iGEM 2023 — Team shanghai-hs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4874/wiki/notebook.png)

**1 Calendar**

**Date:**
4
/
8
/2023

**Event:** Members training (theory)(online)

**Agenda:**The team leader introduced the iGEM competition content and challenge
background to the members.

**Date:**
4
/1
5
/2023
-5/27/2023

**Event:**Members training (theory) (online)

**Agenda:**The instructor introduced the basic research methods of modern
molecular biology to the members.

**Date:6/9/2023****-****6/****1****0/2023**

**Event:** Project
Topic
Brain storm (online)

**Agenda:**
We decided to focus on the diabetes issue.

**Date:** 6/17/2023

**Event:** Project Design Brain storm（online）

**Agenda:** We had a online discussion meeting with teachers
who brought us many practical inspirations.

**Date:** 6/24/2023

**Event:** Project Design Brain storm（online）

**Agenda:** We confirmed the preliminary project design plan.

**Date:** 7/6/2023

**Event:** Project Launch Meeting（online）

**Agenda:**
We confirmed the final project design plan.

**Date:**
7
/
1
1/2023

**Event:** Meeting

**Agenda:** For the first face-to-face meeting, an ice breaking event was
held and a candidate for the leader was selected
.
All members designed the team logo, uniform, and name
together

**Date:** 7/
1
2/2023

**Event:** Members training (practical)

**Agenda:** The teacher conducted laboratory safety training for the
members and led the team to
drafted
a detailed
experiment plan.

**Date:**
7
/
13
/2023

**Event:** Construction of plasmids

**Agenda:** Construction of plastics (TRP promoter UVR8N397 BIC) by enzyme
digestion pBridge CRYN489 Nde1, Bstb1. Amplification of BIC fragments using PCR technology

**Date:**
7
/
14
/2023

**Event:** Construction of plasmids

**Agenda:**Agrose gel electrophoresis(BIC) and DNA gel extraction

**Date:**
7
/
15
/2023

**Event:** Construction of plasmids

**Agenda:** Plasmid extraction (pBridge-BD-CRY2)and Homologous recombination
(exogenous genes)(TRP1, UVR8, BIC2)

**Date:**
7
/
16
/2023

**Event:** Construction of plasmids

**Agenda:** Plasmid extraction (pBridge-BD-CRY2)and Homologous recombination
(exogenous genes)(TRP1, UVR8, BIC2)

**Date:**
7
/
17
/2023

**Event:** Construction of plasmids

**Agenda:** Homologous recombination (exogenous genes)(TRP1, UVR8, BIC2)And
the screening of resistant tablets(Amp)

Date:
7
/
18
/2023

Event: Construction of plasmids

Agenda: Plasmid extraction (pBridge-BD-CRY2)and colony PCR

**Date:**7/
19
/2023

**Event:** Construction of plasmids

**Agenda:** Plasmid extraction (pBridge-BD-CRY2)and Restriction enzyme single
digestion(Nde1)

**Date:**7/2
0
/2023

**Event:** Construction of plasmids

**Agenda:** Restriction enzyme digestion(Nde1 Restriction enzyme single
digestion）and pBridge-BIC2

**Date:**7/
21
/2023

**Event:** Construction of plasmids

**Agenda:** Agrose gel electrophoresis and DNA gel extraction

**Date:**7/
22
/2023

**Event:** Construction of plasmids

**Agenda:** Homologous recombination (exogenous genes)(TRP1, UVR8,
BIC2)

**Date:**7/
23
/2023

**Event:**Members training (practical)

**Agenda:**Taking the CRY2 gene of Arabidopsis as an example, I learned how to
design gene primers using NCBI website and snapgene

**Date:**7/
24
/2023

**Event:** Construction of plasmids

**Agenda:**Detection of plasmid concentration and  Restriction enzyme double
digestion(Nde1 and Bstb1)

**Date:**7/
25
/2023

**Event:** Construction of plasmids

**Agenda:** Agrose gel electrophoresis and DNA gel extraction

**Date:**7/
26
/2023

**Event:** Construction of plasmids

**Agenda:** Shake the bacteria and prepare solid Lysogeny broth

**Date:**7/
27
/2023

**Event:** Construction of plasmids

**Agenda:** Homologous recombination (exogenous genes)(TRP1, UVR8,
BIC2)

**Date:**7/
28
/2023

**Event:** Construction of plasmids

**Agenda:** pBridge AD-CIB1

**Date:**7/
3
1/2023

**Event:** Construction of plasmids

**Agenda:** Prep solid SD(-Trp/-Leu)

**Date:**
8
/
1
/2023

**Event:** Construction of plasmids

**Agenda:**Plasmid extraction (pBridge-BD-CRY2)and Restriction enzyme single
digestion(Nde1)

**Date:**
8
/
2
/2023

**Event:** Construction of plasmids

**Agenda:** S.cerevisiae transformation (PEG/LiAc), pBridge-AD+BD-CRY

**Date:**
8
/
3
/2023

**Event:** Construction of plasmids

**Agenda:** pBridge-AD-CIB+BD-CRY and colony PCR

**Date:**
8
/
4
/2023

**Event:** Construction of plasmids

**Agenda:** Agrose gel electrophoresis and DNA gel extraction

**Date:**
8
/
5
/2023

**Event:** Construction of plasmids

**Agenda:** Plasmid extraction (pBridge-BD-CRY2) and S.cerevisiae
transformation(PEG/LiAc）

**Date:** 8/
6
/2023

**Event:** Meeting

**Agenda:** Teacher leads team members to review recent experimental
results

**Date:**
8
/
7
/2023

**Event:** Meeting

**Agenda:**
Initial Wiki writing task distribution

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering.png)

# 1.  Overview

Light controlled switches regulate gene expression, which is an important tool in synthetic biology, targeted therapy of diseases and other fields. Based on the discipline of optogenetics, light-sensitive cellular proteins can be engineered to act as switches, thus promoting the efficiency of "insulin switch".

Cryptochrome 2, a blue light receptor found in Arabidopsis, is a blue/ultraviolet light receptor that can bind to CIB1 to regulate downstream signaling (primarily the expression of genes). Another protein, BIC (blue light inhibitors of cryptochrome), can bind to CRY2 to inhibit its light activation, but the inhibition speed has proven to be rather low. By fusing the UV receptor UVR8 and BIC for expression, a much more rapid "off" switch can be realized under UV light conditions.

The experiment flow chart can be shown as follow (Fig.1).

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-1.png)

2.
**Cycle1
pBridge-BD-CRY2(UVR8-BIC2)****（****Part
：
BBa\_K4874004****）****Plasmid Construction**

**2.1 Design**

To construct the plasmid pBridge-BD-CRY2(UVR8-BIC2), we selected TRP as the promoter, PGK1 as the terminator of UVR8-BIC2; ADH1 as the promoter, and ADH1 as the terminator of CRY2 within the plasmids. Key genes of the plasmids include UVR8N397 and BIC2 genes. The TRP, PGK1 and UVR8N397 DNA fragments were amplified by PCR. Then we performed homologous recombination to link the 3 DNA fragments to the linear pBridge-BD-CRY2 vector digested by double enzyme.

A schematic diagram of the plasmid construction can be shown as follow (Fig.2).

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-2.png)

**2.2 Build**

**2.2.1 Amplification of the three DNA fragments and
double enzyme digestion of plasmid**

**TRP1 promoter PCR amplification 134bp**

TRP-Forward Primers: CATCCATACAATGGGCCATATGAATTCGGTCGAAAAAAG

TRP-Reverse Primers: CCTCCGCCATACTCCAAGCTGCCTTTGTG

TRP1 Template: pBridge plasmid

**UVR8N397 PCR amplification 1213bp**

UVR8-Forward Primers: CAGCTTGGAGTATGGCGGAGGATATGGCTG

UVR8-Reverse Primers: GTGTTCTTCATCCCTGAAGATGGATCGATA

UVR8 Template: UVR8 plasmid template

**BIC2 PCR amplification 384bp**

BIC-Forward Primers: CATCTTCAGGGATGAAGAACACCAATTTGCC

BIC-Reverse Primers: GGATAGCTAGAAGCCATATGTCAACAAGAACTCTCAAC

BIC2 Template: Arabidopsis genome DNA

**Restriction endonuclease single
digestion**

pBridge-BD-CRYN489——Nde1

The DNA fragments of the TRP promoter, UVR8N397, and BIC were
successfully amplified. These can all be seen from the gel map below (Fig.3.2).

The electrophoretic strip of the TRP promoter fell between 100 bp to
250 bp, which matched the designed length of 134 bp (Fig.3.2).

The electrophoretic strip of the UVR8N397 fell between 1000 bp to 2000
bp, which matched the designed length of 1213 bp (Fig.3.2).

The electrophoretic strip of the BIC fell between 250 bp to 750 bp,
which matched the designed length of 384 bp (Fig.3.2).

**Nde1 enzyme cut results**

The electrophoretic strip of pBridge-BD-CRY2 (Nde1 single digestion) falls above or around 7500
bp, which matches the designed length of 8361 bp (Fig.3.2).

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-3-1.png) |

**Figure 3.1 Nde1 digestion**

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-3-2.png) |

**Figure 3.2 DNA fragment amplification**

**Homologous recombination 1731bp**

Linearized vector, TRP1 product, UVR8 product, BIC2 product

**2.2.2 Transformation**

DH5a Competent Cell

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-4.png)

**Figure 4. Result of transformation with the linearized vector （Nde I digested） and the three DNA fragments.**

**2.2.3 Determination by Colony PCR and
sequencing**

The length after colony PCR should be the sum of TRP UVR8 and BIC2, which equals 1731 bp. On plate 1, electrophoretic strips 2,3, and 4 are successful and on plate 2, electrophoretic strips 1,3, and 5 are successful. (Fig.5) We use length to determine if three fragments connect. And the bright band is a primer dimer.

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-5.png)

**Figure 5. Colony PCR results.**

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-6.png)

**Figure 6. The sequence data of the recombinant plasmid
pBridge-BD-CRY2(UVR8-BIC2).**

We picked our single colony and sent them to company for DNA
sequencing, the final results indicated that there were not genetic mutations on our genes and the
recombinant plasmid in lane 4 of plate 1 was successfully constructed validated by sanger sequencing
(Fig.6).

## 2.3 Test

**Transformation**

The homologous recombination plasmids of pBridge-AD-CIB1, pBridge-BD-CRY2 and UVR8-BIC2 were
transferred into AH109.

**Culture**

We used SD double deficient medium, and yeast without inserted DNA fragments
lacked Leucine and Tryptophan, making it unable to grow. However, yeast that successfully inserted
fragments can grow, thereby achieving the goal of expanding culture. Transferring the plasmid into yeast
can maintain a stable structure.

**Functional test**

Our goal is to examine the whether a greater efficiency of the “off” switch can be achieved under
UV conditions with the insertion of UVR8 fragments, and whether UVR8 genes can work under UV with extra
light conditions (dark or blue light). To visualize the results, we designed the detection of β
galactosidase activity. Essentially, the quick “off” characteristic of UVR8 can be verified if the
enzymatic activity shows a rapid decrease trend, vice versa.

Therefore, we set four different light conditions: dark, dark with UV, blue light, and blue light
with UV, and observed different performances of yeast cells with plasmids transformed.

Table 1 Experimental design form for β galactosidase activity detection and expected outcomes.

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4874/wiki/engineering/9.png) |

As indicated in the experimental design form, the variable between experimental group 1 and 2 is the UVR8 gene. The variable between light condition 1 and 2 (3 and 4) is the UV light. The variable between light condition 2 and 4 is the light condition besides UV light. The expected outcome of each condition is outlined in the design form (Table.1).

![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-7.png)
**Figure 7. Bar chart and line chart of β-galactosidase (β-GAL)
activity assay.**

As indicated in the experimental design form, the variable between experimental group 1 and 2 is the UVR8 gene. The variable between light condition 1 and 2 (3 and 4) is the UV light. The variable between light condition 2 and 4 is the light condition besides UV light. The expected outcome of each condition is outlined in the design form (Table.1).

## 2.4 Learn

 In the process of constructing plasmids, we should pay attention to the steps of extraction
after gelation, increase the concentration of enzyme digestion and insertion fragments, and extract
plasmids.

 In this cycle, we learned how to visualize our results from intrinsic gene regulation to
extrinsic enzymatic activity based on a series of positive correlations: how absorbance levels reflect
the activity of β galactosidase, how activity levels of
β-
galactosidase reflect the availability of BD-CRY2-UVR8-BIC2 on/off switches, and how the working
patterns of on/off switches reinforce or invalidate our original assumptions.

# 3.  **Cycle 2 Detection of β Galactosidase Activity**

## **3.1 Design**

In order to confirm whether the blue-light dependent CRY2/CIB2 system
works, we performed yeast transformation of AD-CIB1 and BD-CRY2. We got these two plasmids from a
company.

## **3.2 Build**

The plasmids were transformed into E.coli DH5a to get more. Then pBridge-AD-CIB1 and
pBridge-BD-CRY2 were transformed into AH109.

## **3.3 Test**

Because SD medium is a double deficient enzyme medium, yeast without AD and BD implantation cannot
grow, so white colonies are positive colonies.

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4874/wiki/engineering/engineering-8.png) |

**Figure 8. Yeast culture results in medium
SD/-Leu/-Trp.**

## **3.4 Learn**

 We learned that CRY2/CIB1 system could work. And compared with BD-CRY2-UVR8-BIC2 on/off switches
system, it could not regulate the off by self. In conclusion, our BD-CRY2-UVR8-BIC2 on/off switches
system could increase the rate of “off”.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4874/wiki/experiments.png)

**Plasmid extraction (DiaSpin Plasmid Mini-Preps
Kit)**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| Buffer S | 500μL |
| Buffer SP1 | 250μL |
| Buffer SP2 | 250μL |
| Buffer SP3 | 350μL |
| Wash solution | 500μL |
| ddH₂O | 50-100μL |
| Bacterial fluid | 1.5-5mL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Spin column | 1 |
| Collection tube | 1 |
| Eppendorf tube (1.5mL) | 2 |
| Centrifuge | 1 |
| Vortex machine | 1 |
| Pipette (range: 1~5mL) | 1 |
| Pipette (range: 100~1000μL) | 1 |

1.
Insert one spin column into the collection tube

2.
Add 500μL Buffer S into the column

3.
Centrifuge the tube at 12000×g for 1 minute and discard the waste
liquid

4.
Put 1.5-5mL overnight cultured (37℃ 12 - 16h) bacteria into an
Eppendorf tube

5.
Centrifuge the tube at 8000×g for 2 minutes and discard the
supernatant

6.
Add 250μL Buffer SP1 to the bacteria precipitate

7.
Blowing and vortex until bacteria are suspended

8.
Add 250μL Buffer SP2 and immediately gently invert the tube
5-10
times

9.
Rest the tube at room temperature for 2-4 minutes

10.
Add 350μL Buffer SP3 and immediately gently invert the tube
5-10
times

11.
Centrifuge the tube at 12000×g for 5-10 minutes

12.
Put the supernatant into the spin column

13.
Centrifuge at 9000×g for 30 seconds and discard the waste
liquid

14.
Add 500μL wash solution into the column

15.
Centrifuge the tube at 9000×g for 30 seconds and discard the waste
liquid

16.
Repeat the steps 14&15.

17.
Centrifuge the tube at 9000×g for 1 minute

18.
Place the spin column into a new Eppendorf tube

19.
Add 50-100 μL preheated ddH₂O  (56℃) on the middle
of
the absorption membrane

20.
Rest the tube at room temperature for 1-2 minutes

21.
Centrifuge the tube at 9000×g for 1 minute

**Restriction enzyme single
digestion**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| 10×H Buffer | 5μL |
| Nde1 enzyme | 1μL |
| pBridge-BD-CRY2 plasmid | 800ng |
| dH₂O | Up to 50μL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| Water bath kettle | 1 |
| Small centrifuge | 1 |
| Vortex machine | 1 |
| Pipette (range: 1~10μL) | 1 |
| Pipette (range: 10~100μL) | 1 |

1.
Add 5μL 10×H buffer, 1μL Nde1 enzyme, 800ng
pBridge-BD-CRY2
plasmid, up to 50μL dH₂O into the Eppendorf tube on ice

2.
Vortex and centrifuge the tube

3.
Place the tube into a water bath kettle at 37℃ for 15
minutes

**Restriction enzyme double
digestion**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| 10×H Buffer | 5μL |
| 10× rCutsmart Buffer | 5μL |
| pBridge-BD-CRY2 plasmid | 1μg |
| Nde1 enzyme | 1μL |
| Bstb1 enzyme | 1μL |
| dH₂O | Up to 50μL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| Water bath kettle | 1 |
| Small centrifuge | 1 |
| Vortex machine | 1 |
| Pipette (range: 1~10μL) | 1 |
| Pipette (range: 10~100μL) | 1 |

**Protocol**

1.
Add 5μL 10×H buffer, 5μL 10× rCutsmart buffer, 1μg
pBridge-BD-CRY2 plasmid, 1μL Nde1 enzyme, 1μL Bstb1 enzyme, up to 50μL dH₂O into
the Eppendorf tube on ice

2.
Vortex and centrifuge the tube

3.
Place the tube into a water bath kettle at 37℃ for 2
hours

**Touchdown PCR**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| 2× phata mix | 25μL |
| Template (TRP1 promoter, UVR8 or BIC2 CDS) | 1μL |
| Forward primer (TRP1-F, UVR8-F or BIC2-F, correspond to the template) | 1μL |
| Reverse primer (TRP1-R, UVR8-R or BIC2-R, correspond to the template) | 1μL |
| dH₂O | Up to 50μL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| PCR tube | 1 |
| Small centrifuge | 1 |
| Vortex machine | 1 |
| PCR thermal cycler | 1 |
| Pipette (range: 0.5-2.5μL) | 1 |
| Pipette (range:10-100μL) | 1 |

**Protocol**

1.
Prepare 3 PCR tubes. Add 25μL phata mix, 1μL forward
primer
and 1μL reverse primer into each PCR tube.

2.
Add template into each PCR tube.

3.
Add water into each PCR tube up to 50μL

4.
Vortex and centrifuge the tube.

5.
Set up a reaction program.

|  |  |
| --- | --- |
| **Temperature** | **Time** |
| 94℃ | 5mins |
| 94℃ | 30s |
| 65℃ | 30s |
| 72℃ | 1kb/min (depends on the template) |
| Repeat 10 cycles, decreasing by 1 ° C each cycle during the 65 ° C stage (65 to 55 ° C) | |
| 94℃ | 30s |
| 55℃ | 30s |
| 72℃ | 1kb/min (depends on the template) |
| Repeat 25 cycles | |
| 72℃ | 10mins |

6.
Start the PCR reaction.

**Colony PCR**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| Taq Mix | 5μL |
| Forward primer (TRP-F) | 0.5μL |
| Reverse primer (BIC2-R) | 0.5μL |
| dH₂O | 4μL |
| Template (incubated DH5α chemically competent cells with plasmids pBridge-BD-CRY2-UVR8-BIC2) | -- |
| Liquid LB medium (amp+) | 1mL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| PCR tube | 1 |
| Small centrifuge | 1 |
| Vortex machine | 1 |
| PCR thermal cycler | 1 |
| Pipette (range: 0.5~2.5μL) | 1 |
| Pipette (range:1~10μL) | 1 |
| Pipette (range:1~5mL) | 1 |

**Protocol**

1.
Add 5μL Taq Mix, 0.5μL forward primer, 0.5μL reverse
primer, 4μL dH₂O into a PCR tube.

2.
Add 1mL ampicillin resistance liquid LB into a 1.5mL Eppendorf
tube.

7.
Pick a single clonal colony with a pipette (range: 0.5-2.5μL),
and
the tip must be blown out in the PCR tube for 3 times.

3.
Vortex and centrifuge the tube.

4.
Remove the tip from the PCR tube and put it into the1.5mL Eppendorf
tube to culture bacteria.

5.
Set up a reaction program.

|  |  |
| --- | --- |
| **Temperature** | **Time** |
| 95℃ | 10mins |
| 94℃ | 30s |
| 55℃ | 30s |
| 72℃ | 1min55s |
| 28 cycles | |
| 72℃ | 10mins |
| 10℃ | 30min |

6.
Start the PCR reaction.

**Homologous recombination**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| 2× Mix Buffer | 10μL |
| pBridge-BD-CRY2 plasmid (8361bp) | 167.22ng |
| TRP1 promoter fragment (134bp) | 2.68ng |
| UVR8-N397 fragment (1213bp) | 24.26ng |
| BIC2-CDS fragment (384bp) | 7.68ng |
| dH₂O | Up to 20μL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| Water bath kettle | 1 |
| Small centrifuge | 1 |
| Vortex machine | 1 |
| Pipette (range: 0.5~2.5μL) | 1 |
| Pipette (range:10~100μL) | 1 |

**Protocol**

1.
Add 10μL 2× Mix Buffer, 167.22ng pBridge-BD-CRY2 plasmid,
2.68ng
TRP1 promoter fragment, 24.26ng UVR8-N397 fragment, 7.68ng BIC2-CDS fragment, up to 20μL dH₂O
into an Eppendorf tube.

2.
Vortex and centrifuge the tube.

3.
Place the tube into a water bath kettle at 50℃ for
25min.

4.
Place the tube on ice.

**1% Agarose gel electrophoresis**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| Agarose | 1% (0.8μL) |
| 1× TAE Buffer | 80ml |
| Nucleic acid dye(1000x) | 8μL |
| 10× loading buffer | 1× in template |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Conical flask | 1 |
| Measuring cylinder | 1 |
| Weighing paper | 1 piece |
| Electronic balance | 1 |
| Spatula | 1 |
| Electrophoresis chamber | 1 |
| Electrodes | 1 pair |
| Gel tray | 1 |
| Comb | 1 |
| UV transilluminator | 1 |
| Pipette (range: 1~10μL) | 1 |

**Protocol**

1.
Place the weighing paper on the electronic balance, weigh 0.8g
agarose
and pour it into the conical flask.

2.
Pour 80mL 1× TAE Buffer into the conical flask.

3.
Heat the conical flask in the microwave oven for 1~2minutes until
the
agarose is well dissolved.

4.
Add 8μL nucleic acid dye into the conical flask and shake
it.

5.
Pour the gel into the gel tray and insert the comb.

6.
Rest the gel in room temperature for 30 minutes until it is
solidified.

7.
Pour about 1000mL 1× TAE Buffer into the electrophoresis
chamber.

8.
Carefully take out the gel and place it into the electrophoresis
chamber.

9.
Use the pipette (range: 1~10μL) to add 5μL
Trans*®️* Marker into the first hole of the gel.

10.
Add loading buffer, 0.1× volume of the template, into the template
using a pipette (range: 1~10μL), mix well by blowing and suction.

11.
Carefully add 10 μL of each template into the following holes
of
the gel

12.
Connect the electrodes, turn on the voltage to 130V and work for 10
minutes.

13.
Place the gel into the UV transilluminator and turn on the UV
light.

14.
Comparing with the marker reference to determine the target
section.

**DNA gel extraction (Diamond
kit)**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| Agarose gel | -- |
| Buffer B2 | 400μL |
| Wash Solution | 500μL |
| ddH₂O | 15μL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Scalpel | 1 |
| Eppendorf tube (1.5mL) | 1 |
| Spin column | 1 |
| Collection tube | 1 |
| Water bath kettle | 1 |
| Centrifuge | 1 |
| Pipette (range: 10~100μL) | 1 |
| Pipette (range: 100~1000μL) | 1 |

**Protocol**

1.
Use the scalpel to cut the target section of the gel and place the
gel
into the Eppendorf tube.

2.
Add 400μL buffer B2, place the tube into a water bath kettle
at
50℃ for 5-10 minutes to dissolve the gel.

3.
Insert a spin column into a collection tube and transfer the
dissolved
gel into the spin column.

4.
Centrifuge the tube at 8000×g for 30s and discard the waste
liquid.

5.
Add 500μL Wash Solution.

6.
Centrifuge the tube at 9000×g for 30s and discard the waste
liquid.

7.
Repeat the steps 5&6.

8.
Centrifuge the tube at 9000×g for 1 minute.

9.
Place the spin column into a new Eppendorf tube
(1.5mL).

10.
Add 15μL ddH₂O into the spin column, rest at room
temperature for 1 minute.

11.
Centrifuge the tube at 9000×g for 1 minute, save the solution in
the
Eppendorf tube.

***E.coli*****transformation**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| pBridge-BD-CRY2-UVR8-BIC2 plasmid | 5~10μL |
| DH5α chemically competent cell | 100μL |
| Liquid LB medium (amp-) | 900μL |
| Solid LB medium (amp+) | 1 plate |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| Water bath kettle | 1 |
| Sterile spreading stick | 1 |
| Petri dish | 1 |
| Incubator | 1 |
| Shaker | 1 |
| Pipette (range: 1~10μL) | 1 |
| Pipette (range: 10~100μL) | 1 |

**Protocol**

1.
Thaw 100μL DH5α chemically competent cells in an Eppendorf
tube on
ice

2.
Add 10μL pBridge-BD-CRY2-UVR8-BIC2 plasmid into the tube,
flick
the tube to mix

3.
Rest the tube on ice for 30 minutes

4.
Place the tube into a water bath kettle at 42℃ for 45 seconds and
immediately place the tube on ice for 2~3 minutes

5.
Add 900μL liquid LB medium (amp-) to the tube

6.
Mix the solution uniformly, and incubate it in the shaker at 37℃,
200~250rpm for 1 hour

7.
Preheat the solid LB medium (amp+) in a Petri dish with an
incubator
for 37℃

8.
Centrifuge the tube at 2500×g for 5 minute, discard 900μL
supernatant

9.
Resuspend the bacteria and coat them uniformly on the solid LB
medium
(amp+) using a sterile spreading stick.

10.
Turn the Petri dish upside down and incubate the cells at 37℃ for
12-16
hours.

***S. cerevisiae*****transformation**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| pBridge-AD-CIB1 plasmid | 2μg |
| pBridge-BD-CRY2 plasmid | 2μg |
| AH109 chemically competent cell | 100μL |
| Carrier DNA (Salmon sperm DNA) | 100μL |
| PEG/LiAc | 500μL |
| Solid LB medium (-Trp/-Leu) | 900μL |
| ddH₂O | 50μL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| Water bath kettle | 1 |
| Sterile spreading stick | 1 |
| Petri dish | 1 |
| Incubator | 1 |
| Pipette (range: 1~10μL) | 1 |
| Pipette (range: 10~100μL) | 1 |

**Protocol**

1.
Place the carrier DNA into a water bath kettle at 95℃ for 3 minutes
and
immediately place the tube on ice

2.
Thaw 100μL AH109 chemically competent cells in an Eppendorf
tube
on ice

3.
Add 2~5μg pBridge-AD-CIB1 plasmid, 2~5μg pBridge-BD-CRY2
plasmid, 10μL carrier DNA, 500μL PEG/LiAc into the tube, mix well by blowing and
suction

4.
Place the tube into a water bath kettle at 30℃ for 30 minutes, turn
the
tube upside down 6~8 times when 15 minutes has passed during the water bath

5.
Place the tube into a water bath kettle at 42℃ for 15 minutes, turn
the
tube upside down 6~8 times when 7.5 minutes has passed during the water bath

6.
Centrifuge the tube at 2500×g for 40 seconds, discard
supernatant

7.
Add 400μL ddH₂O and resuspend

8.
Centrifuge the tube at 2500×g for 30 seconds, discard
supernatant

9.
Add 400μL ddH₂O and resuspend

10.
Coat the mixture uniformly on the solid LB medium (-Trp/-Leu) using
a
sterile spreading stick

11.
Turn the Petri dish upside down and incubate the cells at 29℃ for
48~96
hours until the diameter of single colony reaches 1mm

**β-galactosidase activity assay**

**Materials**

|  |  |
| --- | --- |
| **Reagents** | **Amount** |
| YPD medium | 8mL |
| Liquid LB medium (-Trp/-Leu) | 5mL |
| Positive sample (incubated AH109 chemically competent cells with plasmids pBridge-AD-CIB1 pBridge-BD-CRY2) | 2mL |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| Eppendorf tube (1.5mL) | 1 |
| Water bath kettle | 1 |
| Sterile spreading stick | 1 |
| Petri dish | 1 |
| Incubator | 1 |
| Pipette (range: 1~10μL) | 1 |
| Pipette (range: 10~100μL) | 1 |

**Protocol**

1.
The positive colonies of bacteria grown on the -Trp/-Leu solid SD
medium were cultured in 5ml the -Trp/-Leu liquid SD medium (Amp) at 30℃, 250rpm, overnight (wrapped in
tinfoil). During the period, use the bule light treatment to activate β-galactosidase expression.

2.
Light treatment: Vortex the overnight culture tube for 0.5 - 1 min
to
disperse cell clumps. Transfer 2 ml of the overnight culture to 8 ml of YPD medium Immediately. Incubate
fresh culture at 30°C for 3 - 5 h with shaking (230 -250 rpm) until the cells are in mid-log phase
(OD600 of 1 ml = 0.5 - 0.8). Record the exact OD600 when you harvest the cells. During the period, use
the bule light and UVB treatment to close β-galactosidase expression.

3.
Harvest the cell by centrifuge at 14000 rpm(16000xg) for 30 seconds
and
discard the supernatant carefully. According to the number of bacteria or cells (10⁴): the
extraction liquid volume (mL) is 500~ 1000:1 ratio (it is recommended that 5 million bacteria or cells
be added to 1mL of the extraction solution), and the bacteria or cells are broken by ultrasound (ice
bath, power 20% or 200W, ultrasonic 3s, 10s interval, repeat 30 times); 15000g centrifuge at 4℃ for
10min, take supernatant and put it on ice to be measured.

4.

β-galactosidase activity was detected, and cells were collected: centrifuge
at 700g for 5min, collect yeast cells into a centrifuge tube, and discard the supernatant; Add 1ml
of the extract. Ultrasonic crushing of bacteria or cells (ice bath, power 20% or 200W, ultrasonic
3s, 10s interval, repeat 30 times); 15000g centrifuge at 4℃ for 10min, take supernatant and put it
on ice to be measured.

5.
Treatment of standard liquid: Dilute the standard liquid with
distilled
water to 200, 100, 50, 25, 12.5, 6.25, 0 nmol/ml.

6.
Add each reagent in turn according to the protocol of
kit.

|  |  |  |  |
| --- | --- | --- | --- |
| **Reagents** | **Test tube** | **Control tube** | **Standard tube** |
| Reagent 1 | 200 | - | - |
| ddH₂O | - | 200 | - |
| Reagent 2 | 250 | 250 | - |
| Sample supernatant | 50 | 50 | - |
| Mix quickly and place in a water bath at 37℃ for 30 minutes | | | |
| Standard sample | - | - | 500 |
| Reagent 3 | 1000 | 1000 | 1000 |
| Thoroughly mixed, the absorption value A was measured at 400nm, and ΔA was A value of test tube minus A value of control tube. Each test tube shall be provided with a control tube. | | | |

7.
Develop a standard curve. Determine the β-GAL activity of
sample.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4874/wiki/results/results.png)

**Overview**

To alleviate the pain experienced by diabetes patients, we have devised
a novel CRY2/CIB1 system, which operates via blue-light dependent optogenetics. This innovation aims to
enhance the efficiency of light-controlled "off" switches.

In our study, we combined UVR8, a UVB receptor originally identified in
plants, with the recently discovered blue-light inhibitors of cryptochromes BIC2 to create the
UVR8N-BIC2 fusion protein. Through a series of experiments assessing β-galactosidase activity within a
yeast two-hybrid system, we demonstrated the capability to expedite the deactivation process of the
CRY2/CIB1 system in the presence of UVB light.

**Result**

**1. Construction of pBridge-BD-CRY2(UVR8-BIC2) plasmid
using double digestion**

In order to combine UVR8 with BIC2, we construct a fused protein
UVR8N-BIC2 (Fig.1). We firstly designed primers and amplified TRP promoter fragment from pBridge vector,
UVR8N397 fragment from the plasmid containing UVR8 CDS sequence, and BIC2 fragment from Arabidopsis
thaliana genome cDNA by PCR assay. The results showed that we got the 3 DNA fragments successfully
(Fig.2A). We also constructed linearized pBridge-BD-CRY2 vector by double enzymatic digestion with
*Nde*I and *Bstb*I (Fig.2A). Then we obtained the DNA fragments of TRP promoter
fragment, UVR8N397 fragment and BIC2 fragment and the linearized pBridge-BD-CRY2 vector employing DNA extraction kit. We performed homologous recombination to link
the 3 DNA fragments to the linear pBridge-BD-CRY2 vector. Next we transformed the product into
E.coli and as shown in figure 2B, we obtained the E.coli with the plasmid. To verify whether the
recombinant plasmid presented in the E.col, the colony PCR was performed and the results of the plate
suggested the failure of homologous recombination because of the low quality of the linearized
pBridge-BD-CRY2 vector (Fig.2B-2C).

![](https://static.igem.wiki/teams/4874/wiki/results/1.png)

**Figure 1. pBridge-BD-CRY2(UVR8-BIC2) plasmid
map.**

![](https://static.igem.wiki/teams/4874/wiki/results/result-2.png)

**Figure 2. Construction and identification of
pBridge-BD-CRY2(UVR8-BIC2) plasmid using double digestion.**

(A) The identification of DNA fragments by PCR and the linearized
vector*by Nde*I-*Bstb*I double digestion.

(B) Result of transformation with the linearized vector and the three
DNA fragments.

(C)Colony PCR results of the plates in (B).

**2. Construction of pBridge-BD-CRY2(UVR8-BIC2) plasmid
using single digestion**

To get the high qualitied linearized vector, we transformed
pBridge-BD-CRY2 plasmid into *E.coli* and cultured the *E.coli* to obtain more pBridge-BD-CRY2 plasmid. And we identified the
quality of the plasmid by DNA agarose gel electrophoresis (Fig.3A). As shown in figure 3A, the plasmid
was correct and has high quality. Next, we adapted *Nde*Isingle enzyme digestion to linearize the pBridge-BD-CRY2 plasmid and
amplified the three DNA fragments (Fig.3B-C). We got the 3 DNA fragments and linear
plasmid successfully. Then we transformed the homologous recombination product of the 3 DNA
fragments and linear plasmid into *E.coli* and cultured overnight (Fig 3D). The results of colony PCR showed
that the lane 2, 3, 4 of plate 1 and the lane 3 of plate 2 were shown a positive band which is about
1686 bp, suggesting the colony of *E.coli* contained the recombinant plasmid
pBridge-BD-CRY2(UVR8-BIC2) (Fig.3E).

![](https://static.igem.wiki/teams/4874/wiki/results/result-3.png)

**Figure 3. Construction and identification of
pBridge-BD-CRY2(UVR8-BIC2) plasmid using single digestion.**

(A) The identification of re-extracted pBridge-BD-CRY2
plasmids.

(B) The identification of the linearized pBridge-BD-CRY2 vectors by
*Nde*I single digestion.

(C) The identification of TRP promoter fragment, UVR8N397 fragment and
BIC2 fragment by PCR assay.

(D) Result of transformation with the linearized vector
(*Nde*I digested) and the three DNA fragments in (C).

(E)Colony PCR results of the new plates in (D).

We picked our single colony and sent them to company for DNA
sequencing, the final results indicated that there were not genetic mutations
in
 our genes and the recombinant plasmid in lane 4 of plate 1
(Fig.3E)
was successfully constructed validated by sanger
sequencing (Fig.4).

![](https://static.igem.wiki/teams/4874/wiki/results/result-4.png)

**Figure 4. The sequence data of the recombinant plasmid
pBridge-BD-CRY2(UVR8-BIC2).**

**3. The confirmation of CRY2/CIB1 system in
yeast**

In order to confirm whether the blue-light dependent CRY2/CIB2 system
works, we performed yeast transformation of AD-CIB1 and BD-CRY2. The results showed successful
transformation of yeast after 48h culture (Fig.5)

![](https://static.igem.wiki/teams/4874/wiki/results/result-5.png)

**Figure 5. Confirmation of blue-light dependent
CRY2/CIB1 system.**

Then we did a dot-plate experiment, the results showed that CRY2/CIB1 system
could work.

![](https://static.igem.wiki/teams/4874/wiki/results/result-6.png)

**Figure 6. The results of growth of yeast containing
the plasmids.**

**4. Improvement of switch “off” in UVB regulated
pBridge-BD-CRY2(UVR8-BIC2) system**

We then examined whether UVB exposure could improve the switch “off” in
pBridge-BD-CRY2(UVR8-BIC2) system. AD-CIB1 and BD-CRY2(UVR8-BIC2) were transformed into yeast together
and cultured on -Trp/-His/-Leu/-Ade solid plate for 72 h. The positive colony was picked to enlarge
cultivation in QDO liquid medium under conditions of blue light to allow the β-galactosidase
expression. In order to test whether the switch “off” speed of our system was improved under UVB
irradiation, we divided the medium with galactosidase expression under blue light condition into four
parts to shut down, respectively under dark condition and under dark with UV light condition and under
blue light condition and under blue with UV light condition. As shown in
Table1 &
figure
7
, the activity of β-Gal was low so we believed that
pBridge-BD-CRY2(UVR8-BIC2) system did not work under dark and dark with UV condition. Under blue light
condition, the activity of β-Gal was increased with time. While under blue with UV light condition, the
increasing rate of β-GAL activity in the presence of UV was slower than that in the absence of UV, indicating
that The proteins UVB and BIC2 play the role of self-switch off and improve the switch “off” in
pBridge-BD-CRY2(UVR8-BIC2) system.

**Table1.****The data of β-galactosidase (β-GAL) activity
assay**

![](https://static.igem.wiki/teams/4874/wiki/results/result-table-1.png)

![](https://static.igem.wiki/teams/4874/wiki/results/result-7.png)

**Figure 7.****. Bar chart and line chart of β-galactosidase (β-GAL)
activity assay**

{% endblock %}