Team:CSU CHINA/Results
1.AND part
1.1 Glucose induction
The abnormal of blood glucose is important in many diseases but there are few glucose-induced promoters available. In our project, the first condition to be completed is the threshold of blood glucose. Therefore, we chose CHREBP promoter (BBa_K3734001) and GIP promoters (BBa_3734002) as our promoters and conducted cell culture under different glucose concentration. The expression level of the report gene LUC can be used to represent that these two are controlled by the concentration of glucose.
Result：CHREBP promoter and GIP promoter are induced by glucose and the expression level rises along with the raise of the concentration of blood glucose.
Fig1.The expression level of inducible promoter CHREBP was respectively analyzed at 48h in 25mM, 5.6mm and 0mM glucose cultur.
To eliminate the effects of residual of glucose during transmission, we have conducted a 72-hour group, the result fits the anticipation more.
Fig2.The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose cultur.
Fig3.Changes of LUC expression over time after blue light irradiation with different glucose concentrations
1.2 Blue light control
Only being controlled by the concentration may not be safe enough and it may be hard to reach the goal of precise control. Thus we disighned a pair of light sensitive proteins, GI (BBa_K3734004) and Lov (BBa_K3734006) . Under the exposure of blue light, theses two proteins would have a mutual effect and form a GAL4-GI-LOV-VP16 quadruple. GAL4(BBa_K3734005) will recognize and combine with 9XUAS (BBa_K3734016, makes it possible for VP16(BBa_K3734006) to activate the expression of downstream target genes. We used report gene LUC to represent the expression of blue light-controlled system in the very beginning. During the experiments, the engineered cells were put in the dark for 24 hours after the transfection, then exposed by 450nm(wave length) blue light for 30min and continue to be cultured for 24 hours, detect LUC after the culturing. The result suggests that our blue light system is well controlled by blue light. It hardly ever express under the dark condition, while the expression under the blue light is 200 times more compared to the expression in the dark. Moreover, the expression efficiency can meet the commands of our design.
Fig4.Light controlled system testing experiment 20210619
To eliminate the effects of residual of glucose during transmission, we have conducted a 72-hour group, the result fits the anticipation more.
Fig5.Expression level analysis under blue light irradiation and dark treatment
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
Fig6.Insulin and absorbance standard curve
Fig.7 Insulin concentration in supernatant after blue light irradiation and dark treatment respectively
Figure.8 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively
2.NOT Part
2.1 INSR
Althiugh there are insulin receptors(INSR) on the cell memberane, the amount and the sensitivity are both not high enough for our feedback mechanism. We designed carrier with INSR CMV-INSR-EFIA-ZsGreen to express the membrane protein INSR and represent its expression with green fluorescent protein ZsGreen. The expriments have proved that the INSRs are successfully expressed in our engineered cells
Fig.9 Green fluorescence of INSR expression
2.2 MAPK
After combing with insulin, INSR will activate the downstream MAPK phosphorylation pathway. Despite the short reaction time of this pathway, it is quite complicated and the process is quite long. To make sure this MAPK pathway can work, we chose one spot in it: ERK phosphorylation to test. With the method of Western blot, we have proved that ERK can be phosphorylated as the concentration of insulin rises.
Fig.10 ERK phosphorylation changes with different insulin treatment
Fig.11 ERK phosphorylation changes with different insulin treatment
2.3 Tet-off System
It has been proved above that the MAPK phosphorylation system can work when INSR is combined with insulin. In text-off system, TRE can combine with TetR, phosphorylated ELK activate downstream expression. We designed TRE-mCherry-miR21(BBa_K3734030) to regulate miR21 expression by insulin and form a complete feedback mechanism. One thing to be noticed is that Tet-off system formed by TetR(BBa_K3734010) and TRE(BBa_K3734012)can be blocked by tetracycline. Tetracycline will stop TetR and TRE from combining and stop the system, which provides a mandatory brake system to our system. It is possible to avoid abnormal feed back which would cause hypoglycemia and ensure the security of our system. During experiments, we used laser copolymerization microscope and qPCR to prove our Tet-off system works.
Fig.12 Under Laser confocal microscopy, fluorescence of ZsGreen and mCherry expression downstream of Tet-Off system
Fig.13 Transcriptional level analysis of downstream mCherry in TEt-OFF system without tetracycline
2.4 miR21
We wanted to suppress the insulin expression more thoroughly and rapidly to avoid hypoglycemia. We chose miR21(BBa_K3734013) which can target the target site miR21T to suppress the expression of target genes and speed up the degradation of mRNA of the target gene.
When designing the experiment, we tested the LUC/REN ratio of target gene 48 hours after the transfection, miR21 suppressed 40% of expression, but it is not ideal for our design
Fig14.miR21 inhibited Luc expression 48h after transfection
Considering it might be caused by the accumulation of LUC after the expression, we did a 24-hour group. The result proved that miR21 have suppressed 90% of expression, fits our anticipation. Meanwhile, we used qPCR to test the efficiency of miR21 speeding up the degradation of mRNA of the target gene.
Fig15.miR21 inhibited Luc expression 24h after transfection
Fig.16 miR21 promoted the mRNA degradation efficiency of LUC
So far, our whole loop has been tested and every part can function well to complete the insulin secretion under the condition of hyperglycemia and blue light exposure, suppress the insulin secretion under the condition of high insulin concentration and hypoglycemia to form a close loop.
3.Application part
3.1 Sodium alginate glue
In our design, the device gives blue light is a watch. This watch can detect the concentration of blood glucose and turn on the blue light when the concentration has reached a certain level. Thus, we designed the subcutaneous part of forearms as our implant sites. Compared with deeper implants，subcutaneous implant is more safe and convenient.
We used sodium alginate glue as the material for embedding of the engineered cells. Reasons are as followed:
1. Sodium alginate is a biocompatibility material with weak immunogenicity. There are literatures and experiments use it as embedding material with good results.
2. Sodium alginate has relatively high plasticity and flexibility along with low decomposability.
3. CSU_China 2020 has used sodium alginate to embed algae, we have certain experiences.
However, we don’t know the lifespan and the velocity of insulin secretion. Once the lifespan is too short or the insulin is hard to be secreted, this material would not fit our demand. Therefore, we collaborated with ShanghaiTech and deigned experiments.
3.2 Cell lifespan
This part is completed by ShanghaiTech. They have tested alive cells with FAD dyeing, drew a curve of alive ratio after embedding. The result has been proved the cells embedded can live well.
Fig.17 Sodium alginate encapsulated cells were stained for viability
Fig.18 Survival rate curve of sodium alginate embedded cells
3.3 insulin release experiment
This part is completed by our team. We dissolved insulin in sodium alginate dissolution to have a 0.8ng/ml insulin and 1% sodium alginate dissolution. Cross-link with 5% CaCl2 to form sodium alginate glue and put it into insulin culture medium. The result suggests that insulin ca be released through sodium alginate glue. It fits our demands.
Fig.19 Sodium alginate releases insulin
All in all, our AND, NOT part and the loop that connects both parts have been tested and proved the possibility of our project. Meanwhile, we have tested materials we use and proved these materials fit our demands, made sure the security and reliability of applying these meterials.
Reference
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