iGEM 2023 — Team subcat-peking

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![](https://static.igem.wiki/teams/4904/wiki/notebook.png)

**D****ay1—7/19/2023**

1.
PCR expanding: PCR,
which refers to polymerase chain reaction, is a fundamental technique used to copy or amplify DNA
segments. In this experiment, PCR helps develop three types of elements: GFP, UAS, and SPA, which will
be used in
the following experiments.

2.
Electrophoresis: In this experiment, the tested DNA could be compared with the marker to see whether the
expanding result is successful.

3.
Media preparation
(including the K+ and
amp kind, and -Trp -leu and -Trp -leu -His -Ade kind): The configuration of the culture medium is
beneficial for the growth of E. coli and yeast, which will be used in further experiments and will help to
identify whether the experiment is successful.

![](https://static.igem.wiki/teams/4904/wiki/notebook/1.png)

**D****ay2****~3****—7/20/2023****~****7/2****1****/2023**

1.
Digestion: It prepares for homologous recombination and uses digestion to let the ring shape become a
linear carrier

2.
Homologous
recombination: This method is used to connect UAS and GFP to BD and SPA1 to AD in this experiment.

3.
Electrophoresis: Electrophoresis can make sure that the DNA fragment that has been extracted is working
for the DNA specimen.

4.
Heat-shock: It is to introduce exogenous DNA molecules (AD and BD plasmids) into the receptor cells
(Escherichia coli) so that the receptor cells can obtain new genetic traits and grow and reproduce on a
Petri dish, thus forming an ideal colony with the plasmids needed for the experiment.

![](https://static.igem.wiki/teams/4904/wiki/notebook/2-1.png)
![](https://static.igem.wiki/teams/4904/wiki/notebook/2-2.png)

**D****ay****4~5****—7/2****2****/2023****~****7/2****3****/2023**

1.
Plasmid Isolation: Plasmid
isolation is a way to extract plasmid from the cell. This method is used in this experiment to extract
plasmid AD from E. coli since there is a shortage of this plasmid in the lab.

2.
Construction of a dual enzyme digestion system: None
of the E. coli bacteria coated yesterday grew out, so it must be done again today. However, it was found
that the enzyme used to make the AD part was used up, so a new one had to be made.

3.
homologous recombination: Homologous recombination is a common way to connect a segment to the cloning
vector. This experiment uses this method to connect UAS and GFP to BD and SPA1 to AD.

4.
Heat shock conversion: To introduce exogenous DNA molecules (AD and BD plasmids) into the receptor cells
(DH5α E. coli) so that the receptor cells can obtain new genetic traits and grow and reproduce on
the culture dish, thus forming an ideal colony with the plasmids needed for the experiment. (Because the
E. coli planted on the plate yesterday did not successfully develop, it’s necessary to repeat the
process and try to form the ideal colony with the plasmids needed for the experiment).

|  |  |  |
| --- | --- | --- |
| Sample number | concentration | purity |
| 1 | 1.317 | 1.83 |
| 2 | 0.775 | 1.86 |
| 3 | 0.694 | 1.79 |
| 4 | 1.513 | 1.78 |
| 5 | 1.738 | 1.80 |

![](https://static.igem.wiki/teams/4904/wiki/notebook/3.png)

**D****ay****6****—7/2****4****/2023**

1.
Colony PCR is used to check if the two plasmid systems BD-CRY2-GFP and AD-SPA1 have been successfully
constructed. The
last try to develop E. coli was successful. For further development in the experiment, it is necessary to
preserve strain and save it for later use.

2.
Colony
PCR validation: To
test whether BD, GFP, AD, and SPA1 have successfully linked. Whether BD and AD exist can be seen by
whether E. coli had grown on the K+ and
amp culture dishes.

3.
Electrophoresis:
In this experiment, whether BD and AD are constructed with GFP and SPA1 is shown as whether DNA strands
have appeared.

4.
Validation
plasmid: It is to verify whether the plasmid in E. coli is the target plasmid (BD-CRY2-GFP and
AD-SPA1)

5.
Rocking
incubation: Rocking incubation is commonly used to accelerate the incubation time. In this experiment, the
method is used to incubate E. coli.

![](https://static.igem.wiki/teams/4904/wiki/notebook/4.png)

**D****ay****7****—7/2****5****/2023**

1.
Plasmid isolation: It
is to extract plasmid BD-CRY2-GFP and AD-SPA1. It’s necessary to remove the use-acquired DNA to
continue doing yeast two-hybrid.

2.
Yeast two-hybrid:
BD-CRY2-GFP and AD-SPA1 protein can be transferred into the competent cell and
successfully get to the yeast two-hybrid stage.

3.
Flasks Culture: Coat
the yeast from the yeast two-hybrid system on a 2-deficient plate for cultivation, ensuring that the
shaken colonies contain plasmids BD-CRY2-GFP and AD-SPA1 in their bodies.

![](https://static.igem.wiki/teams/4904/wiki/notebook/5.png)

**Two days off****7/26/2023****~****7/2****7****/2023**

**D****ay****8****—7/2****8****/2023**

1.
Plasmid isolation: Extract the plasmid of BD-CRY2-GFP and AD-CIB1 from E. coli to go on to the yeast
two-hybrid process.

2.
Yeast two-hybrid: BD-CRY2-GFP and AD-SPA1(experimental group), and BD-CRY2 and AD-CIB1(control group)
plasmids can be transferred into the competent cell, and therefore successfully get to the yeast
two-hybrid stage. Then, through coating the yeast from the yeast two-hybrid system on a 2-deficient plate
for cultivation, ensuring that the shaken colonies contain plasmids both BD-CRY2-GFP and
AD-SPA1(experimental group) and BD-CRY2 and AD-CIB1(control group) plasmids in their bodies.

3.
Plasmid Extraction: Preparation
of yeast two-hybrid.

4. Yeast culturing.

![](https://static.igem.wiki/teams/4904/wiki/notebook/6-1.png)
![](https://static.igem.wiki/teams/4904/wiki/notebook/6-2.png)

**D****ay****9****—7/2****9****/2023**

1. Wiki documentation and lab work review with the
instructor.

**D****ay****10****—7/****30****/2023**

1. Carry
out blue light and darkness control test and prepare for test.

2. In the meantime, we also set up different volume concentration of
yeast to compare the sensitivity of two yeast under blue light conditions.

![](https://static.igem.wiki/teams/4904/wiki/notebook/7.png)

**D****ay****11~12****—****8****/****1****/2023****~8****/****2****/2023**

1. We observe the yeast growth against different volume concentration
and easily noticed that at low concentrations of yeast, CRY2/SPA1 yeast continues to grow compared to CRY2/CIBI
yeast, which essentially stops growing.

2. Another group of experiments for blue light and dark condition
comparison also drew the conclusion that the yeast possesses the blue light reactivity.

![](https://static.igem.wiki/teams/4904/wiki/notebook/8-1.png)
![](https://static.igem.wiki/teams/4904/wiki/notebook/8-2.png)

**Day13-8/3/2023**

1. Use fluorescence
inverse microscope to observe the GFP expression.

2. Use
β-gal
to test whether the yeast two-hybrid resulted plasmid is active enough.

![](https://static.igem.wiki/teams/4904/wiki/notebook/9-1.png)
![](https://static.igem.wiki/teams/4904/wiki/notebook/9-2.png)
![](https://static.igem.wiki/teams/4904/wiki/notebook/9-3.png)
![](https://static.igem.wiki/teams/4904/wiki/notebook/9-4.png)

**Day14-8/4/2023**

1. Result analysis and Wiki documentation.

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![](https://static.igem.wiki/teams/4904/wiki/experiments.png)

Construction of recombinant plasmid

**1. Steps of
goal
DNA PCR:**

**1） Building a PCR
system.**

|  |  |
| --- | --- |
| Substance | mass/unit |
| 2\*mix | 25ul |
| Forward primer of SPA1 | 2ul |
| Reverse primer of SPA1 | 2ul |
| ddH2O | 19ul |
| SPA1 | 2ul |
| total | 50ul |

(Repeat
step 1 to build a GFP and UAS DNA PCR system.)

Adding
different DNA to different tubes to separate and then send to the PCR machine, which processes through the
following steps

**2) Perform
PCR
program.**

|  |  |
| --- | --- |
| Temperature | Time/unit |
| 95℃ | 5min |
| 95℃ | 30s |
| 55℃ | 30s |
| 72℃ | 10min |
| 72℃ | 2min |
| 4℃ | forever |

**2.
Electrophoresis
testing and gel recovery**

**1. Configure
electrophoresis gel.**

**2. Drop the
DNA of SPA1****,** **GFP,
and UAS DNA into the opening left by the comb.**

**3. Energize
electrophoresis.**

**4. DNA
extraction from the gel.**

**1)
Configure electrophoresis gel.**

In
this process, use 0.5g of agarose and 50ml of pure water to complete the mixture. The
gel can be extracted by putting the configuration into the laboratory microwave oven and heating it until
boiling, and the process of configuring the electrophoresis gel is finished.

**2) Drop the DNA of
SPA1****,** **GFP,and UAS DNA into
the opening left by the comb.**

Use
the pipette gun to move this DNA from the test tube initially configured to the opening where the
electronic comb had been produced by leaving a mark.

**3)
Energize electrophoresis.**

Place
the electrophoresis into the experimental flumes with the charger to energize it.
After energizing the electrophoresis, the DNA will leave some signs, which can be
compared to the markers to determine whether it is the goal DNA.

**4)
DNA extraction from the gel.**

a.
After DNA electrophoresis, the gel containing the target DNA fragment is quickly cut under the ultraviolet
lamp, and the weight of the gel is weighed (the importance of the empty tube is removed). 100 mg gel is
equivalent to 100 L volume, regarded as a gel volume.

b.
Add equal-volume Buffer GDP. Bathe in 50 to 55C water for 7-10 min to ensure the gel blocks are entirely
dissolved.

c.
Centrifuge briefly to collect droplets from the wall of the tube. The Fast Pure DNA Mini Columns-G
adsorption column was placed in the Collection Tubes2ml collection tube, and the 750ul sol solution was
transferred to the adsorption column and centrifuged at 12,000rpm for 30-60s.

d.
Discard the filtrate and place the adsorption column in the collection tube. Add 700 pl Buffer GW
(containing ethanol) to the adsorption column. Centrifuge at 12,000 rpm for 30-60 s.

e.
Repeat Step 5

f.
Discard the filtrate and place the adsorption column back into the collection tube—centrifuge at
12,000rpm for 2 min.

g.
Place the adsorption column in a 1.5ml sterilized centrifuge tube, add 20-30 dd water drops to the center
of the adsorption column, and place for 2 minute-centrifuge at 12,000rpm for 1min. Discard the adsorption
column and store DNA at -20℃.

**3.
Preparation of
culture medium**

|  |  |
| --- | --- |
| Substance | Mass/unit |
| Yeast extract | 2.5g |
| NaCl | 5g |
| Tryptone | 5g |
| H2O | 500ml |
| total | 500ml |

Add
the following things into a measuring cylinder; the total of 500ml cultural medium consists of 200ml
liquid culture medium and 300ml solid culture medium (each tangible culture medium should add 2g agar) and
then divide every 100ml into a flask and sterilize it in a high-pressure steam cooker. During cooling, fix
the hands of the operating students with alcohol and light the Alcohol burner. Sterilize the mouth of the
flask with an external flame, and finally, pack the culture medium in a Petri dish in a sterile
environment near alcohol, etc., and mark it. After final cooling, seal and invert the solid culture
medium.

**4.
digestion**

**1. Double
digestion AD**

**2. Mix
the
reagents**

**3. Use
PCR to
reaction**

a.
Mix the following material, which ends up with a total volume of 50ul.

|  |  |
| --- | --- |
| Substance | mass/unit |
| NdeI | 1ul |
| BamHI | 1ul |
| BD-CRY2-N489 | 5ul |
| 10×buffer | 5ul |
| dd H2O | 38ul |
| total | 50ul |

|  |  |
| --- | --- |
| Substance | mass/unit |
| NdeI | 1ul |
| BamHI | 1ul |
| AD-CRY2-N489 | 5ul |
| 10×buffer | 5ul |
| dd H2O | 38ul |
| total | 50ul |

b.
Use
a centrifuge to mix reagents.

c.
Those reagents are placed in a PCR reaction at 37 degrees for 20 minutes.

d.
the AD double enzyme digestion system (BD double enzyme digestion system) and marker were injected
successively with a pipette gun in the pre-prepared gel gap. After the injection is complete, cover the
electrophoretic apparatus for 20 minutes.

|  |  |
| --- | --- |
| Substance | mass/unit |
| Agarose | 0.5g |
| 10×TAE | 50ml |
| Nucleic acid dye | 5ul |

e.
After the electrophoresis is complete, the middle part of the gel is lifted out, and a particular
instrument is placed to take pictures and observe the luminescence.

f.
Cut off the luminous part with a blade and melt it in a test tube.

**5. homologous
recombination**

**Steps:**

**1. Calculate
the
volume of vector and DNA segment supposed to be used in this experiment.**

**2. Mix the
reagents.**

**3. Use a PCR
machine to help the reaction go on**

A.
The accurate mass of UAS, GFP is calculated by the equation [0.02\*base pair number of the vector]ng. The
vector BD's mass is calculated using the equation [0.02\*base pair number of the segment]. The vector AD's
mass is calculated using the equation [0.04\*base pair number of the segment]. Since both outcomes are
lower than 1 uL, it is rounded to 1 uL. For the AD group, SPA1 is calculated by the equation [0.02\*base
pair number of the vector]ng.

|  |  |
| --- | --- |
| Substance | Mass/unit |
| BD-CRY2 | 3ul |
| GFP | 1ul |
| UAS | 1ul |
| 2×Mix | 5ul |
| total | 10ul |

|  |  |
| --- | --- |
| Substance | Mass/unit |
| SPA1 | 1ul |
| AD | 4ul |
| 2×Mix | 5ul |
| total | 10ul |

B.
The
above reagents are thoroughly mixed by centrifuge.

C.
Those
reagents are placed in PCR for 50 Celsius, 30 minutes, and preserved at 4 Celsius degrees.

**6. Thermal
shock
conversion**

**Steps:**

**1. Rocking
inoculation**

**2. Coated
plate**

**1. Rocking
inoculation**

1)
Pick
two test tubes, open the lid, and add 5 ml of LB reagent to each test tube (add 1000 microliters five
times).

2)
Add five microliters of BD antibody to each of the two test tubes.

3)
Dip the pipette tip into the Escherichia coli bacteria, then directly insert the pipette tip into two test
tubes.

4)
Put the lid on the test tube and seal the two test tubes.

**2. Coated
plate**

1)Use
a pipette to remove 450 μ L of the supernatant from the solution containing the BD (or AD) plasmid
system, and then extract a portion of the LB solution to flush the plasmid precipitated below the
collection tube.

2)
Subsequently, the solution was repeatedly sucked up and down with a pipette,
resulting in a homogeneous mixture of the plasmid and solution.

3)
Then, use a pipette to transfer the solution above the solid culture medium, and use a sterile coating rod
to continuously apply the solution in circles until resistance from the culture medium is felt, proving
that the culture
medium has fully absorbed the solution.

4)
Incubate upside down in a 37C incubator for 12-16h.

**7. plasmids
isolation**

**Steps:**

**1. Mix the
bacteria solution.**

**2. Thallus
pyrolysis and precipitate.**

**3. Collect
DNA.**

**4. Plasmid
BD-CRY2-GFP and AD-CIB1.**

a.
Use a centrifuge to mix BD-CRY2-GFP solution (10000 rpm centrifuge 1 minute) and make a
precipitate.

b.
Add 250ul buffer P1 into precipitation, use a pipette for intensive mixing, and add 250ul buffer P2 in the
solution prepared in the previous step, slowly upside down to crack the BD-CRY2-GFP. Adding 350ul buffer
P3, repeat the above step to complete dissolution buffer P2, make white precipitate, 12000 rpm centrifuge
10 minutes.

c.
Use
a pipettor to transfer the precipitate solution into the adsorption column (do not touch the sediment),
12000 rpm centrifuge for 1 minute, liquid waste removal. Adding 500ul buffer PW1 into the adsorption
column, repeat the above step. Adding 600ul buffer PW2, repeat the above step. Repeat the step: 12000 rpm
centrifuge 1-minute dry adsorption column, removal of residue. After standing for 10 minutes, add 15ul dd
H2O into the adsorption column, let stand at room temperature for 10 minutes, 12000 rpm centrifuge, and
use DNA extraction. Discard the adsorption column; DNA product save at -20 degrees centigrade.

d.
The plasmid insolation steps of AD-CIB1 are the same as above.

**Yeast
hybrid**

1.
Use a PCR apparatus to heat the carrier DNA at 95 Celsius for 5 minutes. Froze the DNA carrier immediately
by placing the tube in ice for 2 minutes. Then, repeat this step.

2.
Transfer the carrier DNA and another reagent to the clean bench, ensuring the experiment undergoes a
sterilized condition. Add 100 uL AH109 competence cell. Then, add 5uL of AD-SPA1 and 5uL of BD-CRY2-GFP to
the bottom of the tube. Add 10 uL preconditioned carrier DNA.

3.
Use a 30 Celsius metal bath, treat the mixture for 30 minutes, and flip the tube to mix the reagent 15
minutes after it begins. After 30 minutes, use a water bath, treat the mixture with 42 Celsius for 15
minutes, and flip the tube to mix the reagent 7.5 minutes after it begins.

4.
Coated plate

a.
Centrifuge the double hybrid yeast strain 5000rmp for 40s using a centrifuge, then discard the supernatant
on a super purification platform, resuspend 400mL of ddH20, and centrifuge for 30s to discard the
supernatant.

b.
Bring the yeast into the ultra-clean table again, add 50mL of dd H2O resuspend, blow, and mix well.

c.
Then, use a pipette to transfer the solution above the solid culture medium, and use a sterile coating rod
to continuously apply the solution in circles until resistance from the culture medium is felt, proving
that the culture medium has fully absorbed the solution.

5.
Rocking inoculation

Place
the culture substrate coated with yeast on a shaker and incubate at 28 ℃ for 48 to 96 hours.

6.
Select the transformed single colony and culture it in -2 liquid medium at 28℃ and 200rpm for 48h

7.
Take 5μl of the successfully cultured bacterial solution, drop it on -Trp-Leu-His-Ade defect medium and
-Trp-leu defect medium, respectively, and culture it at 28℃ under dark and blue light irradiation for
48-96h

8.
Using the -Trp-leu-his-Ade defect medium to screen the sensitivity of the two systems with different
gradient dilutions under blue light irradiation. Then, the plates of -Trp-leu were simultaneously cultured
in the dark, and finally, the two systems of varying gradient dilution were cultured in the night, and the
point plates were diluted by a 10-fold gradient (1, 10-1,
10-2,
10-4,
10-6,
10-8,
10-10,
10-12).

**GFP Fluorescence****Detection**

1. Focusing specimens
under
bright field microscopy conditions

(1) Turn on the power
switch
(transmission lighting LED on)

(2) Open the transmission
light path baffle (Ts2-FL only)

(3) Set the binocular
optical
path to 100% (only when the image head interface is installed)

(4) Fully open aperture
shutter

(5) Set all ring plates
to
bright field-of-view positions

(6) Placing a 4x
objective in
the optical path

(7) Adjust brightness
(rotate
the projection lighting brightness adjustment knob to adjust the field of view brightness)

(8) Place the specimen
and
place the part to be observed in the optical path

(9) Specimen
focusing

(10) Diopter
adjustment

(11) Adjusting pupillary
distance

2. Close the transmission
light path baffle

Place the transmission
light
way confused in the shaded position.

3. Place the fluorescent
filter block into the optical path

Rotate the fluorescent
block
turntable and place the required fluorescent filter block into the optical path.

4. Turn on fluorescent
lighting

Press the fluorescent
lighting
switch to turn on the light path's LED unit corresponding to the fluorescent filter block.

5. Observation of
specimens

(1) Rotate the
fluorescent
lighting brightness adjustment knob to adjust the field of view brightness

(2) Place the portion of
the
specimen to be observed into the optical path

(3) If the specimen needs
to
be observed and the focus is inaccurate, rotate the fine adjustment knob to focus the sample.

(4) When replacing and
observing specimens, check the focus and brightness and adjust them as needed.

6. Turn off the power
switch

Set the power switch to
the
"O" position and turn off the microscope switch.

**Assay of
β-galactosidase activity**

**Kit
components**

|  |  |  |  |
| --- | --- | --- | --- |
| Ingredients | Quantities | Storage temperature | How to use |
| Extract Solution | 50mL \* 1 bottle of liquid | 2-8 ℃ |  |
| Reagent 1 | Powder \* 2 bottles | - 20 ℃ | Add 2.67mL distilled water to each bottle  before use, dissolve thoroughly for use |
| Reagent 2 | Liquid 15mL \* 1 bottle | 2-8 ℃ |  |
| Reagent 3 | Liquid 80mL \* 1 bottle | 2-8 ℃ |  |
| Standard solution | 1ml \* 1 bottle of liquid | 2-8 ℃ | 5pmol/ml solution of p-nitrophenol. |

**Sample
treatment**

1.
Bacteria or cultured cells: first 1200rmp,2min, discard the supernatant; Add 100μl extract, ultrasonic
break bacteria (ice bath, power 200W, ultrasonic 3s, interval 10s, repeat 30 times);12000g centrifuge at
4℃ for 10min, take the supernatant and put it on the ice to be measured.

2.
Organization: According to the tissue mass (g), extract liquid volume (mL) is 1:5 ~10 ratio, ice bath
homogenization. 12000rmp centrifuge at 4℃ for 10min, take the supernatant, and put it on the ice to be
measured.

**Operation
steps**

1.
the
spectrophotometer preheats 30 minutes or more, adjusts the wavelength to 400nm, and zero the distilled
water.

2.
the
treatment of standard liquid: distilled water gradient dilution to 200, 100, 50, 25, 12.5, 6.25, 0
nmol/ml.

3.
calculate
the gradient dilution concentration.

a.
5u
mol/ml = 5000n mol/ml

b.
x
divided by 5000\*4

4.
Add each reagent in turn according to the operation table

|  |  |  |  |
| --- | --- | --- | --- |
| Reagent name (pL) | Measuring tube | Counter charge | Standard tube |
| Reagent 1 | 200 | - | - |
| Distilled water | - | 200 | - |
| Reagent 2 | 250 | 250 | - |
| Samples | 50 | 50 | - |
| Mix quickly and place in 37℃ accurate water baths for 30min | | | |
| Standard solution | - | - | 500 |
| Reagent Three | 1000 | 1000 | 1000 |
| Mix well, measure absorption value A at 400nm, and calculate ΔA=A determination -A control. Each measuring tube needs to be set up as a rule. | | | |

**Activity
calculation**

1.
Establishment
of the standard curve.

According
to the absorbance of the standard tube (x, minus the OD value of the classic tube with A concentration of
0) and the concentration (y, nmol/ml), the standard curve was established, and △A
was put into the standard curve to calculate the amount of product generated by the sample (nmol/ml).

2.
Calculation
of β-GAL activity.

Calculated
by bacterial or cell density:

Unit
definition: 1nmol p-nitrophenol production per 10,000 bacteria or cells per hour is one unit of enzyme
activity.

β
- GAL activity (U / 104
cell)
= (y \* V reverse total) present sample (500 \* V present V sample total) present T = 0.04 \* y

Cpr:
Sample protein concentration, mg/mL; V inverse total: total volume of the reaction system, 0.5mL; V
sample: the sample volume added to the reaction system, 0.05mL; Total V sample: add extraction liquid
volume, 1mL; W: sample mass, g; 500: total number of cells or bacteria, 5 million; T: reaction time,
0.5h.

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![](https://static.igem.wiki/teams/4904/wiki/results.png)

Overview

Results of Our Work

Future Plans

References

Overview

Our
project combines Optogenetics and photobiology to reach an unharmful control during the synthetic process.
We invented a new blue light GAL4-motivated system. We built the basic CRY2 and SPA1 blue light control in
the yeasts and compared that controlling system with the blue light CRY2/CIB1 controlling system.

![](https://static.igem.wiki/teams/4904/wiki/result/1.png)

Results of Our Work

1.
**P****las****mid
Construction**

**1.1
Polymerase Chain
Reaction (PCR)**

We
performed
PCR to obtain enough target gene fragments for further
construction.
Then, we used Electrophoresis testing and Gel Recovery.
As shown in Fig
1.1,
the gel indicates
the
success of our recovery of the gene fragments,
GFP
(781bp),
UAS
(114bp)
and
SPA1
(1635 bp).

![](https://static.igem.wiki/teams/4904/wiki/result/2.png)Fig
1.1. Electrophoresis gel results
of
three essential genes.

**1.2
Enzyme
Digestion**

We
doubled the AD. Then, we mixed the reagents. After that, we used PCR to make reactions. In Fig 1.2A, the
result of Gel Recovery shows that we have successfully operated double enzyme digestion for the AD gene.
In Fig 1.2B, the development of Gel Recovery shows that we have successfully done the process of single
enzyme digestion for the BD-CRY2 gene
that the strip of AD is 7987 bp and the strp of BD-CRY2 is
6526
bp.

![](https://static.igem.wiki/teams/4904/wiki/result/3.png)Fig
1.2. Electrophoresis gel results
of
AD, BD plasmid.

**1.3
Homologous
Recombination**

We
calculated
the volume of DNA segments.
Then,
we mixed the materials.
In
Fig 1.3A, the Gel Recovery result showed our success in recombining AD-SPA1gene
(1635bp)
and
BD-CRY2-N489-GFP
(781 bp)
gene.
Fig 1.3B is our genetic map of the AD-SPA1 gene and BD-CRY2-N489-GFP gene.

![](https://static.igem.wiki/teams/4904/wiki/bj2-results-update.png)

Fig
1.3. Electrophoresis gel results of AD-SPA1,
BD-CRY2-GFP plasmid.

To
ensure that the plasmid construction is 100% correct, we
sent them for sequencing and the results shown below in Fig 1.4 confirmed that
SPA1 has been successfully attached to the plasmid AD (left) and
the GFP has been successfully attached to the plasmid
BD-CRY2
(right).

![](https://static.igem.wiki/teams/4904/wiki/result/5-1.png)
![](https://static.igem.wiki/teams/4904/wiki/result/5-2.png)

Fig
1.4.
Sequencing result of AD-SPA1
and BD-CRY2-GFP

2.
**Yeast
Hybrid**

**2.1
Yeast
culture**

We
use PCR to heat the carrier DNA and then transfer it with another reagent into the clean bench. Then, we
use the coated plate process and rocking inoculation. After that preparation, we culture those bacteria
using a solution of bacteria and the defect medium. Figure 2.1 shows the defect medium in the
experiment.

![](https://static.igem.wiki/teams/4904/wiki/result/6.png)

Fig
2.1. Yeast growing on the plate.

**2.2****Blue** **L****ight** **Reactivity V****erification**

We
culture the bacteria on -Trp-Leu-His-Ade defect medium and -Trp-Leu defect medium at 28℃ under dark and
blue light condition irradiation for 48-96h. Here is our result of those two kinds of defect medium and
the bacteria on those two kinds of medium.
The
results showed that yeast in the experimental and control groups grew successfully in the -Trp-Leu culture
medium (Fig 2.2A and C) and -Trp-Leu-His-Ade culture medium under blue light conditions (Fig 2.2B). In the
dark, the yeast grew successfully in the -Trp-Leu dish but not in the -Trp-Leu-His-Ade culture medium (Fig
2.2D). The results showed that the two plasmids were successfully combined.

![](https://static.igem.wiki/teams/4904/wiki/result/7.png)

Fig
2.2.
Yeast growing condition on the different plates.

A
and C show the target plasmids BD-CRY2-N489-GFP and AD-SPA1 successfully enter the yeast.

B
and D show the yeast that has turned the blue light switch needs blue light to grow, and the plasmids
BD-CRY2-N489-GFP and AD-SPA1-N545 have successfully fused.

**2****.3****GFP F****luorescence****Expression Detection**

The
image below shows yeast under a fluorescence inverse microscope. It can be seen that yeast containing
CRY2/CIB1-GFP shows green fluorescence, which GFP produces.
When
exposed to blue light, the experimental group could see green fluorescence of GFP, while the control group
had a dark field of vision and no fluorescence. The experimental group fluoresces as evidence that the
proteins can interact to make the GFP gene appear. This image also proves the successful
attachment
and expression
of
AD and BD plasmid from the experimental group.

![](https://static.igem.wiki/teams/4904/wiki/result/8.png)

Fig
2.3.
Image
under fluorescence inverse microscope.

**2.4
Sensitivity****Evaluation** **of two****Blue Light** **“****S****witches****”**

We
test the two different switches during the Yeast Culture process. Here is the result of the traditional
switch CRY2/SPA1 (in Fig 2.4) and CRY2/CIB1, our newly-developed switch (in Fig 2.4).
The
results showed that with the decrease in concentration, basically, the growth of yeast
turns slower even with no growth under
blue light. Under blue light activation conditions, CRY2 interacts with
SPA1
or CIB1, forming a dimer, and in dark conditions, the dimer disassembles,
resulting in slow or halted growth in yeast.

It is worth noting that at low concentrations
(10-4
v/vol to
10-12
v/vol) of yeast, CRY2/SPA1 yeast continues to grow compared to CRY2/CIBI
yeast, which essentially stops growing. This indicates that the binding ability of CRY2 and SPA1 under
blue light is stronger, and they exhibit higher sensitivity to blue light responses.

![](https://static.igem.wiki/teams/4904/wiki/result/9.png)

Fig
2.4.
Concentration gradient test, the volume proportion of yeast solution decreases from left to
right

3.
**3.****β-Galactose Activity
Test**

**3.1
Standard
Curve**

In order to evaluate the engineering of Gal4-activated gene in
CRY2/SPA1
yeast, we conducted β-Galactose activity
test with β-galactosidase (β-GAL) Activity Assay
Kit from Sangon. We
test the activity of β-Galactose for every concentration, from 200
nmol/ml
to
0
nmol/ml. We
calculate the average value and gain the absorbance value. Then, we formed a curve that perfectly fitted
the linear model, like in Figure 3.1.

![](https://static.igem.wiki/teams/4904/wiki/result/10.png)

Fig
3.1. standard curve
of β-Galactose Activity: the horizontal axis is the Absorbance (A) and
the vertical axis is the concentration (nmol/ml) of β-Galactose

**3.****2****β-Galactose****A****ctivity** **with****D****ifferent Time intervals
(Dark and Blue
Light)**

The
β-Galactose activity of CRY2/SPA1
was
then tested from 0 to 3 hours, and the action was tested with an interval of 30 minutes. The result is
shown below in a diagram. It can be seen that the β-Galactose activity of yeast containing novel
blue-lights interaction under blue-light condition
indicates
an increasing trend
through
time. While in a dark state, the activity does not show a significant change
which also proves the engineering success of the blue light
“switch”.

![](https://static.igem.wiki/teams/4904/wiki/result/11.png)

Fig
3.2 β-Galactose
activity
(U) of
the yeast (CRY2/SPA1) under blue light and dark light against hours,
respectively

Future Plans

According
to the literature and experiments, the transplantation of microorganisms is essential in preventing and
treating diseases such as clostridium difficile infection, inflammatory bowel disease, inflammatory bowel
disease, and diabetes [1].

Chimeric
antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment. CAR-T has already
shown a significant benefit in clinical service for patients with terminal cancer. This artificial T cell
can identify cancer cells and use their hinge to identify the antigen of the cancer cell. After that, the
T cell will release cytokine to activate the immune system to fight against the cancer. However, this
treatment faces various challenging problems. The over-release of cytokine can lead to a toxic level, thus
causing several syndromes such as CRS, which refers to cytokine-release syndrome. CRS can be accompanied
by cardiac dysfunction, system failure, and death. A recent method to ameliorate CAR-T toxicity is to use
their gene strategies. However, this method is limited by its efficiency.

Our
new blue light-dependent interaction can add an “off-switch” on CAR-T cells. Since the
interaction is operated by light, the exchange can give an immediate response and prevent the T cell from
expressing more cytokine in time. CAR-T is an emerging approach to tumor treatment, and in a vast recent
scientific breakthrough, optogenetic technology wirelessly manipulates receptor T cells in vitro. And the
switch of our blue light can be applied in this method [2].

References

[1]
M. Cui et al., NIR light-responsive bacteria with live bio-glue coatings for precise colonization in the
gut. Cell Rep 36, 109690 (2021).

[2]
Nguyen, N.T., Huang, K., Zeng, H. et al. Nano-optogenetic engineering of CAR T cells for precision
immunotherapy with enhanced safety. Nat. Nanotechnol. 16, 1424–1434 (2021).
https://doi.org/10.1038/s41565-021-00982-5.

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