iGEM 2022 — Team thinker-china

* [![](https://static.igem.wiki/teams/4153/wiki/des-icons/spaceship.png)

  Week of July 11th to 17th](#general-terms)
* [![](https://static.igem.wiki/teams/4153/wiki/des-icons/document.jpg)

  Week of July 18th to 24th](#Current Detection Methods)
* [![](https://static.igem.wiki/teams/4153/wiki/des-icons/sq.jpg)

  Week of July 25th to 31st](#license)
* [![](https://static.igem.wiki/teams/4153/wiki/des-icons/switch.jpg)

  Week of July 18th to 24th](#hyperlinking)

# Note Book

## Lab Notes——Week of July 11th to 17th

### Prepare culture medium and experimental consumables；Activate E. coli DH5 α/ pSB1A3-mRFP，E. coliDH5 α/ pMD18T-insert.

###### *Participants：Sangji Zhuoma，Li Ruoxi，Fan Yuke，Zhang Haotian，Wang Chengyi，Feng Yihui, Ye Xiru, Chen Guo, Yuan Jiarui, Wang Haopeng, Mai Jiarui, Liu Lexuan*

#### **Experimental Procedures：**

Turn on the super clean table, place the centrifugal tube plate, test tube rack and maker pen, and conduct UV irradiation for 30 minutes.

#### **Sterilization**

Prepare 2 ml Eppendorf tubes, PCR tubes, and 30 ml centrifuge tubes, put them into aluminum boxes, and wrap them with gauze. Fill large (1 ml), medium (200 μl), small (20 μl), and 5 ml, 2 boxes of pipette each type, wrap the shell with kraft paper, and bind the pipette box with rubber band. Prepare ddH2O, 0.1 M CaCl2, 15% 0.1 M CaCl2. Prepare 200 ml LB medium, sub pack 40 test tubes, plug the mouth of the test tubes with test tube plugs, bundle 7 test tubes into one, and wrap the mouth of the test tubes with kraft paper. Prepare 50 mlx2 bottles of LB liquid medium. The above items are sterilized at 121 ℃ for 20 minutes.

**1. Diluting Primer**

Centrifuge the primer dry powder, add the amount of water marked on the tube wall, and obtain the mother liquor (100 mm), which is diluted 10 times with ddH2O.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/1.jpg)

**2. Prepare Amp antibiotic mother liquor (100 mg/ml):**

accurately weigh 1g Amp powder in a glass, add 10 ml dH2O to dissolve it, filter it into a sterile 2 ml EP tube with a filter, and store it at minus 20 ℃.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/2.jpg)

**3. Activate 3-tube DH5 α/ Psb1a3 MRFP, 3 tube DH5 α/ Pmd18t insert, 1 tube BL21:**

Open the super clean table, ventilate for 10 minutes, spray 70% alcohol on the sterilized LB test tube, amp mother liquor, medium gun head and strain preservation glycerol tube, and put it into the table. Light the alcohol lamp, burn the tube mouth and tube plug, and connect 100 μl to each tube preserved bacterial solution (2.5 μl needs to be added Amp antibiotic mother liquor), and then burn the tube mouth and tube plug again. Plug the tube mouth, bind the tube, and place it in a shaking table for overnight culture (150 rpm, 37 ℃).

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/3.jpg)![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/4.jpg)

## Lab Notes——Week of July 18th to 24th

### Plasmid extraction; PCR amplification; Double digestion; Gel recovery experiment.

###### *Participants：Sangji Zhuoma，Li Ruoxi，Fan Yuke，Zhang Haotian，Wang Chengyi，Feng Yihui, Ye Xiru, Chen Guo, Yuan Jiarui, Wang Haopeng, Mai Jiarui, Liu Lexuan*

#### **Experimental Procedures：**

**1. Plasmid extraction**

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/5.jpg)

1. Column balance: add 500 μl balance liquid BL to the adsorption column CP3 (the adsorption column is put into the collection tube), centrifuged at 12000 rpm for 1 minute, poured out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Take 1-5 ml of bacteria cultured overnight, add it to the centrifuge tube, use a Bench Centrifuge, centrifuge at 12000 rpm for 1 minute, and try to suck out the supernatant (when there is more bacterial liquid, the bacterial precipitation can be collected into a centrifuge tube by multiple centrifugations).

3. Add 250 μl solution P1 to the centrifuge tube with bacterial precipitation (please check whether RNase A has been added first), and use a pipette to completely suspend bacterial precipitation.

4. Add 250 μl solution P2 to the centrifuge tube, gently flip up and down 6-8 times make bacteria sufficient cracking.

5. Add 350 μl solution P3 to the centrifuge tube, flip it up and down gently for 6-8 times immediately and mix it well. At this time, white flocculent precipitation will appear. Then 12,000 rpm centrifuge for 10 minutes.

6. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is put into the collection tube), and pay attention not to suck out the sediment as much as possible. Centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

7. Add 600 μl rinse solution PW to adsorption column CP3 (please check whether absolute ethanol has been added first), centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

8. Repeat step 7.

9. Put the adsorption column CP3 into the collection pipe and centrifuge it at 12000 rpm for 2 minutes in order to remove the residual rinsing liquid in the adsorption column.

10. Place the adsorption column CP3 in a clean centrifuge tube and add 50 μl ddH2O drops to the middle of the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes to collect the plasmid solution into the centrifuge tube.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/6.jpg)
![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/7.jpg)

**Solution**

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/8.png)

**2. Prepare 50 ml of 1% agarose gel:**

Insert the comb of 2 μl into the 50 ml dispenser. Dissolve 0.5 g agarose in 50 ml Tae buffer, and use alcohol lamp to dissolve agarose. Cool down (based on the standard of not being hot), add 5 μl 10 mg/ml EB solution, shake evenly, and pour into the mixing plate. Wait about 40 minutes for solidification.

**3. PCR amplification:**

Using high fidelity enzyme PrimeSTAR ® HS amplified the target fragment from pSB1A3-insert, 4x50 μl reaction. 50 μl reaction system is as follows:

Buffer 10 μL

primer 1 1 μL

primer 2 1 μL

Template 2 μL

ddH2O 11 μL

PCR amplification procedure:

1. 98℃，5 min

2. The three reactions are carried out for 30 cycles:

1. 98℃ 10s

2. 55℃ 5s

3. 72℃ (1min/kb) 30s

3. 72℃ 8 min

4. stored in a 4-degree refrigerator

**4. Double digestion:**

The extracted plasmid pSB1A3-mRFP and the amplified target fragment were double digested by EcoRI and Xhoi. 4x50 μl reaction. 50 μl enzyme digestion system is as follows:

DNA 21 μL

EcoRI 2 μL

XhoI 2 μL

Buffer 5 μL

H2O 20

Enzyme digestion at 37 ℃ for 4H.

**4. Gel recovery:**

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/9.jpg)

1. Column balance: add 500 μl balance liquid BL to the adsorption column CA2 (the adsorption column is put into the collection tube), centrifuged at 12000 rpm for 1 minute, poured out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Cut a single target DNA band from the agarose gel (try to cut off the excess part) and put it into a clean centrifuge tube, and weigh it.

3. Add equal volume solution PN to the rubber block (if the gel weighs 0.1 g and its volume can be regarded as 100 µL, then add 100 µL PN solution), place it in a 50 ℃ water bath, and gently turn the centrifuge tube up and down during this period to ensure that the rubber block is fully dissolved. If there are still undissolved rubber blocks, you can continue to stand for a few minutes or add some sol solution until the rubber blocks are completely dissolved (if the volume of the rubber blocks is too large, you can cut the rubber blocks into pieces in advance).

4. Add the solution obtained in the previous step to an adsorption column CA2 (the adsorption column is put into the collection pipe), place it at room temperature for 2 minutes, centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection pipe, and put the adsorption column CA2 into the collection pipe.

5. Add 600 μL rinse solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CA2 into the collection pipe.

6. Repeat step 5.

7. Put the adsorption column CA2 into the recovery header, centrifuge at 12000 rpm for 2 min, and try to remove the rinsing liquid as much as possible. Place the adsorption column CA2 at room temperature for several minutes and dry it thoroughly to prevent the residual rinsing solution from affecting the next experiment.

8. Put the adsorption column CA2 into a clean centrifuge tube, drop an appropriate amount of elution buffer EB in the middle of the adsorption membrane, and place it at room temperature for 2 minutes. The DNA solution was collected by centrifugation at 12000 rpm for 2 min. Preserve DNA solution at -20 ℃.

**5. Activate one tube of E. coli DH5α and E. coli BL21 (for the preparation of competent cells).**

## Lab Notes——Week of July 25th to 31st

### Prepare competent cells, conduct ligation reaction, and transform ligation products

###### *Participants：Sangji Zhuoma，Li Ruoxi，Fan Yuke，Zhang Haotian，Wang Chengyi，Feng Yihui, Ye Xiru, Chen Guo, Yuan Jiarui, Wang Haopeng, Mai Jiarui, Liu Lexuan*

#### **Experimental Procedures：**

**1. Preparation**

Open the super clean table and put 1 ml pipette, 200 μ L pipette, 5 ml pipette, 2ml and 30 ml centrifuge tube, 2 x 50 ml LB medium, UV irradiation. Place 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a 4 ℃ refrigerator for precooling.

2. Prepare DH5 α And BL21 competent cells：

1. Transfer the activated DH5α to 50 ml medium (37 ℃, 150 RPM), and monitor the OD600 value with a spectrophotometer until the OD600 is 0.4-0.6 (use dH2O as the control, about 2h).

2. Divide the bacterial solution into 30 ml centrifuge tubes (25 ml for each tube). Centrifuge 4 degrees precooling. Put 30 ml centrifuge tube into the centrifuge, centrifuge 4000 g for 10 min, discard the supernatant, add 20 ml of 0.1 M CaCl2 solution, and ice bath for 30 min.

3. Divide the competent cells into 2 ml centrifuge tubes on ice, 100 μL for each tube. Mark and store the competent cells in the refrigerator at -80 ℃.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/10.jpg)
![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/11.jpg)

**3. Prepare DH5 α And BL21 competent cells：**

3. Prepare DH5 α And BL21 competent cells：

\*Note: this system is an empirical system, and the best connection carrier / target fragment volume ratio can also be calculated according to the instructions:

T4 DNA ligase 1 μL

5 x DNA ligase buffer 2 μL

Linear plasmid 1 μL

Insert DNA 6 μL

Connect with PCR instrument at 16 ℃ for 1h. The linked products were stored at - 20 ℃.

**4. Preparation of antibiotic plate**

Prepare LB solid medium (add 4.5 g agar to 300 ml LB liquid medium),121 ℃, high temperature and high pressures sterilization for 15 minutes. After cooling to about 60 ℃, add 150 μ L 100mg/ml amp antibiotic, shake it evenly, and pour it into a disposable Petri dish (about 10 plates can be poured).

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/12.jpg)

**5. Preparation of antibiotic plate**

Add 10 μL pSB1A3-target segments to 100 μL competent cells, ice bath for 30min, then 42 ℃ water bath for 60s, then ice bath for 5 min, and finally add 1 ml LB liquid medium, culture for 1h to resuscitate (37 ℃, 150 rpm). In the super clean table, take 100 μL resuscitation solution, use sterile coating rod to coat Amp antibiotic plate.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/13.jpg)
![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/14.jpg)

## Lab Notes——Week of July 18th to 24th

### Obtain positive clones

###### *Participants：Sangji Zhuoma，Li Ruoxi，Fan Yuke，Zhang Haotian，Wang Chengyi，Feng Yihui, Ye Xiru, Chen Guo, Yuan Jiarui, Wang Haopeng, Mai Jiarui, Liu Lexuan*

#### **Experimental Procedures：**

1. Add 2.5 μL amp antibiotics into LB test tube, select 3 tubes of colonies into lb Test tubes, and culture overnight in a shaking table (37 ℃, 150 rpm).

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/15.jpg)

2. Mark 6 DH5α/pSB1A3-target fragment colony. Select half of the colonies as the template, and use Taq enzyme for colony PCR. The primers were VF and VR, and the reaction system and procedure were carried out according to the instructions. Prepare 50 ml agarose gel and use DL 2000 DNA marker for electrophoresis.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/16.png)
![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/17.png)

3. Pick the DH5α/pSB1A3- target fragment colony that is verified to be correct add to LB liquid medium, add 2.5 μ L amp antibiotics, expanded culture.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/18.jpg)

**Transform engineering vector into expression host E. coli BL21, Verify experiment.**

1. Extracted the plasmid was from the bacterial solution that verified the correct DH5α/pSB1A3-target fragment.

2. Transform the pSB1A3-target fragment into the E. coli BL21.

3. Fluorescence microscope verification experiment:

Basic operation steps of fluorescence microscope:

(1) Install UV protective cover.

(2) Turn on the power control box switch of the high-pressure mercury lamp.

(3) Insert the light barrier and interrupt the light path.

(4) Warm up for 5 minutes.

(5) Place the glass slide containing the sample on the stage.

(6) Select the objective lens.

\*(in the order of low power first and then high power)

(7) Rotate the rotating disc of the spectroscope assembly and select the required spectroscope assembly:

\*( "O" is used to observe the transmitted light; "Wu" is used to observe blue fluorescence (such as dapl); "WB" is used when observing green fluorescence (such as FITC); "WG" is used for observing red fluorescence (such as TRITC).)

(8) Use barrier filter

(Most of which are built into fluorescence microscope at present)

(9) Adjust the focal length through thick and thin screws.

(10) Turn on the computer connected to the microscope, click the digital imaging system software, and collect digital images.

![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/19.jpg)
![table](https://static.igem.wiki/teams/4153/wiki/photos/notebook/20.png)

* [![](https://static.igem.wiki/teams/4153/wiki/des-icons/spaceship.png)

  Cycle 1](#general-terms)
* [![](https://static.igem.wiki/teams/4153/wiki/des-icons/document.jpg)

  Cycle 2](#Current Detection Methods)

# Engineering Success

![table](https://static.igem.wiki/teams/4153/wiki/photos/engineer/cycle.jpeg)

## Cycle 1

#### Design

Our initial notion is to design a complete circuit that allows visible color change of different greyscale values, for us to measure the intensities of target molecules. First and foremost, we designed a module that allows stable expression of b-galactosidase. Bacterial endogenous ß-galactosidase can catalyze the chromogenic reaction of the colorless substrate x-gal or oNPG, and in combination with IPTG inducers, the chromogenic reaction will demonstrate a clear pattern of color change on different greyscale values.

The second module, the lysis module, is responsible for the visibility of our chromogenic reaction. We couldn’t observe the color change inside the cell’s cytoplasm through the naked eye, therefore, a lysis gene sequence comes into place. Phages contain a specific SRRz lysis gene in their gene sequence, the expression of which has the effect of lysing the bacterial cell wall. SRRz lysis gene is inserted into the bacterial plasmid, then, when the bacteria receive a signal from a specific molecule, the operon inserted upstream of the SRRz gene leads to its rapid expression, resulting in the lysis of the bacterial wall.

![table](https://static.igem.wiki/teams/4153/wiki/photos/engineer/000.png)

#### Build

We selected two inducible promoters, the pBad/araC promoter (BBa\_10500), also known as the arabinose promoter, and the copper-sensitive promoter (BBa\_1760005), to activate our gene circuit. The RBS we used in our circuit is BBa\_B0034, a relatively strong RBS used by many previous iGEM teams. Right after the b-galactosidase expression gene, the RBS is implemented again, followed by the SRRz lysis gene. Finally, we selected a double terminator BBa\_B0015 to stop transcription. BBa\_B0015 terminator is a double terminator, composed of BBa\_B0010 and BBa\_B0012. It is the most frequently used terminator in the community, emphasizing its high efficiency.

#### Test

In the first version of our design, we succeeded in obtaining a colour change reaction. This proves that when either arabinose or copper ions were present in the solvent, the process was initiated and b-galactosidase was translated, and the SRRz lysis gene was successfully translated at the RBS. Hence, b-galactosidase flowed out to the surrounding solution and reacted sufficiently with X-gal to produce the colour change. However, we found that not only did the colour change fail to meet expectations, but that the gene circuit did not work efficiently, leading to bacterial death before sufficient b-galactosidase was produced.

#### Learn

After talking to our advisors, we found out that this was due to the fact that the lysis gene started working shortly after the activation of b-galactosidase production, so the bacteria died before adequate galactosidase could be produced. Through discussions among group members and arguments from the data, with the help of our advisors, we decided to apply an individual circuit for galactosidase expression to improve the effectiveness of the colour change.

## Cycle 2

#### Design

![table](https://static.igem.wiki/teams/4153/wiki/photos/engineer/0000.png)

For our new circuit, we adjusted the design of galactosidase expression. With the addition of a double-terminator and an inducible promoter, the galactosidase expression circuit became independent, allowing sufficient amount of galactosidase to be produced before the bacteria was triggered to death.

#### Build

We have carried out the following improvements:

1. The addition of T7 promoter from T7 phage, which reacts specifically to the T7 RNA polymerase.

2. A double terminator was added prior to the lysis reaction to prevent RNA polymerase from continuing transcription. With the help of the terminator, RNA polymerase will be shedded from the DNA strand, thus terminating transcription.

The T7 promoter is used to translate galactosidase at the RBS, and then the reaction is stopped at the first terminator. For the lysis sequence, arabinose or copper ions are used as promoters to translate the lysis gene at the RBS, and when the cell is lysed, the reaction is halted by the terminator. With the addition of X-gal, a colour change is induced.

#### Test

![table](https://static.igem.wiki/teams/4153/wiki/photos/engineer/3.jpeg)

The results suggested that the lysis circuit works regularly, as the rapid decline of OD600 at 10^-5 mol/L indicates lysis of bacterial wall, which proves that our modified gene circuit could function normally and continue to work in a relevant context.

![table](https://static.igem.wiki/teams/4153/wiki/photos/engineer/4.png)

After a round of improvements to our detection system, our advisors collected samples of soil and river water from Dongdagou, Baiyin city, to test and verify the results of our cycle 2 gene circuit. The samples successfully undergo chromogenic reactions, demonstrating a series of blue, each in a different shade.

With the addition of the T7 promoter and the double terminator, the bacterium is able to produce galactosidase continuously before activation including the fermentation phase, the subsequent colour development with X is optimised and the bacterium is produced much more efficiently.

#### Learn

Our improved gene circuit functions more efficiently and conducts successful chromogenic reaction, as shown by the results of our experiments (See: Cycle 2-Test). For future improvements, we aim not only to maintain stability, increase efficiency and add new components to our gene circuit that aids regulation, but looking forward to innovative possibilities of inserting more operons into our bacterial plasmid. We will carry out more experiments, evaluating effectiveness of future modified plasmids, developing new concepts of multi-operon, and hence multi-functioning genetic sequence, balancing advantages to its trade-offs.

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}