iGEM 2024 — Team csu-china

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## Notebook

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[1.Continuous glucose monitoring system （Hardware）](#continuous)
[2.Insulin secretion system](#insulin)
[3.Luciferase verification system](#ver)

Engineering success

1. Continuous glucose monitoring system
（Hardware）

We designed an instrument to sense glucose concentrations in real time. When the blood glucose concentration
exceeds a certain value, the switch can be triggered to activate the blue light. We have performed real-time
detection of its concentration in glucose solution.

1.1 Design

In our design, the role of the hardware instrument is to control whether the blue light is irradiated or not
based on the change in glucose concentration. From this, we logically came up with three components together:
the Glucose Detection Device, the Monitor, and the Instrument Emitting Blue Light.

1.2 Conduction

To achieve our ideal design above, we learned about the biochemical principle of the glucose concentration test
strip method. Ideally, glucose reacts with glucose oxidase on the test strip. And during the reaction, glucose
is oxidized to gluconic acid, while hydrogen peroxide is generated. The produced hydrogen peroxide reacts with
an indicator on the test strip, resulting in a color change, and the extent of the color change is proportional
to the glucose concentration, which can be determined by comparing it to a standard color chart or using a
measuring instrument. These are the principles that we conduct the detecting system of our hardware.

1.3 Test

To visualize the test results, we utilize a display screen that showcases the glucose concentration. By
comparing the measured concentration with predefined threshold values, our instrument will emit blue light if
the concentration exceeds the threshold, and vice versa.

1.4 Learn

In the future, we plan to anticipate the use of the device in mice for minimally invasive blood glucose
concentration monitoring and the wristband in humans to monitor changes in blood glucose concentrations in real
time to achieve our desired results.

1.4.1 Continuous
glucose monitoring (CGM) for Detecting Blood Glucose

A glucose sensor with a soft needle will be implanted under the skin, and glucose oxidase will be used to
continuously measure the consumption of liquid oxygen or the production of hydroxide ions in the subcutaneous
tissue every 10 seconds, and then convert it into a glucose value. The glucose value will be sent to the main
control module through Bluetooth for data processing, and the blue light device will be switched and controlled
according to the received glucose value and glucose threshold. When the glucose value reaches the threshold, the
blue level will be opened, and vice versa.

![](https://static.igem.wiki/teams/5156/wiki/eng/en1.png)

Fig 1. The glucose sensor

![](https://static.igem.wiki/teams/5156/wiki/eng/en5.png)

Fig 2. The master control module

1.4.2 Near-Infrared
Spectroscopy (NIR) for Detecting Blood Glucose

After the near-infrared light is irradiated into the human body, the near-infrared light will be transmitted
and diffused in the human tissues, and the sensor will be used to receive the spectrum passing through the human
body, and these spectra contain information about the concentration of glucose in the human body.

Based on near-infrared spectroscopy and stoichiometric analysis, we established a mathematical model of the
measured spectral data and the corresponding blood glucose samples of known concentration, and the intensity of
the outgoing light after passing through the human body conformed to Lambertbeer's law:

A=lg(1/T)=Kbc

![](https://static.igem.wiki/teams/5156/wiki/eng/en2.png)

Fig 3. the Near-infrared optical glucose sensor

2.Insulin secretion system

2.1 Method

We transfected the plasmids encoding the complete insulin sequence into HEK 293T cells and nurtured them for a
duration of 36 to 48 hours. Consequently, these modified cells released the protein into the surrounding medium,
known as the supernatant. This represents the tailored system we devised for the secretion of insulin.To prevent
excessively low blood glucose levels resulting from the continuous action of insulin, we linked sponge to the
GIP promoter element to facilitate its expression. The multiple miRNA binding sites contained within sponge bind
to various miRNAs, thereby inhibiting the negative regulatory effect of miRNAs on insulin mRNA.

2.2 Result

miRNA effectively inhibits the expression of insulin, while upon the addition of sponge, the inhibitory effect
of miRNA is significantly suppressed.

![](https://static.igem.wiki/teams/5156/wiki/eng/eng3-1.png)

Fig 4. Regarding some of the experimental results
of our system, more data can be found on the results wiki

3.Luciferase verification system

3.1 Method

To ascertain the expression level of insulin, we employed the luciferase gene, which was potentiated by 5xUAS,
as a substitute for the insulin gene. By quantifying the bioluminescence intensity during the oxidation process
of luciferin, we aimed to mirror the cellular secretion of luciferase, ultimately assessing the capability of
our biological system under investigation to modulate luciferase secretion in response to varying blood glucose
concentrations.

3.2 Result

![](https://static.igem.wiki/teams/5156/wiki/eng/en4-1.png)

Fig 5.  the structure of the
pcDNA3.1-LucER plasmid, more data can be found on the parts wiki

![](https://static.igem.wiki/teams/5156/wiki/eng/eng6.png)

Fig 6.  the structure of the
5XUAS-Luc-miR-BS plasmid, more data can be found on the parts wiki

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[1.Plasmid experiments
1.1 Plasmid extraction](#pla)
[1.2 PCR](#pcr)
[1.3 Enzyme cutting](#enz)
[1.4 Connecting](#con)
[1.5 Transform](#tra)
[1.6 Pick a single coenobium to clone
1.7 Cautions of germ experiments](#pic)
[2.Cell experiments
2.1 Cell revival](#cell)
[2.2 Cell fluid exchange](#fluid)
[2.3 Cell passage](#pass)
[2.4 Cell transfection](#trans)
[2.5 Dual luciferase reporter gene detection](#dual)
[2.6 Cautions of cell experiments](#cautions)

Plasmid experiments

1.1 Plasmid extraction

1.1.1 Preparation before the
experiment

(1)Before use, add all RNaseA to Solution I and save it at 4 degrees.

(2)DNA Wash Buffer, was diluted with anhydrous ethanol as following the table and stored at room temperature.

1.1.2 Cells were cultured

(1)add 500 ul LB medium containing screening antibiotics to volumes of 2ml centrifuge tube , with target
species 2-5ul, 37℃ oscillatory culture for 16 hours.

(2)After 16h, the bacterial fluid was relatively cloudy.If the lysis
can not be performed immediately, it can be temporarily stored at 4 degrees.

1.1.3 Lysed the bacteria

(1)The bodies were collected at 20 ml and collected by centrifugation at 12000r/min at 10min, at room
temperature.

(2)The medium was abandoned.Add 10 ml Solution I/RNaseA mixing liquid to the precipitation and mix with a
pipette gun to completely resuspend the cells.

(3)Add 10 ml Solution II, and the centrifuge tube was gently reversed up and down 8 – 10 times to obtain
clarified lysates.After reversal, the lysate was resting at room temperature for 2 – 3 min.

(4)The lid was covered with 5 ml precold N3 Buffer, and gently reverse up and down 10 times until a white
flocculation precipitate was formed and quietly incubated for 2min at room temperature.

(5)Prepare a syringe filter, pull out the piston, place the syringe vertically on a test tube rack, and place
a 50 ml centrifuge at the lower outlet of the syringe with the syringe opening upward.Immediately pour the
lysate into the syringe of the filter. Cell lysates were maintained in the syringe for 5min.The white
flocculation will float on the surface of the lysate.Cell lysates may have flowed from the filter syringe
mouth.

(6)Cell lysates were collected using a new 50-ml tube.The syringe piston was inserted into the syringe and
slowly pushed to flow the lysate into the 50 ml centrifuge tube.

(7)Add 1 x volume of ETR Solution (blue) to the outgoing filtered lysate and the tube was reversed 10 times
and then resting in 10min in an ice bath.

(8)The above lysate was placed in a water bath at 42℃ for 5min.The lysate will appear cloudy again.At this
time, centrifugation at 25℃, 4,000xg for 5min,ETR Solution would form a blue stratification at the bottom of
the tube.

(9)The supernatant was moved to another new 50 ml tube, adding 5 x the volume of anaqueous ethanol at room
temperature, and the tube was gently reversed 6-7 times for 1-2min. at room temperature.

1.1.4 Activated columns

(1)A HiBind DNAMaxi binding column was embedded into a 50 ml collection tube, and 20 ml superfiltrate was
added to the HiBind DNAMaxi binding column.The cells were centrifuged at 4,000xg for 3min. at room
temperatureAbandon filter.

(2)The HiBind DNAMaxi binding column was embedded into the same collection tube, and step 11 was repeated
until all the remaining overfiltrate was bound to the HiBind DNAMaxi binding column and centrifuged under the
same conditions.

(3)The HiBind DNAMaxi binding column was embedded into the same collection tube, added to 10 ml HBC Buffer to
HiBind DNAMaxi binding column and centrifuged at 4,000xg for 3min, at room temperature.

(4)The HiBind DNAMaxi binding column was embedded into the same collection tube with 15 ml DNA Wash
Buffer(diluted with anhydrous ethanol) to the HiBind DNAMaxi binding column and the filtrate was discarded by
centrifugation at 4,000xg for 3min, at room temperature.

(5)The HiBind DNAMaxi binding column was embedded into the same collection tube with 10 ml DNA Wash
Buffer(diluted with anhydrous ethanol) to the HiBind DNAMaxi binding column and the filtrate was discarded by
centrifugation at 4,000xg for 3min, at room temperature.

(6)6000xg was emptied to dry the substrate of the HiBind DNAMaxi binding column for 10min.

(7)The HiBind DNAMaxi binding column was placed on a clean 50 ml centrifuge tube, adding 1 – 3 ml Endo-Free
Elution Buffer to the HiBind DNAMaxi binding column matrix (added amount depending on the expected final
product concentration) and resting for 5min at room temperature.

(8)It was centrifuged at 14,000xg for 5min to elute the DNA.

(9)The columns were discarded and the DNA product was stored at-20℃.

1.2 PCR

1.2.1 PCR system
experiment

![](https://static.igem.wiki/teams/5156/wiki/ex/t1.png)

1.2.2 PCR process
experiment

![](https://static.igem.wiki/teams/5156/wiki/ex/t2.png)

1.3 Enzyme cutting

Enzyme cutting system

(1)Establish the following reactions on the ice. (Note: Add the enzyme to the reaction at last)

![](https://static.igem.wiki/teams/5156/wiki/ex/t3.png)

(2) Slowly blow and mix the reaction liquid and conducted
instant centrifuge.

(3) Thermal insulation below 37°C for 5-15 minutes.

(4) Thermal inactivated in 20 minutes at 800 °C.

1.4 Connecting

According to the electrophoresis diagram, the appropriate target fragment is mixed with the carrier (1:1-8:1
ratio of molecular weight) and connected by Infusion method. Connected at 37°C for 2 hours. If T4 joinase is
used, stay overnight at 4°C.

1.5 Transform

(1) -80°CDH5α receptor cells melt on ice.

(2) 10μl bacterial liquid +0.5μl plasmid.

(3) Mix well and take an ice bath for 30 minutes.

(4) 42°C Heat for 90s.

(5) Ice bath for 5 minutes.

(6) Add 500μl nonresistant LB and put it into a 37°C rocker
for 40 minutes.

(7) Centrifuge under 8000rpm for 5 minute.

(8) Inhale the upper clearance and leave 40μl heavy suspension.

(9) Coating board.

1.6 Pick a single coenobium
to clone

Select a single colony, lift it with the tip of a pipette, and mark it on a new LB plate.

1.7 Cautions of germ experiments

(1)In step 6 of lysis of bacteria, inverted the centrifuge tube
on clean absorbent paper allowed the removal of the media
more thoroughly.

(2)In step 7 of the lysis of bacteria, to avoid intense mixing and lysates, the resting time should not exceed
5min, or otherwise break chromosome DNA and reduce the resulting plasmid purity.Exlonged resting time may lead
to breakage of plasmid DNA.(When using SolutionII, cover the cap).

(3)The solution must be thoroughly mixed.If the mixture remains viscous and brown spherical, mixing needs to
continue until full solution neutralization, which is essential to obtain a high yield.

(4)At steps 9 and 10 of the lysis of bacteria, a small fraction of the lysates may remain in the flocculation
precipitation, do not reluctantly push the residual lysates into the filter, otherwise a small portion of the
precipitation will flow into the collection tube.If any precipitation flowed into the overfiltrate, the
precipitation was removed by centrifugation.

(5)Upon addition of ETR Solution, the lysates may appear turbidity, but will gradually turn to clarification
after an ice bath.

(6) Concentrated DNAWashBuffer must be diluted in ethanol according to the instructions prior to use.If the
DNA washing
buffer is placed in the refrigerator before use, it must be taken
out at room temperature.

(7) Enzymatic cutting can be done overnight.

Cell experiments

2.1 Cell revival

1. Remove the bacterial strain from the -80 ℃ refrigerator and immediately shake it in a 37 ℃ water bath
until the liquid begins to melt and flow.

2. Prepare 1ml of DMEM culture medium and add it to a clean centrifuge tube. Also, add about 1ml of bacterial
solution and dilute the DMSO cryopreservation solution.

3. Centrifuge (5 minutes, 800rpm), discard the supernatant, and resuspend the cells in 1ml of culture medium.

4. Take a culture dish (10cm) and add an appropriate amount of culture medium (about 6ml). Add the resuspended
cells to it for propagation, and pass them once before use.

2.2 Cell fluid exchange

1. Discard the old culture medium and add 1-2ml PBS to wash the cells. (optional).

After discarding PBS, add 6ml of new culture medium to the culture medium.

2.3 Cell passage

(Observing cell growth status and density under a microscope, generally 80%
-90% passage is preferred)

1. Discard the old culture medium, add 1-2ml trypsin (cover the cells), and digest for 1 minute.

2. Add twice the volume of trypsin to the culture medium to terminate digestion, carefully blow it into a cell
suspension, and transfer it into a centrifuge tube.

3. Centrifuge (800rpm, 5min)
Add new culture medium to the new culture dish while centrifuging
Large dish (10cm) 5-6ml
Medium dish (65mm) 3-4ml
Small dish (35mm) 1-2ml
The thickness of the culture medium should be 2-3mm

4. Discard the supernatant, add 1ml of culture medium, and gently blow the cells with a pipette to resuspend
them.

5. Load the cell suspension into 2-3 new culture dishes according to the culture plan and continue culturing
in the incubator.
(Generally, the same size of culture dish is passed from one to three, or the large dish is 400ul, the medium
dish is 200ul, and the small dish is 100ul)

2.4 Cell transfection

1. Lay the plate one day in advance, and when the cell density reaches 70% -80% under the microscope,
transfection
can be performed.

2. Dilute 5ng of plasmid with Opti-mem200 μ l and 15 μ l of lipoMax separately, mix well and leave at room
temperature for 5 minutes.

3.After 3.5 minutes, mix the diluted plasmid with lipoMax and leave it at room temperature for 15-20 minutes
to form a complex.

4. Drop the mixture into the cell culture dish and gently shake the dish to mix well.

5. Observe the transfection efficiency after culturing in the incubator for 36-48 hours.

2.5 Dual luciferase reporter gene detection

When using it for the first time, prepare LAR II, which is the substrate of luciferase. Dissolve LAR II in
LAR II buffer and store separately at -80 ℃ in the dark.

(1) Take a 24 well plate for cell microscopy and remove the cell chamber for subsequent operations;

(2) Take 100 µ l PBS and carefully add it to each well for cleaning. Vacuum pump suction, then add 100 µ l PBS
along the hole wall;

(3) Add 100 µ l of luciferase reaction solution to each well, mix well with a shaking plate, blow and digest
the cells, transfer to an empty EP tube and let it stand for 7-8 minutes. Measure the fluorescence value of
luciferase on a Sirius instrument and try to complete the detection within 30 minutes;

(4) After measuring Luc, add 100 'µ l of Renilla reaction solution to each tube (1ml of Renilla detection
solution mixed with 10ul of substrate, freshly prepared), mix well, let it stand for 7-8 minutes, and detect
the fluorescence value of Renilla. The detection should be completed within 30 minutes as much as possible.
After completion, export the data using a USB flash drive and close the software and instrument.

2.6 Cautions of cell experiments

1.When the cell adhesion is not firm, try to digest at room temperature for 10-30 seconds, or reduce the
concentration of trypsin to 0.05% for digestion.

2. Do not blow or beat cells when washing or digesting them with PBS or trypsin.

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[General information](#general-information)
[The AND circuit](#the-and-circuit)
[The NOT circuit](#the-not-circuit)
[Conclusion](#conclusion)

Results

General
information

The aim of our project is to construct a designer cell that expresses insulin in response to high
glucose level. The expression of insulin is controlled by an AND circuit, a glucose-dependent promoter
and a glucose-dependent switch for blue light. In addition, we also designed a NOT circuit to prevent
the excessive insulin expression and subsequent hypoglycemia when glucose levels drop. The NOT
circuit includes a miRNA-binding element at the 3’-UTR of insulin coding region, miRNA and sponge. To
easily monitor the precise expression of insulin, we used luciferase reporter in the experiments. The
expression plasmids for insulin and luciferase were shown Figure 1.

![](https://static.igem.wiki/teams/5156/wiki/results/r1-2.png)

![](https://static.igem.wiki/teams/5156/wiki/model/sugar0.png)

Figure 1. The expression plasmids for insulin
(A) and luciferase (B)

The AND
circuit

Glucose activated the GIP promoter in a dose-dependent manner

In this project, we chose GIP promoters to control the expression of galactose-responsive transcription
factor GAL4 [Saccharomyces cerevisiae S288C], VP16 [Human alphaherpesvirus 2], and sponge, as it was
reported that GIP promoter can be activated by glucose.

To confirm that the GIP promoter can be activated by glucose, we constructed a plasmid (2401), in which
the expression of luciferase is under the control of GIP promoter. We then transfected  2401
pGIP-luciferase plasmid into 293T cells cultured under different glucose concentration (0, 1, 5, and 25
mM), and the enzymatic activity of luciferase was measured. As shown in Figure 2, the luciferase
activity increased in response to higher glucose levels.

![](https://static.igem.wiki/teams/5156/wiki/results/r2-1.png)

![](https://static.igem.wiki/teams/5156/wiki/improvement/doc.png)

Figure 2. Glucose activated the GIP promoter
in a dose-dependent manner

Activation of 5XUAS promoter by the GAL4-VP16 transcription factor

As shown in Figure 1, the expression of insulin/luciferase was controlled by a 5XUAS promoter, which can
be activated by GAL4-VP16 transcription factor. To confirm the activation of 5XUAS promoter by GAL-VP16,
we transfected 5XUAS-Luciferase (2409) in the absence or presence of plasmid expressing GAL4-VP16 (2404)
into 293T cells, and the enzymatic activity of luciferase was measured. As shown in Figure 3, there is
about 2000 fold increase of luciferase activity after expression of GAL4-VP16. Therefore, our data
verified the activation of 5XUAS promoter by the GAL4-VP16 transcription factor.

![](https://static.igem.wiki/teams/5156/wiki/results/r4.png)

![](https://static.igem.wiki/teams/5156/wiki/description/doc2.png)

Figure 3. Activation of 5XUAS promoter by the
GAL4-VP16 transcription factor

Blue light stimulates the formation of an active transcription factor from GI-Gal4 and LOV-VP16

In our design, the DNA-binding domain (Gal4) and transactivation domain (VP16) of transcription factor
Gal4-VP16 were separated and fused to GI and LOV, respectively. It is proposed that the GI-Gal4 (2406)
and LOV-VP16 (2407) will form an active transcription factor in the presence of blue light, to activate
the expression of proteins under the control of 5XUAS. To test this, we co-transfected 5XUAS-Luciferase
and plasmids expressing GI-Gal4 and LOV-VP16 into 293T cells, and cells were cultured in the absence or
presence of blue light. As shown in Figure 4, there is about 300-fold increase in luciferase activity
when the cells were exposed to blue light for 15 minutes. Our data thus verified that blue light can
stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16.

![](https://static.igem.wiki/teams/5156/wiki/results/r9.png)

![](https://static.igem.wiki/teams/5156/wiki/description/sugar4.png)

Figure 4. Blue light can stimulate the
formation of an active transcription factor from GI-Gal4 and LOV-VP16

![](https://static.igem.wiki/teams/5156/wiki/results/ball1.png)
![](https://static.igem.wiki/teams/5156/wiki/results/ball2.png)

A blue light switch under the control of glucose concentration

As GIP promoter responses to an wide range of glucose levels, we sought to design a switch in response
to a specific glucose concentration. To this end, we have collaborated with Luzai Technology Company to
construct a blue light switch under the control of glucose concentration. As shown in Figure 5, a
digital glucometer is modified to a switch, when the glucose concentration reached certain level (in
this case, it is 10mmol/L), the switch was turned on and the blue light was subsequently turned on,
which stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16 (Figure 5).

![](https://static.igem.wiki/teams/5156/wiki/results/r5.png)

![](https://static.igem.wiki/teams/5156/wiki/description/doc1.png)

Figure5. Blue light strument under the control
of glucose concentration

The small box below the picture is a glucose detection instrument. When a test strip is inserted and
immersed in a glucose solution, it can detect the concentration of the glucose solution and display it
on the monitor screen on the left side of the picture. On the right side of the picture is a blue light
instrument that can control the blue light switch based on glucose concentration.

The NOT circuit

Inhibition of luciferase expression by miRNA

In the above AND circuit, we demonstrated the feasibility of insulin expression when glucose
concentration increases. However, using the previous circuit alone may lead to excessive insulin
expression and the risk of hypoglycemia, as it takes for hours for Gal4-VP16 to degrade. To solve this
problem, we designed a miRNA system to inhibit the expression of insulin/luciferase. In this system, the
miRNA-binding site was placed within the 3’-UTR of insulin/luciferase. As shown in Figure 6,
co-transfection of a miRNA-expressing plasmid significantly inhibited the expression of luciferase
induced by Gal4-VP16.

![](https://static.igem.wiki/teams/5156/wiki/results/r6.png)

![](https://static.igem.wiki/teams/5156/wiki/model/sugar0.png)

Figure6. Inhibition of luciferase expression
by miRNA

The disinhibition
effect of sponge

In order to prevent miRNA from exerting inhibitory effects on insulin/luciferase expression, we
introduced sponge, which can bind miRNA and disinhibit the miRNA effect. As shown in Figure 7,
co-transfection of sponge attenuated the inhibitory of miRNA on the expression of luciferase.

![](https://static.igem.wiki/teams/5156/wiki/results/r10.png)

![](https://static.igem.wiki/teams/5156/wiki/description/sugar4.png)

Figure 7. The disinhibition effect of sponge

The sponge exerts its function in a glucose-dependent manner

Ideally, the sponge functions when the glucose level is high and is dysfunctional when the glucose level
drops. To this end, we placed the sponge under the control of GIP promoter. As shown in Figure 8, the
disinhibitory effects of the sponge is significantly higher in 25 mM glucose than 1mM glucose.Therefore,
The sponge RNA exerts its function in a glucose-dependent manner.

![](https://static.igem.wiki/teams/5156/wiki/results/r8.png)

![](https://static.igem.wiki/teams/5156/wiki/description/doc2.png)

Figure 8. The sponge exerts its function
in a glucose-dependent manner

Conclusion

In summary, we have validated the AND circuit and NOT circuit in 293T cells. Both circuits function as
expected. We are thus able to synthesize insulin when the glucose level reaches a specific level by
using blue light together with a GIP promoter. We are also able to stop insulin expression when glucose
level drops by using a miRNA-sponge system.

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