Team:CSU CHINA/Design
1.Overview
Type I diabetes is this kind of disease that due to the destruction of Islet b cells, the secretion of insulin is absolute deficient, which directly leads to Type I diabetes. It usually flares up in patients’ adolescence, the “3 more 1 less” symptom is consists of eating more, drinking more, urinating more and less body weight. In some serious conditions, patients directly develop in a more serious disease state of ketoacidosis.
Current efficient treatments for T1DM patients are mostly controlling the blood glucose with strict diet and lifelong insulin injection. As for patients, whether it is the inconvenience of carrying the injection devices or the pain of multiple injections everyday, or complications including the subcutaneous lipid hyperplasia, it can be a big disturbance for patients. However, current methods like stem cell transplantation are still holding disadvantages like the difficulty of regulation. When it comes to the situation that most patients get sick in their teenage time, it will definitely influence their daily life.
To solve the problems of current therapy and improve patient welfare, we designed our synthetic biology project of the year: “Sweet Guard”. It is consists if AND and NOT part. Parts we use in the AND part can secrete insulin when the hyperglycemia and blue light exposure are both presence. Meanwhile, parts in NOT part ca n be activated by the high insulin concentration and inhibit the secretion of insulin in a feedback way to prevent the oversecretion of insulin, forming a close loop.
The application scene in our envision is that the patient is wearing a smart phone that can detect the glucose concentreation and the watch will send the alarm when the after-meal blood glucose concentration reaches the threshold. The engineered cell embedded with sodium alginate glue under the skin will secrete insulin after the patient turning on the blue light. When the level of insulin is high enough, the feedback inhibition will be activated to stop the secretion.
Compared with traditional therapy, there are many advantages in our design. For example, the system with 2 switches: blood glucose concentration and blue light exposure can realize the auto/semi-auto regulation, with higher security level. Feedback inhibition can prevent the hypoglycemia caused by the oversecretion of innsulin and improve the sensitivity of the system.
2.miRNA
Type I diabetes is this kind of disease that due to the destruction of Islet b cells, the secretion of insulin is absolute deficient, which directly leads to Type I diabetes. As one kind of diabetes, the amounts of Type I is only 5% of total diabetes patients. However, as we can see from this figure, most of Type I patients get sick at their childhood or adolescence, parts of adult can also have an attack. Approximately 80 to 90 percent child diabetes patients are patients of Type I. The global incidence rate of Type I is on a rise
A typical symptom of T1DM is called “3 more 1 less”, which means eating more, drinking more, urinating more and less body weight.. Sometimes , patients directly develop in a more serious disease state of ketoacidosis.
Various of factors can be dangerous factors of Type I including Genetic, toxins, immune system disorder and viral infection.Genetically, the consistency of type I in identical twins can be up to 50%, if one of the parents is the patient of type I, the risk their offspring should take is only 2 to 5 percent. Toxins like Rodeticide and viruses like mumps virus can also increase the possibility of getting sick. Meanwhile, due to the disorder of immune system , islet beta cells are severely destroyed, insulin secretion is in absolute deficiency, thus the blood glucose rises, then leads to type I.
Multiple complication can be caused by type I including acute ketoacidosis and chronic diabetic foot and other complications implicating other systems and organs, which will seriously effect the quality of patients’ life.
3.Engineering design
Fig.1 The model diagram of engineering cell
3.1 AND part
3.1.1 Glucose induction
The abnormality of blood glucose concentration is important in various diseases but existing glucose-induced promoters are relatively few. In our project, the first condition to be met for secreting insulin is that the blood glucose concentration has reached a certain level. Thus we chose 2 glucose-induced promoter CHREBP promoter (BBa_K3734001) and GIP promoter (BBa_K3734002) to conduct cell culture under different glucose concentration and reflected these 2 promoters are regulated by glucose via the expression level of report gene LUC.
Fig.2 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T
3.1.2 Blue light control
In order to increase the safety of patients' use, it may be difficult to achieve the expected goal of accurate regulation by relying
on the regulation of blood glucose concentration alone, so we designed a pair of photosensitive proteins of GI (BBa_K3734004)
and LOV (BBa_K3734006). Under blue light irradiation, the Protein structure changes and interacts with each other to form a GAL4-GI-LOV-VP16 quadruple, while GA
L4 (BBa_K3734005) identifies and combines 9XUAS (BBa_K3734016), so that VP16 can activate the gene of downstream expression.
Fig.3 The model diagram of LOV-VP16
3.1.3 Insulin secretion
Naturally, a mature insulin molecule should go through precursors like preproinsulin and proinsulin to be secreted and make a difference. This means our cells need to be able to:(1) process insulin; (2) secrete insulin. Based on these two demands, we chose 293T. Meanwhile, we used gene Insulin(BBa_K3734015) to replace the report gene LUC(BBa_K3734014) we mentioned above. During the experiment, we used the specific ELISA tool kit for human mature insulin to test the supertanant and conducted experiments by deciding into two groups: dark and light group. The experiment proves that cell 293T can secret mature insulin outsides and is controlled by blue light.
Fig.4 The model diagram of 9XUAS-Ad-Insulin
3.2 NOT Part
3.2.1 INSR
Although there is insulin receptor (INSR)on the cell membrane, the quantity and the sensitivity can not meet our requirement for feedback mechanism very well.
Fig.5 The model diagram of INSR
3.2.2 Tet-off System
TetR can combine with TRE in Tet-off system, phosphated ELK would activate the expression of downstream target gene. We have designed TRE-mCherry-miR21(BBa_K3734030) to make the expression of miR21 is regulated by insulin, forming a complete feedback mechanism. What is worth noticing is that the Tet-off system consists of TetR(BBa_K3734010) and TRE(BBa_K3734012) can be blocked by tetracycline. Tetracycline will stop the combination between TetR and TRE, stop the system from working. This provides a mandatory brake system for our loop. We can make sure the security and prevent the hypoglycemia caused by abnormal feedback through exogenous tetracycline.
Fig.6 The model diagram of TetR-ELK
Fig.7 The model diagram of TRE's work
3.2.3 miR21
When designing the loop, we wanted to suppress the insulin exoression in a more root and efficient way to prevent hypoglycemia caused by the oversecretion of insulin. We used NO21 miRNA (miR21), it can not only suppress the expression of target gene by targeting miR21T, but also speed the degradation of mRNA up
Fig.8 The model diagram of TetR-ELK
4.Application part
4.1 Sodium alginate glue
The application scene in our imagination is that a patient is wearing a smart phone that can detect the glucose concentreation and the watch will send the alarm when the after-meal blood glucose concentration reaches the threshold. The engineered cell embedded with sodium alginate glue under the skin will secrete insulin after the patient turning on the blue light. When the level of insulin is high enough, the feedback inhibition will be activated to stop the secretion. Compared with deeper implantation, this is safer and more convenient.
We chose sodium alginate as the embedding material for engineering cells for the following reasons: (1) sodium alginate is a biological affinity material, which is not easy to cause allogeneic immune reactions. It is used to embed cells in many literatures and experiments and has a good effect. (2) sodium alginate has good plasticity and flexibility and is not easy to decompose, which well meets the requirements of our subcutaneous embedding. (3) The 2020 CSU_China team once used sodium alginate to embed algae, which can inherit certain experimental experience.
4.2 Watch
The application scene in our imagination is that a patient is wearing a smart phone that can detect the glucose concentreation and the watch will send the alarm when the after-meal blood glucose concentration reaches the threshold. The engineered cell embedded with sodium alginate glue under the skin will secrete insulin after the patient turning on the blue light. When the level of insulin is high enough, the feedback inhibition will be activated to stop the secretion. Compared with deeper implantation, this is safer and more convenient.
The watch device we envisioned with engineering cells can monitor the blood glucose concentration of the human body, prompt when the blood glucose concentration reaches the threshold, and turn on the blue light irradiation switch to illuminate blue light.
Fig.9 The model of watch device
Reference
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