4 is an enzyme destroying a gastrointestinal hormones called incretins which stimulates insulin production and inhibits the gluconeogenesis by liver. Blocking DPP-4 function by the inhibitor may reduce blood glucose level and lose weight. But DPP-4 inhibitor has severe side effects of nausea, diarrhea and stomach pain, etc.
Exercise to burn off the calories you take from food is not realistic. Artificial sweeteners and diet drinks are considered bad to your health and have higher risk to some diseases. Medicine is the bottom line and you never want to crave food followed by taking pills with side effects. Then, what should you do to deal with the excess glucose?
What have iGEMers done to solve the obesity and diabetes problems?
Problem-solving iGEMers
What have iGEMers done to solve the obesity and diabetes problems?
iGEMers are always tackling real problems happening in the world and seeking a potential solution based on synthetic biology. In the issues of obesity and diabetes, iGEM teams NTU-LIHPAO-Taiwan and Stony_Brook in 2015 contributed to the work of body weight control and diabetes treatment, respectively.
NTU-LIHPAO-Taiwan has developed Appetite Controller to reduce appetite so as to control body weight. They engineered a probiotic, Lactobacillus casei, to produce CPP-PYY fusion polypeptides. Peptide YY (PYY) is one of the gastrointestinal hormones which controls appetite. The PYY is carried by Cell Penetrating Peptide (CPP) to penetrate into the intestinal cells. The products would keep the body fit by lowering appetite when people crave foods but aren’t hungry.
Stony_Brook has engineered a QSP tripeptide secreting E. coli to regulating blood glucose level. The QSP tripeptide is encoded by RSC1A1 gene and acts on kidney cells to inhibit the expression of a glucose transporter (SGLT2) on the cell membrane which facilitates the glucose retrieval. Therefore, the product acts like anti-diabetic drug canagliflozin and would help people with high blood glucose excrete the glucose via urination.
However, if you take NTU-LIHPAO-Taiwan’s product, you may no longer enjoy delicious food. And if you need Stony_Brook’s solution, you’re probably getting some troubles with high blood glucose. Is there any better way to control glucose and enjoy the sweets?
- Reference -
- 1. Type 3 Diabetes: Cross Talk between Differentially Regulated Proteins of Type 2 Diabetes Mellitus and Alzheimer's Disease. Sci Rep. 2016;6:25589
- 2. Ten calorie dense food and their activity equivalence. Royal Society for Public Health Publication, 2016.
- 3. Sugar- and Artificially Sweetened Beverages and the Risks of Incident Stroke and Dementia: A Prospective Cohort Study. Stroke. 2017;48(5):1139-1146.
- 4. Nonnutritive sweeteners and cardiometabolic health: a systematic review and meta-analysis of randomized controlled trials and prospective cohort studies. CMAJ. 2017;189(28):E929-E939.
- 5. The Role of Sodium-Glucose Cotransporter 2 Inhibitors in the Management of Type 2 Diabetes. Can J Diabetes. 2017;41(5):517-523.
- 6. SGLT2 inhibitor/DPP-4 inhibitor combination therapy - complementary mechanisms of action for management of type 2 diabetes mellitus. Postgrad Med. 2017;129(4):409-420.
DESIGN
- BioBrick Blueprint & Prototype Design -
CP29-RBS-aeBlue-RBS-STM1128-TT/pSB1C3 (BBa_K2230028)
A facilitated sodium/glucose cotransporter encoded by STM1128 was cloned out from Salmonella typhimurium LT2. Promoter CP29 is a strong and constitutive promoter working in both E. coli and Lactobacillus spp. The device also displays blue color to demonstrate the engineered bacteria.
Pcar-wRBS-PhlF-T-Pr-sRBS-GFP-sRBS-lysis-sRBS-NucA/pSB1C3 (BBa_K2230017)
The device includes a glucose responsive promoter (Pcar), a repressor circuit (PhlF & its repressed promoter), and a suicide switch composed of lysis protein (lysis) and nuclease (NucA) to destroy cell membrane and chromosomal DNAs. In the presence of glucose, the repressor PhlF is expressed and inhibit the corresponding promoter. When glucose runs out, the PhlF is gradually degraded and the suicide circuit will then turn on to kill the host. The device also carries GFP for detection and measurement.
RBS-EmR-CP29-RBS-aeBlue/pLBA169 (BBa_K2230004)
pLBA169 was designed as vector for transforming Lactobacillus acidophilus thru chromosome homologous recombination. The gene cassette will be inserted to the location at the downstream of slpA (LBA0169) which encodes a surface layer protein. EmR (erythromycin resistance gene) is driven by the upstream promoter of slpA and acts as a selection marker. Promoter CP29 drives gene expression of aeBlue in Lactobacillus.
Our products will be encapsulated into the pill. Microencapsulation process combines sodium alginate and calcium chloride to form microspheres. This tiny spheres are acid resistant and don’t release probiotics in gastric fluid until passing into higher pH (pH=7~9) in the environment of the intestinal tract.
DEMONSTRATION
Glucose is transported into the small intestine and from there into the blood. Na+-glucose cotransporter SGLT1 is involved in intestinal sugar absorption, and Glucose transporter 2 (GLUT2) facilitate glucose transportation from intestine to blood stream.
Credit: American Physiological Society, 1998
Which glucose transporter should we use?
In order to compete the absorption of glucose with intestine, we need a better efficiency of glucose uptake. The value of Km of an enzyme was utilized as a parameter to select our target. Km (Michaelis constant) is determined by the concentration of substrate which permits the enzyme to achieve half Vmax. The lower Km means a higher substrate affinity.
As you can see in the below table, based on our research, the glucose transporter of Salmonella typhimurium has lowest Km, meaning the highest affinity of glucose bound to the transporter. So we decided to choose glucose transporter system of Salmonella as our target.
FACT
Interestingly, Salmonella is an intracellular intestinal pathogen. To survive in the small intestine epithelial cells, Salmonella has to utilize and metabolize available and limited glucose in a multiple and efficient way. Not surprisingly, Salmonella has higher affinity of glucose transporter compared to human small intestine. And it all makes sense to our assumption.
- - REFERENCE -
- 1. Glucose Galactose Malabsorption. American Journal of Physiology - Gastrointestinal and Liver Physiology 1998;275:G879-G882
- 2. Functional Properties and Genomics of Glucose Transporters. Curr Genomics. 2007;8(2): 113–128.
- 3. The SLC2 (GLUT) Family of Membrane Transporters. Mol Aspects Med. 2013;34(0): 121–138.
- 4. Glucose and Glycolysis Are Required for the Successful Infection of Macrophages and Mice by Salmonella enterica Serovar Typhimurium. Infect Immun. 2009;77(7): 3117–3126.
Gene Cloning
Salmonella typhimurium LT2 has two glucose-specific transporter systems, PTS system and sodium/glucose cotransporter. PTS system contains two subunits IIA encoded by crr and IIBC by ptsG which are assembled to a high-affinity active transporter. The other is a Na+/glucose cotransporter encoded by STM1128 that contributes to facilitated transport with lower glucose affinity. We decided to genetically engineer microbes with these two systems.
In order to express the genes in E. coli for demonstration and in probiotics for proof-of-concept in a real world. We chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp1. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280) was used and to be assembled with the transporter genes.
Demonstration
Experiment
iGEMers are always tackling real problems happening in the world and seeking a potential solution based on synthetic biology. In the issues of obesity and diabetes, iGEM teams NTU-LIHPAO-Taiwan and Stony_Brook in 2015 contributed to the work of body weight control and diabetes treatment, respectively.
NTU-LIHPAO-Taiwan has developed Appetite Controller to reduce appetite so as to control body weight. They engineered a probiotic, Lactobacillus casei, to produce CPP-PYY fusion polypeptides. Peptide YY (PYY) is one of the gastrointestinal hormones which controls appetite. The PYY is carried by Cell Penetrating Peptide (CPP) to penetrate into the intestinal cells. The products would keep the body fit by lowering appetite when people crave foods but aren’t hungry.
Stony_Brook has engineered a QSP tripeptide secreting E. coli to regulating blood glucose level. The QSP tripeptide is encoded by RSC1A1 gene and acts on kidney cells to inhibit the expression of a glucose transporter (SGLT2) on the cell membrane which facilitates the glucose retrieval. Therefore, the product acts like anti-diabetic drug canagliflozin and would help people with high blood glucose excrete the glucose via urination.
However, if you take NTU-LIHPAO-Taiwan’s product, you may no longer enjoy delicious food. And if you need Stony_Brook’s solution, you’re probably getting some troubles with high blood glucose. Is there any better way to control glucose and enjoy the sweets?
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