iGEM 2019 — Team NUDT_CHINA

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# Team:NUDT CHINA/Notebook

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* [SAFETY](https://2019.igem.org/Team%3ANUDT_CHINA/Safety "SAFETY")
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  + [Human Practices](https://2019.igem.org/Team%3ANUDT_CHINA/Human_Practices "HP")
  + [Education&Engagement](https://2019.igem.org/Team%3ANUDT_CHINA/Public_Engagement "Public_Engagement")

# NoteBook

## Tying it all together

#### Archives

* [Monthly progress & Milestones](#Monthly progress & Milestones)
* [Personal Contribution](#Personal Contribution)

## NoteBook

We would like to share with you what we have done for our project.

### Monthly progress & Milestones

#### 2018 November

● Summary for 2018 NUDT\_CHINA in iGEM.

#### 2018 December

● 2019 NUDT\_CHINA team set-up.

#### 2019 January

● Brainstorm for 2019 team project. Anchor T2D as the field we want to anchor our project.

● Background information gathering.

● Team member split into groups: Wet Lab, Human Practice, Wiki design, Model.

#### 2019 February

● Nurse interview and visit in geracomium to gather realistic T2D information and publicity of good lifestyles for the elders to avoid or retard T2D complication.

![](https://static.igem.org/mediawiki/2019/9/94/T--NUDT_CHINA--notebook-nurse2.svg)

● Determine 2019 project --- continue with PR Predator in 2018, but focus on the degradation of glucagon receptor in hepatocytes.

● Look up for authority articles.

● Experimental skills and safety training for team members in Wet Lab.

![](https://static.igem.org/mediawiki/2019/4/41/T--NUDT_CHINA--2019picnb1.png)

#### 2019 March

● Crucial parts construction.

#### 2019 April

● First milestone in project --- designed gene circuit works well in degrade GCGR in HepG2 cell line.

● Diabetes Knowledge Popularization Activity.

● Public questionnaire about diabetes.

![](https://static.igem.org/mediawiki/parts/4/43/T--NUDT_CHINA--notebook-3.svg)
![](https://static.igem.org/mediawiki/parts/6/63/T--NUDT_CHINA--notebook-8.svg)

#### 2019 May

● Expert interview on our subject. Question raised: What if the CMV promoter keeps working, would this circuit result in hypoglycemia in human body?

● Internet articles research.

● Improvement of project: involvement of glucose sensing promoter.

![](https://static.igem.org/mediawiki/parts/7/7f/T--NUDT_CHINA--notebook-4.svg)

#### 2019 June

● Experiment protocol gathering and design.

● Glucose sensing promoter related circuit construction.

● Model preparation.

#### 2019 July

● Glucose sensing promoter functional test in HepG2.

#### 2019 August

● 2019 iGEM Chongqing Meet-up.

●Second milestone: Result for glucose sensing promoter functional test.

●Determination: Mouse primary hepatocytes as platform for continuous experiments of designed GCGR degradation circuit. Adenovirus as circuit transport tool.

●Research, discussion and review for safety concerns on adenovirus and mouse primary hepatocytes.

![](https://static.igem.org/mediawiki/parts/a/ab/T--NUDT_CHINA--notebook-6.svg)
![](https://static.igem.org/mediawiki/parts/2/2a/T--NUDT_CHINA--notebook-5.svg)
![](https://static.igem.org/mediawiki/parts/f/f6/T--NUDT_CHINA--notebook-7.svg)

#### 2019 September

● Collaboration with CSU\_CHINA about current gene delivery methods and new ways to improve delivery efficiency as well as decrease cell toxicity.

●Third milestone: Result for glucose sensing enabled GCGR degradation circuit in HepG2.

![](https://static.igem.org/mediawiki/parts/8/8c/T--NUDT_CHINA--notebook-9.svg)

#### 2019 October

● Fourth milestone: Result for mouse primary hepatocytes.

●Fifth milestone: Model for evaluate the effectiveness of initial design and indicate the potentials of circuit in long-term in vivo uses.

●Preparation for team wiki, poster, presentations and the Giant Jamboree!

![](https://static.igem.org/mediawiki/2019/c/ce/T--NUDT_CHINA--notebook-10.svg)

### Personal Contribution

| Name | Project Design | Human Practice | Lab | Model | Art Design | Wiki Construction |
| --- | --- | --- | --- | --- | --- | --- |
| Jiaxin Ma | √√ |  | √√ |  |  |  |
| Chaodong Zhang |  | √ |  | √ | √ |  |
| Hanxiao Feng |  |  | √ |  |  |  |
| Qinrui Jiang |  |  |  |  |  | √ |
| ZhenYu Huang |  |  |  |  |  | √√ |
| Zuyu Dai |  | √√ | √ |  |  |  |
| Jie Cai |  |  | √ | √ |  |  |
| Xinyu Su |  |  | √ |  |  |  |
| Yating Long |  |  | √ |  |  |  |
| Qianqian Tao |  | √ |  |  | √ |  |
| Liang Shengpeng |  |  |  |  |  | √ |
| Liu Meiqi |  | √√ |  |  |  |  |
| Mufei Guo |  | √ |  |  |  |  |
| Yiyun Zhang |  |  |  | √ |  |  |
| Wang Zhenyuan |  |  |  | √ |  |  |
| Yanchen Gou |  |  |  | √√ |  |  |
| Yuhang Ye |  | √ |  |  |  |  |
| Hanxiao Feng |  | √ |  |  | √ |  |
| Qiang Yi |  | √ |  |  | √ |  |

✔: involved

✔✔: made main contribution


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NUDT CHINA/Experiments

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  + [Human Practices](https://2019.igem.org/Team%3ANUDT_CHINA/Human_Practices "HP")
  + [Education&Engagement](https://2019.igem.org/Team%3ANUDT_CHINA/Public_Engagement "Public_Engagement")

# Experiments

## To create, to dream

## Protocols and Experiments

[Protocol for Agarose Gel Electrophoresis](https://static.igem.org/mediawiki/2019/1/18/T--NUDT_CHINA--Protocol_for_Agarose_Gel_Electrophoresis.pdf)

[Protocol for Annealing](https://static.igem.org/mediawiki/2019/2/23/T--NUDT_CHINA--Protocol_for_Annealing.pdf)

[Protocol for Culture and Subculture](https://static.igem.org/mediawiki/2019/3/31/T--NUDT_CHINA--Protocol_for_Cell_Culture_and_Subculture.pdf)

[Protocol for Colony PCR](https://static.igem.org/mediawiki/2019/a/ae/T--NUDT_CHINA--Protocol_for_Colony_PCR.pdf)

[Protocol for Double Digestion](https://static.igem.org/mediawiki/2019/c/c2/T--NUDT_CHINA--Protocol_for_Double_Digestion.pdf)

[Protocol for Gel Purification with OMEGA Gel Extraction Kit](https://static.igem.org/mediawiki/2019/c/cb/T--NUDT_CHINA--Protocol_for_Gel_Purification_with_OMEGA_Gel_Extraction_Kit.pdf)

[Protocol for Western Analysis](https://static.igem.org/mediawiki/2019/2/2a/T--NUDT_CHINA--Protocol_for_Western_Analysis.pdf)

[Protocol for Transformation](https://static.igem.org/mediawiki/2019/c/cf/T--NUDT_CHINA--Protocol_for_Transformation.pdf)

[Protocol for Ligation](https://static.igem.org/mediawiki/2019/a/a7/T--NUDT_CHINA--Protocol_for_ligation.pdf)

[Protocol for Adenovirus Infection in Mouse Primary Hepatocytes](https://static.igem.org/mediawiki/2019/1/19/T--NUDT_CHINA--Protocol_for_Adenovirus_Infection_in_Mouse_Primary_Hepatocytes.pdf)

[Protocol for Amplification of cDNA with NovoScript® Plus 1st Strand cDNA Synthesis SuperMix(gDNA Purge)](https://static.igem.org/mediawiki/2019/1/13/T--NUDT_CHINA--Protocol_forAmplification_of_cDNA_with_NovoScript%C2%AE_Plus_1st_Strand_cDNA_Synthesis_SuperMix%28gDNA_Purge%29.pdf)

[Protocol for Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit](https://static.igem.org/mediawiki/2019/2/2d/T--NUDT_CHINA--Protocol_forBeyotime%E2%84%A2_RG027_Dual_Luciferase_Reporter_Gene_Assay_Kit.pdf)

[Protocols of transferrin-lipoplex transfection into mouse primary hepatocytes](https://static.igem.org/mediawiki/2019/0/01/T--NUDT_CHINA--protocol_for_transferrin.pdf)

[Protocol for cell mRNA extraction with Trizol](https://static.igem.org/mediawiki/2019/8/80/T--NUDT_CHINA--Protocol_for_cell_mRNA_extraction_with_Trizol%281%29.pdf)

[Protocol for Glucose Concentration Test with Solarbio™ Glucose Detection Kit](https://static.igem.org/mediawiki/2019/e/e0/T--NUDT_CHINA--Protocol_for_Glucose_Concentration_Test_with_Solarbio%E2%84%A2_Glucose_Detection_Kit.pdf)

[Protocol for Glucose Sensing Promoter Test in HepG2 Cell Line](https://static.igem.org/mediawiki/2019/8/8d/T--NUDT_CHINA--Protocol_for_Glucose_Sensing_Promoter_Test_in_HepG2_Cell_Line.pdf)

[Protocol for Glycogen Test with Solarbio™ Glycogen PAS Dye for Cell](https://static.igem.org/mediawiki/2019/d/d3/T--NUDT_CHINA--Protocol_for_Glycogen_Test_with_Solarbio%E2%84%A2_Glycogen_PAS_Dye_for_Cell.pdf)

[Protocol for Immunoprecipitation with Beyotime™ Protein A+G Agarose](https://static.igem.org/mediawiki/2019/8/88/T--NUDT_CHINA--Protocol_for_Immunoprecipitation_with_Beyotime%E2%84%A2_Protein_A%2BG_Agarose.pdf)

[Protocol for lipo3000 transfection with Lipofectamine™ 3000 Reagent](https://static.igem.org/mediawiki/2019/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf)

[Protocol for Plasmid Extraction with Sangon Biotech™ Sanprep Plasmid Extraction Kit](https://static.igem.org/mediawiki/2019/4/49/T--NUDT_CHINA--Protocol_for_Plasmid_Extraction_with_Sangon_Biotech%E2%84%A2_Sanprep_Plasmid_Extraction_Kit.pdf)

[Protocol for Real-time PCR with NovoStart® SYBR qPCR SuperMix Plus](https://static.igem.org/mediawiki/2019/b/b9/T--NUDT_CHINA--Protocol_for_Real-time_PCR_with_NovoStart%C2%AE_SYBR_qPCR_SuperMix_Plus.pdf)

## Method for Glucose sensing promoter Function Validation (luciferase)

### Material

1. Complete Cell Culture media(400ul/well):Hyclone™ High Glucose Dulbecco's Modified Eagle Medium( DMEM ) + 10% PAN™ Fetal Bovine Serum(FBS) + 1% Hyclone™ 100X Penicillin-Streptomycin Solution(P/S).

2. Glucose-free Cell Culture Media:Gibco™ glucose free Dulbecco's Modified Eagle Medium( DMEM ) +10% PAN™ Fetal Bovine Serum(FBS)+1%Hyclone 100X Penicillin-Streptomycin Solution(P/S).

3. Glucose Stork Solution:1.25mol/L D-Glucose stork solution made of MilliQ™ Deionized Water and Sangon™ Glucose.

4. (Beyotime)Dual Luciferase Reporter Gene Assay Kit

5. Invitrogen LipofectamineTM 3000 Reagent

### Method

![](https://static.igem.org/mediawiki/2019/3/3f/T--NUDT_CHINA--experiment1.png)

Figure 1. The simple chart of the experimental step

1. 2 hours before transfection, starve cells using Dulbecco’s Modified Eagle Medium( DMEM ) be cultured in 24-well plate. Before this, it had has 90% HepG2;

2. Dilute 0.75μL lipofectamine3000TM Reagent in 24.25 μL DMEM pre well;

3. Dilute 600 ng DNA and 1ul of P3000 in DMEM and make a total volume of 25 ul per well;

4. Add diluted DNA to diluted LipofectamineTM 3000 Reagent (1:1 ratio);

5. Incubate for 10-15 minutes at room temperature;

6. Change cell culture into complete cell culture medium, add DNA-lipid complex to a well of the 24-well plate;

7. Incubate cells for 12 h at 37°C, 5% CO2, change cell medium into glucose-free complete culture medium.

8. Incubate cells for 6 h at 37°C, 5% CO2, add glucose stock solution and give glucose stimulation.

9. Incubate cells for 6/20 h at 37°C, 5% CO2, collect data by Dual Luciferase Reporter Gene Assay Kit.

## Method for Glucose sensing promoter Function Validation（GFP as reporter）

### Material

1. Complete Cell Culture media(400ul/well): Hyclone™ High Glucose Dulbecco's Modified Eagle Medium( DMEM ) + 10% PAN™ Fetal Bovine Serum(FBS) + 1% Hyclone™ 100X Penicillin-Streptomycin Solution(P/S).

2. Glucose-free Cell Culture Media: Gibco™ glucose free Dulbecco's Modified Eagle Medium( DMEM ) +10% PAN™ Fetal Bovine Serum(FBS)+1%Hyclone 100X Penicillin-Streptomycin Solution(P/S).

3. Glucose Stork Solution: 1.25mol/L D-Glucose stork solution made of MilliQ™ Deionized Water and Sangon™ Glucose.

4. (Beyotime)Luciferase Reporter Gene Assay Kit.

5. Invitrogen LipofectamineTM 3000 Reagent

### Method

![](https://static.igem.org/mediawiki/2019/6/62/T--NUDT_CHINA--experiment2.png)

Figure 2. The simple chart of the experimental step

1. 2 hours before transfection, starve cells using Dulbecco’s Modified Eagle Medium( DMEM ) be cultured in 24-well plate. Before this, it had has 90% HepG2;

Dilute 0.75μL lipofectamine3000TM Reagent in 24.25 μL DMEM pre well;

2. Dilute 600 ng DNA and 1ul of P3000 in DMEM and make a total volume of 25 ul per well;

3. Add diluted DNA to diluted LipofectamineTM 3000 Reagent (1:1 ratio);

4. Incubate for 10-15 minutes at room temperature;

6. Change cell culture into complete cell culture medium, add DNA-lipid complex to a well of the 24-well plate;

7. Incubate cells for 12 h at 37°C, 5% CO2, change cell medium into glucose-free complete culture medium.

8. Incubate cells for 6 h at 37°C, 5% CO2, add glucose stock solution and give glucose stimulation.

9. Incubate cells at 37°C, 5% CO2, collect data of efficiency of transfection via fluorescence microscopy at continous time set after glucose stimulation of 6, 18, 30, 42 and 54 hours.

## Documents for Project Progress

#### 2019 January

● Brainstorm for 2019 team project. Anchor T2D as the field we want to anchor our project.

● Background information gathering.

#### 2019 February

● Nurse interview and visit in geracomium to gather realistic T2D information and publicity of good lifestyles for the elders to avoid or retard T2D complication.

● Determine 2019 project --- continue with PR Predator in 2018, but focus on the degradation of glucagon receptor in hepatocytes.

● Look up for authority articles.

● Experimental skills and safety training for team members in Wet Lab.

#### 2019 March

● Crucial parts construction.

#### 2019 April

● First milestone in project --- designed gene circuit works well in degrade GCGR in HepG2 cell line.

#### 2019 June

● Glucose sensing promoter related circuit construction.

#### 2019 July

● Glucose sensing promoter functional test in HepG2.

#### 2019 August

● Result for glucose sensing promoter functional test.

●Determination: Mouse primary hepatocytes as platform for continuous experiments of designed GCGR degradation circuit. Adenovirus as circuit transport tool.

#### 2019 September

●Third milestone: Result for glucose sensing enabled GCGR degradation circuit in HepG2.

#### 2019 October

● Fourth milestone: Result for mouse primary hepatocytes.

●Fifth milestone: Model for evaluate the effectiveness of initial design and indicate the potentials of circuit in long-term in vivo uses.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NUDT CHINA/Results

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  + [Composite Parts](https://2019.igem.org/Team%3ANUDT_CHINA/Composite_Parts "Composite Parts")
  + [Part Collection](https://2019.igem.org/Team%3ANUDT_CHINA/Part_Collection "Part Collection")
* [SAFETY](https://2019.igem.org/Team%3ANUDT_CHINA/Safety "SAFETY")
* [MODEL](https://2019.igem.org/Team%3ANUDT_CHINA/Model "MODEL")
* [HUMAN PRACTICES](https://2019.igem.org/Team%3ANUDT_CHINA/Human_Practices "HP")
  + [Human Practices](https://2019.igem.org/Team%3ANUDT_CHINA/Human_Practices "HP")
  + [Education&Engagement](https://2019.igem.org/Team%3ANUDT_CHINA/Public_Engagement "Public_Engagement")

# Results

#### Archives

* [GCGR Predator Module](#GCGR Predator Module)
* [Glucose Sensing Module](#Glucose Sensing Module)
* [Integrated Analysis](#Integrated Analysis)
* [Future Plan](#Future Plan)
* [Reference](#Reference)

# GCGR Predator Module

[(See the design of this module here)](https://2019.igem.org/Team%3ANUDT_CHINA/Design#GCGR%20degrading%20module)

    The corner stone of the GCGR Predator design is to assume that the ligand-IgG FC fusion protein and endogenous receptors interact within the endomembrane system. Such intracellular ligand-receptor interaction should also be spatiotemporally correct to trigger the Trim21 based protein degradation cascade. To test the such hypothesis, we first performed immunoprecipitation assay to determine if the intracellular ligand-receptor interaction occur. For such matter, HepG2 cells were transfected with Gcg-Fc expressing cassette or GCGR Predator (GCGR Pr) expressing cassette. Immunoprecipitation was performed with IgG-Fc targeting Protein A/G beads 48 h posttranscription followed up by Western blotting analysis probing GCGR and the Gcg-Fc domain of the GCGR Predator (Figure 1a). Results showed that interaction signal between Gcg-Fc and GCGR could be observed on Gcg-Fc transfected group while the non-transfected group showed no corresponding signal. Surprisingly Cells transfected with GCGR Pr plasmids showed significantly weaker interaction signal together with a significantly lower GCGR abundance, we reasoned that might due to the Trim21 mediated GCGR degradation. We then performed Western Blotting to further proof that such interaction is capable of triggering GCGR degradation. Results indicated that GCGR Pr transfected cells showed ~40% lower GCGR level comparing to the vector control (Figure 1c). For a closer mimic to the *in vivo* application condition, we packed GCGR Predator into Adenovirus for circuit delivery into mouse primary hepatocytes. Western Blotting showed that a 50% GCGR decrease 48 hours post-infection comparing to cells infected by the control Ade-GFP virus (Figure 1d). These results implied that GCGR Predator mediates *in vitro* and *ex vivo* GCGR degradation via physically interact with endogenous GCGR.

![](https://static.igem.org/mediawiki/2019/7/73/T--NUDT_CHINA--2019_Results_Fig1.png)

**Figure 1. GCGR Predator mediates GCGR degradation by direct interaction with endogenous GCGR.** (a). Schematic representation of co-IP assay; (b). co-IP-blotting analysis; (c). Lipofectamine delivered GCGR Predator mediates GCGR degradation in HepG2 cells. Left panel: representative blotting of GCGR degradation; right panel: statistical gray scale analysis of blotting assays; (d). Adenovirus delivered GCGR Predator mediates GCGR degradation in primary mouse hepatocytes. Left panel: representative blotting of GCGR degradation; right panel: statistical gray scale analysis of blotting assays. “GCGR Pr” stands for GCGR Predator. Relative protein expression levels were calculated by using β-actin expression level as initial normalization and then set the protein expression level in the control groups arbitrarily as 1.0. Error bar represents SD of at least 3 biological replicates, \*\* *p*<0.01.

    Since changes on glucose metabolic program is expected under GCGR depletion1, we performed metabolic analysis to determine if our GCGR Predator could generate similar phenotype changes. For such assay, cells were transfected with GCGR Predator by adenovirus (for mouse primary hepatocytes) or lipofectamine (for HepG2 cell line) and stimulated with Glucagon in DMEM medium without glucose (Figure 2a). To measure the overall glucagon sensitivity, glucose concentration in the medium was evaluated by glucose oxidase assay. As shown in Figure 2b, GCGR predator carrying cells showed significantly lower glucose concentration (~20% and ~60% decrease in HepG2 cells and mouse primary hepatocytes respectively) in the culture medium comparing to the control group (vector control for HepG2, and Ad-GFP control virus for mouse primary hepatocytes), indicating a significantly impaired overall glucagon sensitivity mediated by GCGR Predator. To further reveal the metabolic changes generated by GCGR Predator, we used quantitative PCR analysis and Periodic Acid-Schiff (PAS) staining Assay to evaluate the cellular gluconeogenesis and glycogenolysis process. Results showed that GCGR Predator transfection significantly down-regulates the expression of key enzymes in gluconeogenesis pathway, Glucose 6-phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (PEPCK) (Figure 2c). Also, significantly higher amount of glycogen could be observed in the GCGR Predator transfected cells under glucose starvation, indicating lower glycogenolysis activity (Figure 2d). Generally, these results indicated that GCGR predator significantly impairs hepatocyte glucagon sensitivity *in vitro* and *ex vivo* by affecting metabolic programs downstream of GCGR.

![](https://static.igem.org/mediawiki/2019/3/3a/T--NUDT_CHINA--2019_Results_Fig2.png)

**Figure 2. GCGR predator impairs hepatocyte glucagon sensitivity *in vitro* and *ex vivo* by affecting GCGR-downstream metabolic programs.** (a). Schematic representation of the functional assay workflow; (b). Glucose production analysis of HepG2 cells or mouse primary hepatocytes transfected with GCGR predator; (c). Quantitative PCR assay of G6Pase and PEPCK in HepG2 cells or mouse primary hepatocytes transfected with GCGR predator; (d). Representative glycogen staining microscopy of GCGR predator transfected HepG2 cells or mouse primary hepatocytes. “GCGR Pr” stands for GCGR Predator. Relative gene expression was calculated using the 2-ΔΔCT method, with initial normalization of genes against Hprt1 mRNA within each group. The expression levels of each gene in the control groups were arbitrarily set to 1.0. Error bar represents SD of at least 3 biological replicates, \* *p*<0.05, \*\* *p*<0.01.

# Glucose Sensing Module

[(See the design of this module here)](https://2019.igem.org/Team%3ANUDT_CHINA/Design#Glucose%20sensing%20module)

    To optimize the design of our glucose sensing module, four promoters were designed with different number of CHREBP binding motif (Figure 1a). To characterize the CHREBP activation, Glucose sensing promoters (GSPs) were cloned into pGL3 vector to control the expression of firefly luciferase. Corresponding plasmids were co-transfected into HepG2 cell line with SV40-rluc internal control plasmid. As is shown in Figure 3b, comparing to mini-Promoter, 3x GSP, 6x GSP and 9x GSP all showed significant increase of luciferase signal. The transcriptional strength showed a significant or margnially significant improvement of 9×GSP comparing to that of 3×GSP or 6×GSP counterparts. To validate the glucose responsiveness, we challenged the 9x GSP-luciferase carrying cells by culturing cells in 5mM or 20mM glucose after overnight starvation. Results showed a ~1.5-fold increase of luciferase signal in 20 mM treatment group comparing to the 5 mM group.

    To further characterize the glucose sensing module, we generated a 9x GSP-GFP reporter plasmid to increase the detection throughput. By using CMV-mcherry to normalize the effect of glucose on general exogenous gene expression level, we demonstrated that the 9x GSP is capable of activating gene expression in a glucose dose dependent manner (Figure 3d).

![](https://static.igem.org/mediawiki/2019/e/e5/T--NUDT_CHINA--2019_Results_Fig3.png)

**Figure 3. Glucose sensing by CHREBP binding hybrid promoter.** (a) Schematic representation of the promoter design; (b). Transcriptional strength analysis of different GSPs; (c) Glucose response of 9x GSP-luc cassette; (d) Glucose response profiling of 9x GSP-GFP cassette. Fluorescent intensity of GFP or RFP was calculated by imaging gray scale analysis.  Relative luminance was calculated by normalizing to Renilla luciferase signal, relative Fluorescent Intensity was calculated normalizing RFP signal. To calculate fold changes, levels of each control groups (minip or low glucose groups) were arbitrarily set to 1.0. Error bar represents SD of at least 3 biological replicates, \* *p*<0.05, \*\* *p*<0.01.

# Integrated Analysis

[(See the design of this module here)](https://2019.igem.org/Team%3ANUDT_CHINA/Model)

    To further understand how our two modules may work together and to validate the *in vivo* performance of our circuit, we developed a set of mathematical models on molecular, cellular and whole-body level. To be specific, a cellular glucose sensing model, a protein degradation model, and a whole-body glucose-insulin-glucagon model were built based on fundamental biochemical and physiological principles (Figure 4a). By integrating three models, we obtained glycemia simulation results under different conditions. Healthy object showed stabled glycemia level at around 96 mg/dL. By decreasing insulin sensitivity by 50%, object showed significant hyperglycemia at around 133 mg/dL. When GCGR-Predator is introduced into the model with a strong constitutive promoter, object showed mild hypoglycemia (~53 mg/mL), which is consistent with the suggestions provided by HP expert interview as well as previous literatures1-3. However, when GCGR Predator system is controlled by glucose sensing device, which provides feed-back control over the degradation of GCGR, simulation showed that the glycemia level would be stabled at around 86.7 mg/dL, indicating that the close-loop controlling system may provide a more robust control over the glycemia level (Figure 4b).

![](https://static.igem.org/mediawiki/2019/3/36/T--NUDT_CHINA--2019_Results_Fig4.png)

**Figure 4. Glucose sensor-GCGR Predator chimera restores glucose homeostasis of T2D patients *in silico*.** (a). Schematic representation of the modeling structure; (b). Glycemia simulation under different conditions.

# Future Plan

* Further characterize and optimize glucose sensing devices;
* Combine the glucose sensing module with the GCGR predator module in wet lab, test the combined system on HepG2 and mouse primary hepatocytes;
* We’re currently applying for animal use and ethical consent from IACUC for animal usage and *in vivo* experiments.

# Reference

1 Christine, L. *et al.* Liver-specific disruption of the murine glucagon receptor produces α-cell hyperplasia: evidence for a circulating α-cell growth factor. *Diabetes* **62**, 1196-1205 (2013).

2 Lok, S., . *et al.* The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization. *Gene* **140**, 203-209 (1994).

3 Rivero-Gutierrez, B. *et al.* Deletion of the glucagon receptor gene before and after experimental diabetes reveals differential protection from hyperglycemia. *Molecular metabolism* **17**, 28-38, doi:10.1016/j.molmet.2018.07.012 (2018).