iGEM 2022 — Team jiangsu-united

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# iGEM Notes

## 7/9

* Ice-breaking and self-introductions.
* Designing the team name, team logo, and the products we are ordering for charity selling in the near
  future.

## 7/10

![](https://static.igem.wiki/teams/4516/wiki/notebook/t-jiangsu-united-notebook-01.png)

### Wet group:

* Lab safety training.
* FoxO1 gene PCR (failed, insufficient yield).

### Dry group:

* Finishing the buying of all products on online suppliers with low bulk buying prices.
* Brainstorming the questions that can be included, potentially, in the online questionnaire.
* Starting the writing of online articles by first assigning clearly the labor division.
* Designing the offline interview question booklet.

## 7/11

![](https://static.igem.wiki/teams/4516/wiki/notebook/t-jiangsu-united-notebook-02.png)

### Wet group:

* Cultivate HepG2 cells in MEM-FBS liquid culture.
* CR to amplify PGL3 -3IRF, SV40, and FoxO1 genes.
* Electrophoresis & validate the correct fragments (1988 bps).
* Exaction of the hFoxO1 DNA fragment from the agarose gel.

### Dry group:

* Offline interview
* Learning and starting the preparation of Education and Communication

## 7/12

![](https://static.igem.wiki/teams/4516/wiki/notebook/t-jiangsu-united-notebook-03.png)

### Wet group:

* Transfer HepG2 cells into new MEM-FBS culture.
* Take a sample (20ul) to count the density of cell culture.

### Dry group:

* Business Plan Workshop and brainstorming every part of the business plan.
* Distributing the work to three dry groups.
* Finishing the interview conclusion writing.

## 7/13

![](https://static.igem.wiki/teams/4516/wiki/notebook/t-jiangsu-united-notebook-04.png)

### Wet group:

* Repeat FoxO1 DNA PCR.
* Plasmid transfection using LipoFiter 3.0 kit.
* Set up 48 well plates, 12 positive control & 12 experimental groups.
* Add AS1842856 & compound 355 with varying dosages.

### Dry group:

* Writing BP first draft and finishing it at midnight.
* Interview with Mr. Lu Jian, the first expert we reached out to.
* Learning Collaboration and preparation for the collaboration with ATCG Beijing Group.

## 7/14

### Wet group:

* Test protein activity using Dual-Luciferase Reporter Assay System.
* Isolate cells lysate using lysis buffer (both AS1842856 & compound 355).
* Verify the expression of hFoxO1 by Western blot.
* Add Firefly & Renilla luciferase to cell lysate solution.
* Test luciferase activity in a microplate reader.

### Dry group:

* Refining the first business plan draft.
* Collaboration with ATCG group.
* Writing conclusion for the ATCG collaboration.
* Interview with Mr. Lu Chen, the second expert we reached out to. Discussion with Mr. Zhang, the business
  angel we reached out to due to his unique perspective as a business investor.

## 7/15

### Wet group:

* Data collection & analysis.
* Verification of the construction of plasmids.
* Prep glucose & protein activity test groups: set up 18 well plate
  + 6 positive control & 6 experimental groups, each adding 1ul of glucose solution
  + Add varying dosages of AS1842856 (positive control) & compound 355 (experimental group)

### Dry group:

* Education sharing conference
* Collaboration with the Shanghai SHSID team.
* Writing conclusion for this time collaboration.
* Interview with Mr. Ye Mao, our third expert interviewee.

## 7/16

### Wet group:

* Test glucose & protein activity of both positive control groups and experimental groups.
  + Add glucose & protein mixture.
  + Detect the intensity of luciferase using a microplate reader.
* Data collection & analysis.
* Presentation preparing.

### Dry group:

* Offline Fundraising.
* Refining the second edition of our business plan.

## 7/18

* Learning about wiki writing and preparing for gathering the materials.
* We are going to use the wiki page for manifestation.
* Making promotion video
* Learning about public speaking skills.
* Finishing the shooting of our formal pictures.

## 7/19

* Writing Wiki and gathering the materials.
* Making the promotion video
* Finishing the last conclusions of the items we done during the days.

## 7/20

* Mock Thesis examination.

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# Engineering

## Introduction

Type 2 diabetes accounts for 90% of all kinds of diabetes caused by a declining response to insulin, the
victims suffer from huge side effects. And the number of cases is increasing rapidly. Moreover, it is
gradually spreading among younger crowds. Therefore, we find it helpful to many people to design a drug used
to prevent symptoms of type 2 diabetes.

Forkhead box protein (Fox) is a family of transcription factors. The DNA binding region of this family of
proteins has a conserved winglike helix structure. There are currently 17 subfamilies in this family, of which
the FoxO subfamily is the most well studied. There are four subtypes: FoxO1, FoxO3, FoxO4, and FoxO6 in
mammals. FoxO1 has four domains, which are DNA-binding domain, nuclear localization domain, nuclear export
sequence, and transcriptional activation domain. It binds with IRE sequence and plays a role in regulating
downstream genes. FoxO1 is mainly expressed in insulin-responsive tissues, The main role of FoxO1 is to
regulate downstream target genes, such as PEPCK, G6Pase, PGC1-α, and PDK-4 to promote gluconeogenesis and can
regulate cell proliferation, gluconeogenesis, and energy metabolism. Thus, we intend to reduce the expression
of the two enzymes by regulating the expression of FoxO1.

## How we design our plasmid

In order to construct a FoxO1 expression plasmid that can duplicate both in *E.coli* and HepG2 cells, we
designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector
(Fig.1), and transfect the HepG2 cells with the recombinant plasmid and set up our experiment platform.

![](https://static.igem.wiki/teams/4516/wiki/engineering/t-jiangsu-united-engineering-01.jpg)
Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1

## How we build our plasmid

To build the plasmid, we use PCR to amplify the hFoxO1 gene from template DNA (HepG2 cell genome), and
extract the target fragment (Fig.2). At the same time, we did the plasmid extraction to obtain the plasmid
pcDNA3.1. The second step was double-enzyme digestion with XhoI and KpnI. The goal of digestion was to get the
linearized pcDNA3.1 vector and inserted DNA fragments of hFoxO1. The third step was to ligate the inserts and
linearized vector and transfer the ligation product into *DH5α* competent.

![](https://static.igem.wiki/teams/4516/wiki/engineering/t-jiangsu-united-engineering-02.png)

Fig.2 Agarose gel electrophoresis of PCR product.
(A)Lane 1 is the hFoxO1 target band.

We send the constructed recombinant plasmid to a sequencing company for sanger sequencing. The returned
sequencing comparison results showed that the plasmid was successfully constructed (Fig.3). Then we extract
the recombinant plasmid from *E.coli* *DH5α* and transfect it into HepG2 cells to express hFoxO1
proteins.

![](https://static.igem.wiki/teams/4516/wiki/engineering/t-jiangsu-united-engineering-03.jpg)

Fig.3 Agarose gel electrophoresis diagram of the clone.

(A) Verify the colony in lanes 1-6
(B) Sequence comparison results of successful gene editing

## How we test our hFoxO1

### a) Protein expression and verification

In order to verify if hFoxO1 protein was successfully expressed in HepG2 cells which were transfected with
the correct recombinant plasmid after 12 hours, we did Western blot (Fig.4).
The result shows that the protein expression level of hFoxO1 increased accompanied by the increase of
plasmid concentration, indicating that hFoxO1 was expressed successfully in the cell.

![](https://static.igem.wiki/teams/4516/wiki/engineering/t-jiangsu-united-engineering-04.png)

Fig.4 Protein determination of HepG2 cells after transfection.

(A) GAPDH was used as control; The numbers on the X-axis represent the different concentrations of
hFoxO1 plasmids.

### b) Functional test through luciferase activity detection

To confirm the activity of hFoxO1 protein to activate downstream gene expression, we chose two kinds of
luciferase for detection. What’s more, we also chose AS1842856, which is a small component that can inhibit
the activity of hFoxO1, as control group.

![](https://static.igem.wiki/teams/4516/wiki/engineering/t-jiangsu-united-engineering-05.jpg)
Fig.5 hFoxO1 activity verification through luciferase activity detection

As shown in Fig.5, after the addition of different concentrations of positive control (AS1842856), we could
see a gradient of luciferin activity and concentration dependence, proving that positive control has an
inhibitory effect on hFoxO1 transcriptional activation. This means the hFoxO1 transcription activation
platform constructed by us is successful and can be used for follow-up experiments.

## How we learn from our project

We have already collected the data and figures from our experiments. hFoxO1 plays a role in regulating
downstream genes, especially in gluconeogenesis. And we find out that within a certain range, we could see a
gradient of luciferin activity and concentration dependence.

After expressing hFoxO1 protein in HepG2 cells, we can easily detect the downstream gene expression through a
fluorescence reporting system. By the way, when we compared these data with the positive control group, we
find that the activity of the hFoxO1 is easily regulated by special small components.

Because of the great effect of the hFoxO1, we believe that if we can regulate the expression of hFoxO1 in the
future and promote it in the market, it will become a great power to fight against type 2 diabetes, and it may
be applied to clinical disease treatment, improve the quality of life of patients, and even reduce the number
of diabetes patients.

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# Experiments

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# Results

## 1. PCR amplifies DNA fragments & Construction of hFoxO1 expression plasmid

FoxO1 is a member of the Forkhead Box protein family. It has an important role in gluconeogenesis, cell
proliferation, and energy metabolism. To obtain the target hFoxO1 gene, PCR and agarose electrophoresis were
performed.

![](https://static.igem.wiki/teams/4516/wiki/results/t-jiangsu-united-results-01.png)

Fig.1 Agarose gel electrophoresis of PCR product.

(A)Lane 1 is the hFoxO1 target band.

In order to obtain the target fragment, we used PrimeStar high-fidelity polymerase to amplify hFoxO1 DNA
products. Then we obtained the target DNA fragments **(Fig.1)**. Afterward, we cloned the corresponding
plasmids
and used DNA sanger sequencing to verify the construction.

## 2. Identification of plasmid construction

Under the premise of having extracted the target DNA fragment, we selected the appropriate endonuclease and
digested both the DNA fragments and plasmid carrier simultaneously. The correct fragments are recovered by
electrophoresis. In addition, the T4 DNA ligase, viscous terminal carrier, target fragment, and buffer were
all added proportionately for conversion.

![](https://static.igem.wiki/teams/4516/wiki/results/t-jiangsu-united-results-02.png)

Fig.2 Agarose gel electrophoresis diagram of the clone.

(A) Verify the colony in lanes 1-6
(B) Sequence comparison results of successful gene editing

In Fig.2A, we can see that there are obvious bands in the range of 1900-2000bp in 1-6 lines, proving that our
recombinant cloning products were constructed successfully. In Fig.2B, we can see that there is no difference
in the result of template and construction, which represents the success of construction. This meant that we
can carry out subsequent cell transfection and characterization qualitative detection.

## 3. Construction of transcriptional activation platform

In order to construct a transcriptional activation platform of hFoxO1, we transfected HepG2 cells with hFoxO1
plasmids, which were verified by Western Blot.

![](https://static.igem.wiki/teams/4516/wiki/results/t-jiangsu-united-results-03.png)

Fig.3 Protein determination of HepG2 cells after transfection.

(A) GAPDH was used as control; The numbers on
the X-axis represent the different concentrations of hFoxO1 plasmids.

After transfected HepG2 cells with different concentrations of hFoxO1 plasmids, the protein expression level
of hFoxO1 increased accompanied by the increase of plasmid concentration, indicating that hFoxO1 was expressed
successfully in the cell **(Fig.3A)**. By comparing the expression level of internal reference proteins, we
could
obtain hFoxO1 plasmids with the appropriate concentration. We can construct a transcriptional activation
platform and perform tests on it.

## 4. Transcriptional activation platform test

To test if the hFoxO1 transcriptional activation platform we constructed worked well, we tested it with three
plasmids: pcDNA3.1-hFoxO1, pGL3-3IRE, pRL-sv40 **(Fig.4A, 4B, 4C)**, a transcriptional regulation-hFoxO1,
and a
positive control-AS1842856

![](https://static.igem.wiki/teams/4516/wiki/results/t-jiangsu-united-results-04.jpg)

Fig.4 Plasmid profiles and test results

(A) pcDNA3.1-hFoxO1 plasmid
(B) pGL3-3IRE plasmid
(C) pRL-sv40 plasmid
(D) hFoxO1 transcriptional activation platform test results
(E) Effect of positive control (AS1842856) on hFoxO1 transcriptional activation inhibition

In **Fig.4D**, “+” represents the presence of the corresponding plasmid (or transcriptional regulation) in
the sample, and “-” represents the negative control. The results indicated that luciferase activity was only
affected by pcDNA3.1 plasmid and hFoxO1 transcription regulator. **Fig.4D** also showed that hFoxO1 had a
good luciferase activation effect. After the addition of different concentrations of positive control
(AS1842856), we could see a gradient of luciferin activity and a concentration dependence **(Fig.4E)**,
proving that positive control has an inhibitory effect on hFoxO1 transcriptional activation. This means the
hFoxO1 transcription activation platform constructed by us is successful and can be used for follow-up
experiments.

## 5. Representational qualitative result

After the feasibility of the hFoxO1 transcription activation platform was confirmed, the original positive
control (AS1842856) was replaced by different small molecular compounds from the database to examine their
effects on gluconeogenesis (Table1). Compound 335 was found to have a significant role in reducing glucose
concentrations in HepG2 cells **(Fig.5A)**.

Table 1. The effects of different small molecular compounds on gluconeogenesis.

| Compound | Inhibitory rate (%) |
| --- | --- |
| AS1842856 | 50.13 |
| 177 | 49.54 |
| 222 | 63.2 |
| 240 | 38.12 |
| 340 | 31.91 |
| 355 | 97.17 |
| 389 | 45.31 |
| 391 | 40.28 |
| 392 | 77.57 |
| 396 | 50.06 |
| 460 | 25.27 |
| 548 | 57.52 |
| 550 | 41.47 |

In Fig.5A, in the third column of the histogram, the luciferase activity level was still low in the presence
of the hFoxO1 transcriptional activator under the action of compound 355, so it could be concluded that
compound 355 has an obvious inhibitory effect on hFoxO1 transcriptional activation.

![](https://static.igem.wiki/teams/4516/wiki/results/t-jiangsu-united-results-05.jpg)

Fig.5 Plasmid profiles and test results

(A) Compound 355 inhibits hFoxO1 transcription activation
(B) Effect of compound 355 on gluconeogenesis in hepatocyte
(C) Effect of compound 355 on reducing the mRNA level of key gluconeogenesis enzymes G6Pase
(D) Effect of compound 355 on reducing the mRNA level of key gluconeogenesis enzymes PEPCK

Compound 355 was also used to inhibit both glucose concentration in HepG2 cells and mRNA abundance of key
enzymes of gluconeogenesis. In Fig.5B, we can find that with the gradual increase of the concentration of
compound 355, the glucose level showed a counter gradient change and was concentration-dependent, indicating
that compound 355 could significantly inhibit gluconeogenesis in hepatocytes.

G6pase is an enzyme that releases glucose into the blood by hydrolyzing glucose-6-phosphate in liver tissue.
PEPCK is a gluconeogenic enzyme that allows hepatic parenchymal cells to produce glucose from pyruvate derived
from amino acid metabolism. In Fig. 5C and 5D, when the volume of compound 355 increases gradually, the mRNA
levels of G6pase and PEPCK gradually decreased in a counter gradient and were also concentration-dependent,
indicating that compound 355 could significantly reduce the mRNA levels of key enzymes in gluconeogenesis

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