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.
Problem 1.
– Optimized DNA sequences can’t be synthesized
Integrated DNA Technologies, Inc. (IDT) contributes to synthetic biology community and is kindly providing free DNA synthesis service for iGEM teams every year. We first designed the genes by optimizing the gene sequence for expression in both E. coli and Lactobacillus spp. and expected to acquire gene synthesis from IDT.
Three weeks passed, IDT has tried hard but not made the synthetic gene successfully. In the end, we knew that it’s probably because the overexpression of glucose transporter genes are somehow toxic to E. coli. Therefore, IDT can’t synthesize the genes which are linked to a strong promoter.
Trouble-shooting 1.
– Amplification of genes by PCR with gDNA
We found a way to directly clone the genes out from Salmonella. Unfortunately, Salmonella is a human pathogen and classify as Biosafety Level 2 agents. First of all, we check the virulence factors (e.g., invasion/adhesion proteins, fimbriae, flagella, type I and III secretion systems)2 and confirm the glucose transporter genes are out of the list.
We sought collaboration with other iGEM teams to got the genes. Luckily, the place of iGEM team CSMU_NCHU _Taiwan is just near our school campus and working in a lab with the materials of Salmonella. They helped us amplify the genes of crr, ptsG and STM1128 by PCR using genomic DNA of Salmonella typhimurium LT2 as template (the gDNAs are recognized as Biosafety Level 1 agents, according the information provided by ATCC).
Now we could move forward with the PCR-amplified DNA fragments of glucose transporter genes of Salmonella.
Problem 2.
– Promoter was gone in the recombinant DNA
We soon assembled RBS-crr and RBS-ptsG to Double terminator/pSB1C3 (BBa_B0015) to make RBS-crr-RBS-pstG-TT/pSB1C3, so did to RBS-STM1128 to make RBS-STM1128-TT/pSB1C3. But something unexpectedly was happening in the next step.
We are unable to assemble both of the transporter composite parts with Double terminator under CP29 promoter.
A paper demonstrated growth inhibition by elevated transport of sugar phosphates in E. coli with overexpression of a glucose transporter (uhpT) gene3. Sugar phosphates transported through the transporter directly enter into glycolytic pathway resulting in the accumulation of toxic metabolite, methylglyoxal which causes the bacterial growth inhibition or cell death. It could also occur in our situation.
Although different and regulated promoters like PBAD or Plac could save our gene cloning, we still continued to focus on the CP promoter because a feasible promoter working both on E. coli and Lactobacilus spp. and easily applied to industrial manufacture without addition of any chemicals (inducers) are what we want.
Trouble-shooting 2.
– Different culture media replacing LB
Based on the fact that excess glucose entering bacterial cell at a time stresses the survival of E. coli, we were working in other strategies or with substitutes as energy source.
First, we tried to use agar plates made of M9 minimal salt media with a series of reducing concentration of glucose (i.e, 20mM, 10mM, 5mM, 2.5mM, 1.25mM, 0mM). The results showed that the colonies formed are either carrying CP29 promoter only or containing RBS and transporter genes without any promoters.
Secondly, we learned that MacConkey broth replace glucose with lactose for fermentation studies. We transformed E. coli with recombinant DNAs and grew them on MacConkey agar plate. We screened dozens of colonies by PCR and luckily found one containing CP29-RBS-aeBlue-RBS-crr-RBS-ptsG/pSB1C3. The plasmids were extracted and checked with restriction enzyme, as well as further confirmed by sequencing.
Finally, we thought another carbon sources in addition to glucose and lactose. This time, M9 media with glycerol were used. Luckily again, one colony carrying CP29-RBS-aeBlue-RBS-STM1128 was picked up by us. And we’ve check it with PCR, restriction enzyme and sequencing.
For more gene cloning detail, please go to our PARTS section.
- - REFERENCE -
- 1. The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters. Appl Environ Microbiol. 1998;64(1): 82–87.
- 2. Salmonella – the ultimate insider. Salmonella virulence factors that modulate intracellular survival. Cell Microbiol. 2009;11(11): 1579–1586.
- 3. Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli. J Gen Microbiol. 1992;138(10):2007-14.
Demonstration
Experiment
To measure glucose uptake by the engineered E. coli expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34μg/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD600 = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.
Fig 1. Overview of glucose uptake assay
Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.
Result
As shown in Fig. 3, the growth of E. coli expressing the PTS reporter (i.e., crr and ptsG genes) was seriously retardant. As I mentioned earlier, the E. coli overexpressing glucose transporter may lose viability because of the toxic metabolites produced in glycolytic pathway. Not surprisingly, the PTS overexpression bacteria was almost unable to absorb glucose.
Fig 3. The cell growth overnight and glucose uptake of E. coli expressing the PTS transporter in 4 hours
Fig. 4 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3rd hour. The glucose uptake efficiency was greater in Na+/Glu group than in control group with 1.2 times difference.
Fig 4. The cell growth overnight and glucose uptake of E. coli expressing the Na+/Glu transporter at different time point
Discussion
We’ve successfully genetically engineered E. coli expressing high-affinity active PTS transporter system and low-affinity facilitated Na+/glucose cotransporter system by screening in MacConkey and glycerol agar plate with chloramphenicol, respectively. Our data is consistent with the previous study that overexpressing glucose transporter genes do really harm E. coli partly due to disturbing glycolytic pathways.
PTS expressing bacteria grew difficultly in LB broth but Na+/glucose transporter expressing bacteria grew comparably with general E. coli, suggesting that high-affinity active transporter did interact with sugar-related substances in LB culture media and harm the bacterial survival.
In our results, glucose absorption efficiency was just slightly enhanced in Na+/glucose transporter expressing bacteria compared to general E. coli. Therefore, to increase glucose uptake, it is necessary to think about the glucose metabolism or conversion to other materials when entering into the cell.
Imagine an undercover agent exposing his id, who is going to commit suicide by taking a poisonous substance. What components in the substance could be and in what situation the agent would trigger the suiciding process.
Back to the view of a microbial cell, normally, a cell has a programmed suicide or defending mechanism to response to unfavorable environments or to outcompete other organisms.
What have iGEMers done and who are the "killers" ?
Lysis gene [BBa_K117000] created by NTU-Singapore in 2008 encodes Lysis protein which could not only lyse bacterial cell membrane but also activate the endonuclease of Colicin E7 (ColE7). The lysis-colicin is one class of bacteriocins which are produced to response to worsening environmental conditions and outcompete other bacteria1
NucA [BBa_K1159105] created by TU-Munich in 2013 from Staphylococcus aureus produces a thermostable exo- and endo-nuclease that is able to degrade genomic DNAs2. NucA also has a role in the cleavage of extracellular DNAs and preventing biofilm formation.
IMPROVE EXISTING PARTS
Part Name [Number]:
- Pcar, synthetic promoter repressed by CRP [BBa_K861171]
- RBS + PhlF repressor + terminator [BBa_K1725041]
- PhlF repressible promoter + strong RBS + GFP [BBa_K1725001]
Triggering the suicide circuit
In our case of sugar hijacking, ideally, the tiny, living “agents” are supposed to kill themselves in the story to make a happy ending. Therefore, we introduced a glucose responsive elements and a repressor circuit to be connected to suicide genes (lysis and NucA).
Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.
PhlF repressor system contains the repressor PhlF [BBa_K1725041] and the PhlF repressible promoter [BBa_K1725001] created by Glasgow in 2015. PhlF could repress GFP fluorescence intensity by 83-fold according to the study of Glasgow’s work.
Glucose responsive repressor device
We’ve innovated this year a novel glucose responsive repressor system (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 [BBa_K2230012]) by connecting these two system and extend the function of them.
Please go to DEMONSTRATION section below to check the function of this device in our experimental results.
- - REFERENCE -
- 1. Amount of colicin release in Escherichia coli is regulated by lysis gene expression of the colicin E2 operon. PLoS One. 2015;10(3):e0119124.
- 2. Characterization of a nuclease produced by Staphylococcus aureus. J Biol Chem. 1967;242(5):1016-20.
Gene Cloning
Our final composite parts (device) were listed in the table below. The basic and composite parts composed of the devices were briefly described in the following.
Problem 1.
– BioBricks are unvailable
The part of PI promoter was not in stock. Therefore, we are unable to request from iGEM HQ. We asked and got the plasmid DNA from team NCKU_Tainan. But unfortunately, we can’t re-transform E. coli with materials we got from them.
Trouble-shooting 1.
– Synthesis of promoter sequences on primers
It’s good news that the glucose responsive promoter region contains only 36 bp (PI and Pcar). Therefore, we designed the sequences on the forward primer. The cloning was going smoothly in the following process. And DNA sequences are all confirmed by sequencing.
Problem 2.
– The part of lysis gene was flanked by a wrong BioBrick Prefix
We’ve finished the gene cloning for lysis genes. Unfortunately, according to our sequencing data, we found a mistake that the previous team added the wrong Prefix in front of the part. The part starts with ATG, but unfortunately, the team took the wrong Prefix “for all the other parts”.
Trouble-shooting 2.
– PCR using a primer with correct Prefix
We designed new primers with the correct Prefix “for the coding region parts” along with a strong RBS / a weak RBS, and performed PCR to get the part. This approach successfully solved the previous problem we met.
Demonstration
Experiment
Result
First of all, we’d like to know how glucose responsive promoters (i.e., PI and Pcar) are induced in response to different concentration of glucose (GROUP 1) as shown in in Fig. 1. RFP intensity will be represented as promoter activity and measured at Ex/Em = 584nm/607nm.
Secondly, the glucose responsive repressor system (GROUP 2) will be tested in response to various concentration of glucose. GFP intensity will be measured at Ex/Em = 488nm/518nm as reporter showing the response of repressible promoter in the presence of glucose.
Finally, the glucose responsive suicide circuit (GROUP 2) will be examined in response to decreasing concentration of glucose. OD600 and cell numbers will be calculated to understand the killing efficacy of suicide circuit in the loss of glucose.
The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD600 was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. RFP, GFP or OD600 were indicators of promoter activities as mentioned. For suicide assay, the culture media were taken out and diluted 106 times following by spreading onto LB Cm agar plate at 37°C overnight. The third day, the numbers of colonies were counted and bacterial viability was calculated.
Result
The result shown in Fig. 2 indicated that PI promoter has significant activity in LB culture media. However, the activity of Pcar promoter is greater than negative control but much smaller than PI and positive control. It’s consistent with the properties of PI and Pcar promoters just mentioned previously in GENE CLONING section and described previously in Part Resgistry [Part: BBa_K861170] by team WHU-China in 2012 who designed the promoters.
In our experiment as presented in Fig. 3, PI and Pcar promoters just responded to various concentrations of glucose with a very slight dose-dependent increase. This phenomenon didn’t correspond to the data provided by team WHU-China in 2012 and team NCKU_Tainan in 2016. Maybe our measurement was not in an optimized condition. Or the reporter of RFP activity was not sensitive enough to respond this difference.
In the assay for repressor system, the data in Fig. 4 gave the similar results as team Glasgow did in 2015, in which the strong activity of the repressible promoter was significantly repressed in the presence of PhlF repressor.
Furthermore, we modified the expression of PhlF under the glucose responsive promoter (Pcar-PhlF-T-Pr-GFP/pSB1C3 [BBa_K2230012]). And the E. coli carrying this plasmid cultured in LB broth overnight was transferred to M9 minimal media with decreasing concentrations of glucose. The result in Fig 5 clearly indicated that the GFP activity driven by the repressible promoter was gradually increased in response to the loss of glucose to 1.88 folds compared to the initial GFP activity at the beginning culture in M9 media, suggesting that the level of expression of PhlF was positively corresponding to the concentration of glucose.
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