iGEM 2024 — Team rainsaca-china

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{% block title %}Notebook{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5488/cover/{{self.title()|lower}}.png {% endblock %}
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{{self.title()}}

[Calendar: February - April 2024](#a1)
[**Project launch - team member recruitment**](#b1)
[Calendar: April - June 2024](#a2)
[**Team building and knowledge learning**](#b2)
[Calendar: April - July 2024](#a3)
[**Project planning**](#b3)
[Calendar: July - August](#a4)
[**Laboratory Activities**](#b4)

# {{self.title()}}

Calendar: February - April 2024

**Project launch - team member recruitment**

Team recruitment goal: to form a diverse, interdisciplinary team whose members have different professional skills and knowledge backgrounds, who can take on different roles and tasks in the project to ensure the smooth progress of this year's iGEM project. We will publish recruitment information through school clubs and social media accounts.

Calendar: April - June 2024

**Team building and knowledge learning**

1. After all team members are determined, we formulate a learning plan based on their professional background and project requirements. At a fixed time every week, team members study and discuss relevant knowledge and skills, make continuous progress, and prepare knowledge for the completion of various tasks in subsequent projects.

2. Organize team building activities every two weeks, including sharing of learning problems, time management discussions, sharing of innovative ideas, sharing of interesting life stories, etc., to promote mutual understanding and trust among members and enhance team cohesion.

Calendar: April - July 2024

**Project planning**

1. 6th, April

**Meeting topic:** Project kick-off meeting

**Meeting objectives:** Team members get to know each other, understand the team of instructors, and clarify the iGEM competition requirements and project goals.

**Meeting agenda:**

Introduction of team members and instructors;

Each member briefly introduces himself to prepare for the subsequent division of labor.

Instructors introduce their research fields, professional experience, and roles and responsibilities in the project.

Emphasis the importance of the project and the goals to be achieved.

The iGEM competition requires analysis and planning for the new season, and clear goals, including the work content and expected results to be completed at each stage.

2. 20th , April

**Meeting topic:** Discussion meeting on the application of synthetic biology technology and global issues

**Meeting objectives:** Understand the current status of the application of synthetic biology technology in different industries, and team members share the global issues they are concerned about, and prepare for the inspiration of the topic.

**Meeting agenda:**

Members collect relevant information in advance to understand the application of synthetic biology technology in different industries. The team discusses these cases together, summarizes the application advantages and limitations of synthetic biology technology, and thinks about how to learn from and innovatively apply these technologies in the project.

And share the potential connection between the global issues they are concerned about and synthetic biology technology, and think about how to use synthetic biology technology to solve these problems.

3. 28th, April

**Meeting topic:** Project inspiration meeting

**Meeting objectives:** Based on the previous discussion, preliminarily determine the direction of the project topic.

**Meeting agenda:**

Team members brainstormed and sorted out several research directions closely related to humans, such as environmental protection, agriculture, food and nutrition, and proposed various possible project ideas. For example:

1. Use gene editing technology to edit key genes of crops to make them resistant to specific pests and diseases and reduce the use of chemical pesticides.

2. Use gene editing technology to transform plants or microorganisms so that they can synthesize a large amount of specific nutrients needed by the human body, providing raw materials for the development of functional foods.

After the meeting, search and review literature independently to understand feasibility and innovation.

4. 4th ,May

**Meeting topic:**  Conceptualization of the topic

**Meeting objectives:**  Determine the research direction and key content of the topic.

**Meeting agenda:**

The tutor analyzed and guided, combining environmental protection, agricultural sustainability, food and nutrition, and focusing on the plant itself - plant metabolism.

Analyzed the role of plant metabolism, a physiological process of its own, in growth and development support, environmental adaptation and defense, and contribution to the ecosystem.

5. 5th, May - 20th, May

**Task:**  Literature search and reading

**Objective:**  To gain in-depth knowledge about natural plant secondary metabolites, including their production mechanism, types, characteristics, research status, and their effects and potential value on human health.

6. 25th, May

**Meeting topic:**  Literature survey summary

**Meeting objectives:** Sort out the results of literature reading

**Meeting agenda:**

The key research results were sorted out and analyzed, and the appropriate research plant - cucumber was selected after brainstorming.

The next step plan was clarified, and team members were required to conduct further research on the plant metabolism of cucumber after the meeting, including in-depth understanding of the metabolic pathways, gene expression and types of secondary metabolites of cucumber, so as to prepare for the determination of specific research methods and experimental designs.

7. 1st, June

**Meeting topic:** Deepening of the topic

**Meeting objectives:** Further clarify the research content and methods of the topic.

**Meeting agenda:**

The secondary metabolites flavonoids in cucumber epidermal hairs were determined as the research object, and the key genes for flavonoid synthesis were determined.

Hypothesis: Can the key gene CsTBH and flavonoid metabolism gene (PAL) for the initiation of cucumber epidermal hairs be modified to produce more flavonoids in cucumbers by using synthetic biology?

8. 2nd, June

**Meeting topic:** Project division meeting

**Meeting objectives:** Make reasonable project division according to the research content and members' skills and expertise.

**Meeting agenda:**

The instructor introduces the principles of project division to ensure that each member can give full play to their strengths. Emphasize the importance and necessity of division of labor, as well as the key role of teamwork in the project.

According to the principle of division of labor, the research tasks of the project are broken down into specific subtasks, and the dry and wet team members are assigned. The work responsibilities and task objectives of each member are clarified, and effective communication and collaboration are carried out in the team WeChat group.

9. 22nd, June

**Preliminary experimental design**

Under the guidance of our instructor, we defined the experimental objectives: to construct a co-expression vector of CsTBH and CsPAL, verify the gene interaction, and explore its effect on the synthesis of flavonoids in cucumber epidermal trichomes.

The CsTBH and CsPAL genes were obtained through gene cloning, and related vectors were constructed. The gene interaction was verified by yeast single hybridization, and the constructed vector was transformed into cucumber plants using Agrobacterium-mediated transformation. Finally, the experimental results were verified by phenotypic observation and flavonoid content determination (liquid chromatography-mass spectrometry and fluorescence staining). It is expected that the expected results will be achieved in terms of gene cloning and vector construction, gene interaction verification, plant transformation phenotype and flavonoid content determination.

Calendar: July - August

**Laboratory Activities**

Experimental safety training

Note: General laboratory safety knowledge includes rules and regulations, personal protective equipment, use of laboratory instruments and equipment, biosafety knowledge including biohazard identification, operating specifications, and reagent classification and storage and use. Master operating procedures and emergency measures to ensure your own safety and the laboratory environment.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img1.png)

Acquisition of CsTBH and CsPAL genes

RNA was extracted from cucumber plants and reverse transcribed into cDNA using reverse transcriptase.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img2.png)

Specific primers were designed based on the known CsTBH and CsPAL gene sequences, and PCR amplification was performed using cDNA as a template to obtain the target gene fragments.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img3.png)![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img4.png)

TOPO vector cloning verification

The PCR products were ligated to the TOPO vector and transformed into Escherichia coli. Colony PCR and sequencing were used to verify the consistency of the cloned CsTBH and CsPAL genes with those in the cucumber genome database to ensure that the selected cucumber materials had not mutated in the natural state.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img5.png)

Construct overexpression vector and yeast one-hybrid vector

Construct PCY-CsTBH and PCY-CsPAL overexpression vectors and CsTBH-PLacZi vector (for activating CsPAL promoter) respectively.

Verify the constructed vectors by colony PCR and sequencing to ensure that the target gene fragment is correctly inserted into the vector.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img6.png)![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-new-img7.png)

Yeast competent cell transformation

Extract the plasmid DNA of the constructed CsTBH-PLacZi vector and related control vectors.

Transform the plasmid DNA into yeast competent cells (such as EGY48) and perform operations according to specific transformation methods, including adding Carrier DNA, PEG/LiAc and other reagents, water bathing, centrifugation and other steps, and finally spread the transformed cells on SD plates for culture.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img8.png)

Chromogenic culture and result observation

Prepare color development medium, inoculate yeast cells grown on SD plates into the color development medium, and culture at a specific temperature (such as 29°C) for a specific time (such as 48-96h).

Observe the color development of yeast cells. If yeast cells containing CsTBH-PLacZi vector appear blue in the color development medium, while control cells do not show color, it proves that CsTBH can bind to CsPAL promoter and there is an interaction between the two.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img9.png)

Agrobacterium-mediated plant transformation

The constructed PCY-CsTBH and PCY-CsPAL overexpression vectors were transformed into Agrobacterium (GV3101) and transformed by freeze-thaw method or electroporation method.

The Agrobacterium containing the overexpression vector was infected into cucumber cotyledons, and transgenic cucumber plants were obtained through tissue culture steps such as co-cultivation, induction of adventitious buds, and rooting.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img10.png)

Greenhouse cultivation

Cucumber plants are cultivated in artificial greenhouses, and environmental conditions such as light, temperature, and water are artificially controlled to monitor plant growth in real time and improve the survival rate of genetically modified plants.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img11.png)

Phenotypic observation

The transgenic cucumber plants and wild-type cucumber plants were observed and their fruit thorn morphology was compared. If the transgenic plants had many small black thorns on their fruit thorns, it might be related to the production of flavonoids, which needs further verification.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img12.png)

Flavonoid content determination

The results of LC-MS analysis showed that the flavonoid content in the fruits of transgenic cucumber plants was significantly higher than that in wild-type plants, and the fluorescence staining results assisted in proving the changes in flavonoid content.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img13.png)![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img14.png)

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{% block title %}Engineering{% endblock %}
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{{self.title()}} Success

[Overview](#a1)
[Cycle1 Construction and preservation of CsTBH and CsPAL overexpression vectors in E.coli.](#a2)
[Design](#b1)
[Build](#b2)
[Test](#b3)
[Learn](#b4)
[Cycle2 Validation of CsTBH regulation of CsPAL expression in yeast system](#a3)
[Design](#b5)
[Build](#b6)
[Test](#b7)
[Learn](#b8)
[Cycle3 Design of CsTBH -CsPAL Module in Plant Live Bioreactors](#a4)
[Design](#b9)
[Build](#b10)
[Test](#b11)
[Learn](#b12)

# {{self.title()}} Success

Overview

We follow a Design-Build-Test-Learn cyclic pattern to move the project forward. This year's project was roughly divided into three cycles. In the first cycle, we constructed CsTBH and CsPAL overexpression vectors, which are the prerequisites for the synthesis of flavonoids in plants, and stored the constructed CsTBH-PCY and CsPAL-PCY in E. coli; in the second cycle, we constructed the CsTBH-PJG and CsPAL-lacZi vectors, which were used to verify the regulatory relationship between CsTBH and CsPAL, and to clarify the relationship between CsTBH and CsPAL. the regulatory relationship between CsTBH and CsPAL, and after clarifying that CsTBH positively regulates CsPAL, we entered the third cycle; we designed the bioreactor based on the results of the first two parts. CsTBH-PCY/CsPAL-PCY was cotransformed into cucumber plants, and the phenotypes of cucumber fruit spines were observed for the quantitative assay of in vivo plant bioreactors.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img1.png)

Fig.1: DBTL (design-build-test-learn) schema diagram for this project

Cycle1 Construction and preservation of CsTBH and CsPAL overexpression vectors in E.coli.

Design

In biological studies, E. coli often serves as a host for functional vectors. We constructed vectors for CsTBH-PCY, CsPAL-PCY by cloning the sequences of the coding regions of the genes CsTBH and CsPAL, ligating them into the overexpression vector PCY, and then preserving their plasmids in E. coli (DH5α).

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img2.png)

Fig.2: Mapping of PAL-LacZi, TBH-PJG, PAL-PCY and TBH-PCY plasmid vectors

Build

1. Normal PCR

2. Gel Electrophoresis

3. Assembly

4. E. coli Transformation

5. Colony PCR

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-new-img-3.png)

Fig. 3: Sequencing results of CsTBH-PCY vector

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-new-img-4.png)

Fig. 4: Sequencing results of CsPAL-PCY vector

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img5.png)

Fig. 5 PCR results of CsTBH-PCY E. coli colonies

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img6.png)

Fig. 6 PCR results of CsPAL-PCY E. coli colonies

Test

The constructed PCY-TBH as well as PCY-PAL can grow on E. coli.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/new-img-7.png)

Fig.7 Growth of constructed E. coli colonies of TBH-PCY and PAL-PCY vectors on culture medium

Learn

Successful chassis synthesis vectors were found to be constructed by cloning the gene and culturing.

Cycle2 Validation of CsTBH regulation of CsPAL expression in yeast system

Design

To verify that the TBH gene is able to interact with the cis-acting regulatory elements on the promoter of the PAL gene, and thus to demonstrate that the two interact.

Build

1、Vector digestion

2、PCR cloning

3、Homologous recombination

4、Plasmid extraction

5、Yeast cell transformation

Test

Demonstrate the reliability of results using the blue color development system

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img8.png)

Fig.8 Yeast color development in chromogenic plates and analysis of interactions

Learn

The yeast one-hybrid assay was able to demonstrate that CsTBH binds the promoter of CsPAL, theoretically validating the feasibility of using the CsTBH-CsPAL module as a flavonoid bioreactor.

Cycle3 Design of CsTBH -CsPAL Module in Plant Live Bioreactors

Design

In order to design a plant bioreactor that produces flavonoids as expected, CsTBH-PCY and CsPAL PCY were co expressed in plants using a system of Agrobacterium mediated infection of cucumber cotyledons based on the results of the first two experiments. The purpose is to expect that our bioreactor can truly produce more flavonoids, which can be better applied in human life.

Build

1、Fluorescence color development

2、Tissue culture

3、Phenotypic investigation

4、Liquid chromatography

Test

Validation of flavonoid production using transgenic phenotypes, substance assays

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img9.png)

Fig.9 Fruit phenotypes of CsTBH overexpressing transgenic plants

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img10.png)

Fig.10 Mass spectra and bar graphs of total flavonoids content in the fruits of CsTBH overexpression plants by LC-MS

Learn

The CsTBH-CsPAL module is feasible as a flavonoid bioreactor. Providing a basis for large-scale production in future plant factories.

{% endblock %}

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{% block title %}Experiments{% endblock %}
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{{self.title()}}

Experiment Overview

[Procedure](#a2)
[Experimental Steps:](#a3)
[RNA extraction](#b1)
[Reverse Transcription](#b2)
[Cloning primer design](#b3)
[RCR amplification](#b4)
[Agarose gel electrophoresis](#b5)
[PCR product purification and recovery](#b6)
[Connection of transformed E. coli sensory state](#b7)
[Pick bacteria, bacteria P and sequencing to protect bacteria](#b8)
[Plasmid extraction](#b9)
[Plasmid digestion](#b10)
[Yeast Receptor Transformation](#b11)
[Yeast color development](#b12)
[Overexpression primer design](#b13)
[Transformation of Agrobacterium tumefaciens receptor cells](#b14)
[Genetic transformation](#b15)
[Fluorescence quantification (qRT-PCR)](#b16)
[LC-MS (Liquid Chromatography-Mass Spectrometry)](#b17)

# {{self.title()}}

Experiment Overview

This experimental page is about all the experimental protocols used in our dry lab and the breakdown of each
specific experimental step, where the target gene fragment is transformed into E. coli to extract the
plasmid, and then the plasmid is used to construct the gene overexpression vector, after which
Agrobacterium-mediated methods are used to obtain the overexpression plants, and finally the flavonoid
content is detected by EC-MS.

Procedure

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img1.webp)

Experimental Steps:

RNA extraction

1、Sample handling:

① A 50-100 mg plant sample was taken from a centrifuge tube treated with RNAase scavenger using RNAase
scavenger and loaded into steel beads burned with alcohol;

② Liquid nitrogen grinding. Add 1000ul of cell lysis solution A and place in a vortex shaker for 30s to fully
lysed.

2、Add 300ul of Deproteinized Liquid B and 200ul of chloroform to the centrifuge tube and mix well with rapid
shaking, at this time the solution is uniformly turbid, and leave it at room temperature for 2 minutes.

3、Centrifuge for 5-10 min at room temperature using a centrifuge at 12,000 rpm, and aspirate the supernatant
(no more than 700ul) into another clean 1.5 ml centrifuge tube, in which the lower and middle layers contain
mainly DNA, proteins, and other impurities, so be careful not to aspirate them.

4、Add an equal volume of rinse solution C, mix well by inversion, and then the resulting mixture was divided
into two times if the same adsorption column, centrifuged at 12000 rpm for 1 min, and poured off the waste
liquid.

5、Then add 500ul of column wash solution D to the adsorption column, centrifuge at 12000rpm for 1min at room
temperature, pour off the waste liquid, add another 500ul of column wash solution, repeat once, and finally
leave the column in vacuo for 1min to remove the residual liquid.

6、RNA elution: transfer the adsorption column into RNase-free 1.5ml centrifuge tube, which can be divided
into two times by adding 30ul of RNA eluent F each time, leaving it at room temperature for 3-5min, and
centrifuging it at 12000rpm for 1min, then total RNA of plant body can be obtained.

Reverse Transcription

1、To calculate the volume of RNA required, since a typical experiment requires 2ug of RNA, divide the RNA
concentration by 2 to obtain the volume of RNA required.

2、Sequentially add RNase free ultrapure water, 1ul gDNA Remove, 4ul 5×TURE RT Mix, RNA.

3、PCR program: 42°C 15min , 85°C 5s , 12°C ∞ .

4、The cDNA obtained after reversal was placed in a 4°C refrigerator for backup.

Cloning primer design

1、Open the DNAMAN software, select the Primer option Load Primer, and then select From input, at this time, a
dialog box will appear, and then in our coding region of the sequence of the most upstream in accordance
with the principles of primer design to pull the 20-30bp fragment sequence, imported into the dialog box,
click ok.

2、Then in the selection of Primer options in the Melting Temperature, at this time you can observe the
annealing temperature, GC content of the fragment we pull the value.

3、According to the principle of primer design, we keep testing the sequence fragments until we find a
compatible one that can be used as our F primer.

4、R primer design steps and F primer consistent, the final choice of primer fragments for reverse
complementation can be.

RCR amplification

1、First of all, prepare the cDNA template, primers, droplet water, and M5-Taq enzyme.

2、According to the following indicators in the PCR tube to add the prepared materials: 1.25 μl F primer, 1.25
μl R primer, 2.5 μl cDNA, 25 μl M5-Taq enzyme, 20 μl droplet water.

3、After adding the PCR system, set up the PCR program according to the following parameters: (1)
pre-denaturation: 95℃ for 3 minutes.

(2) Denaturation: 94℃ 25s (3) Annealing: 58℃ 25s (4) Extension: 72℃ 1min (5) End extension: 72℃ 5min (6) ∞:
4℃. (In which steps 2-4 set 34 cycles, the annealing time can be adjusted by the transcription speed of the
enzyme used and the maximum length of the fragment to be copied.)

4. At the end of PCR, use gel electrophoresis to verify that the length of the sample is correct.

Agarose gel electrophoresis

1、Preparation of TAE buffer: according to the ratio, dilute 50×TAE solution into 1×TAE solution.

2、Preparation of agarose gel mixture: mix 0.9g agarose with 60ml 1×TAE solution in a conical flask.

3、Dissolve agarose: Heat the agarose gel mixture in a high-power microwave oven. When the liquid begins to
boil, remove the conical flask and gently stir with gloves on. Then return the conical flask to the
microwave and repeat heating and stirring until the agarose powder is completely dissolved.

4、 Add Nucleic Acid Dye: After cooling slightly, add nucleic acid dye to the gel mixture at a ratio of
1:10,000, stirring or gently rotating to mix completely.

5、 Pour into gel mold: Pour the liquid gel into the gel mold and wait for about 20 minutes until the gel is
completely dry, then take out the gel from the mold (make a gel plate, choose a large hole or a small hole
comb as needed).

6、Prepare the electrophoresis tank: Pour TAE solution into the electrophoresis tank and put in the gel. Add
more TAE solution until the gel is completely covered by TAE solution.

7、Add PCR product: Add PCR product and Marker into the wells of the gel. The amount added can be determined
according to the size of the gel.

8、Start running the gel: run the gel at 150V for 15 minutes. If needed, increase the running time until the
product runs at least to the middle of the gel. For clearer results, reduce the voltage and extend the run
time.

PCR product purification and recovery

1、Under the long-wave ultraviolet light or blue light, use a clean blade to cut off the DNA bands to be
recovered, try to excise the gel without DNA, and get the gel volume as small as possible.

2、Put the cut off gel containing DNA bands into a 1.5ml centrifuge tube and weigh it.

3、For every 100mg of 1% agarose gel, add 100μl of lysis/binding solution HB (high concentration). For high
concentration agarose gels, the amount of lysate/binding solution HB (high concentration) added should be
increased proportionally. In order to simplify the experiment, it is recommended that the amount of
lysate/binding solution HB (high concentration) added is uniformly 400 μl.

4、Place in a water bath at 56°C for 5-10 minutes (or until the gel is completely dissolved). Vortex shaking
every 2-3 minutes to help accelerate dissolution.

Column equilibration: Take a new silica gel membrane adsorption column EC in a collection tube, draw 100μl of
equilibration buffer into the column. centrifuge at 12000rpm for 1 minute, pour out the waste liquid in the
collection tube and put the adsorption column back into the collection tube. At this time, the equilibrium
solution pretreatment column is completed.

5、Add the solution obtained in the previous step to the adsorbent column EC (the adsorbent column is put into
the collection tube), leave it at room temperature for 1 minute, centrifuge at 12,000 rpm for 30-60 seconds,
and pour out the waste liquid in the collection tube.

6、Add 600μl of rinsing solution WB (please check if you have added anhydrous ethanol first!) Centrifuge at
12,000pm for 30 seconds and discard the waste liquid.

7、Repeat step 6 again.

8、Place the column EC back into the empty collection tube and centrifuge at 12,000 rpm for 2 minutes to
remove as much rinse solution as possible.

9、Take out the adsorbent column EC and put it into a clean centrifuge tube, add 50 μl of elution buffer EB to
the middle of the adsorbent membrane, leave it at room temperature for 2 minutes, and centrifuge it at
12,000 rpm for 1 minute. The final liquid in the centrifuge tube is the PCR purified product.

Connection of transformed E. coli sensory state

1、Room temperature (25 ℃ -37 ℃) set up 10 μl ligation system. The system is: purified PCR product 8μl, pTOPO
Vector 1μl, 10 × Enhancer 1μl.

2、After adding the reagents, use a pipette to gently blow and mix or flick the bottom of the tube to mix, and
then centrifuge at low speed to collect all the liquid at the bottom of the tube.

3、Room temperature (25℃-37℃) ligation 5-10 minutes (recommended to put on the PCR instrument temperature
control). Place the ligation product on ice and set aside.

4、Take out the E. coli receptor cells from -80℃, quickly insert them into the ice bath, thaw and melt (about
3 hours).

5、Immediately add 10 μl of ligation product, gently mix by hand by dialing the bottom of the centrifuge tube
(avoid sucking and beating with a gun), and place in the ice bath for 30 min. The 30-minute ice bath will
help the effective binding between the ligation product and the E. coli cells.

6、Warm up the centrifuge tube at 42℃ for 90 seconds, then put it back to the ice bath and leave it for 2-3
minutes, do not shake the tube during the process.

7、Add 300μl LB medium (without antibiotics), 37 ℃ 200rpm shaking culture 1h.

8、Take 100-200μl of bacterial solution with a coater plate (ampicillin plate). When coating the plate must
make the bacterial liquid evenly distributed in the ampicillin plate, and must be blown dry before sealing
the plate, the plate inverted culture in 37 ℃ biochemical incubator.

Pick bacteria, bacteria P and sequencing to protect bacteria

1、Generally in the bacterial solution coated ampicillin plate into the biochemical incubator 12-16h, the
plate will grow single colonies, at this time you can carry out the operation of picking bacteria.

2、Fifteen minutes in advance, the gun head, pipette gun, LB liquid medium, 2ml centrifuge tube, ampicillin
antibiotics, etc. into the ultra-clean bench for ultraviolet sterilization (not less than 15min).

3、After spraying the plate full of single colonies with alcohol, take it into the ultra-clean bench.

4、Add one thousandth of ampicillin antibiotic into LB culture medium (Example: 50ml culture medium + 50μl
ampicillin antibiotic).

5、Add the mixed medium into 2ml centrifuge tube for spare (each tube + 1ml).

6、Take a small gun tip to pick the single colony on the plate, and then hit the gun tip into the prepared
centrifuge tube.

7、Followed by the centrifugal tube into the 37 ℃ shaker for shaking bacteria, 4-5h or so.

8、At this time, the bacterial liquid has been shaken mixed, you can use the bacterial liquid as a template
for PCR amplification, and then run the gel to initially determine whether it is consistent with the length
of our target fragment.

9、According to the following indicators in the PCR tube in order to add the prepared materials: 0.5 μl F
primer, 0.5 μl R primer, 1 μl of bacterial liquid, 5 μl M5-Taq enzyme, 3 μl of droplet water (of which the
F, R primer is the primer used in the gene cloning).

10、After adding the PCR system, set up the PCR program according to the following parameters: (1)
pre-denaturation: 95℃ for 3min

(2) denaturation: 94 ℃ 25s (3) annealing: 58 ℃ 25s (4) extension: 72 ℃ 1min (5) end extension: 72 ℃ 5min (6)
∞: 4 ℃.

11、After the PCR program is finished, start to run the gel. If the gel electrophoresis results show a roughly
similar bp number to that of our target fragment, then the bacterial fluid can be sent to the company for
sequencing.

12、Compare the sequencing results of the company, the sequence is correct, let the company return to the
sample, keep bacteria. That is, take the same volume of glycerol and E. coli bacterial fluid in the
ultra-clean bench, mix well, and put it into -80℃ refrigerator for storage.

Plasmid extraction

1、Take 1.5-4.5ml of overnight culture, centrifuge at 12,000rpm for 30s, pour dry supernatant as much as
possible and collect the bacterial bodies. Collect more than 1.5 ml of bacterial fluid, you can centrifuge
and discard the supernatant, then add more bacterial fluid in the same 1.5 ml tube and repeat step 1 until
enough organisms are collected.

2、Resuspend the bacterial body precipitate with 250 μl of solution P1 and vortex and shake until thoroughly
suspended.

3、Add 250μl of solution P2, gently turn up and down 6-8 times to make the bacterial body fully lysed, and
leave it at room temperature for 4min. mix gently, do not shake vigorously to avoid the plasmid being
damaged.

4、Add 350 μl of solution P3, immediately turn up and down gently 6-8 times, white flocculent precipitate will
appear when mixing thoroughly, centrifuge at 12,000rpm for 10min, take the supernatant carefully. The
supernatant should be removed carefully. Solution P3 should be mixed immediately after addition to avoid
localized precipitation of SDS.

Column equilibration: Take a new silica membrane adsorption column in a collection tube, draw 100 μl of
equilibrium solution into the column. centrifuge at 13,000 rpm for 1 min, pour out the waste liquid in the
collection tube, and put the adsorption column back into the collection tube. At this point, the equilibrium
solution pretreatment of the column is completed. Then proceed to the next step.

5、Add the supernatant obtained in the previous step to the column (the column is put into the collection
tube), centrifuge at 12,000rpm for 30-60s, and pour out the waste liquid in the collection tube.

6. Add 600μl of rinse solution WB (please check if anhydrous ethanol has been added first!) ), centrifuge at
12,000rpm for 30s, discard the waste liquid.

7. Repeat step 6.

8、Place the adsorption column back into the empty collection tube and centrifuge at 12,000rpm for 2min to
remove as much rinse solution as possible.

9、Take out the adsorption column AC, put it into a clean centrifuge tube, add 50-100 μl of elution buffer EB
to the middle part of the adsorption membrane (the elution buffer can be heated up in a 65-70℃ water bath
for better effect), leave it at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1
minute, and the liquid obtained in the centrifuge tube is the desired plasmid.

Plasmid digestion

1、According to the mapping of different vectors to determine the required endonuclease, and then according to
the nature of the endonuclease for enzymatic operation.

2、The three vectors to be cut in this experiment are the overexpression vector pCY-35S, yeast monohybrid PJG
and PlacZi vector.

3、The endonucleases required for pCY-35S are BamHI and PmlI, and the endonucleases required for PJG and
PlacZi are XhoI and EcoRI-HF.

4、Add 50ul of digestion system, 1ul of endonuclease 1, 1ul of endonuclease 2, 5ul of rcutSmart, 8ul of uncut
empty plasmid, 35ul of ddH2O.

5、37 ℃ reaction 2h, add 6 × loading buff running gel to see the band to determine whether the cut.

Recover the purified plasmid after cleavage.

Yeast Receptor Transformation

1、Before transforming the yeast, the plasmids of PJG+ , PlacZi +, PJG-, PlacZi -, PlacZi (carrying cis-acting
regulatory elements of downstream target genes), PJG (carrying transcription factors) and other bacterial
fluids should be extracted out in advance.

2、Take 100 μl of EGY48 receptor cells melted on ice, and add 2-5 μg of pre-cooled target plasmid, 10 μl of
Carrier DNA (95-100 degrees for 5 min, rapid ice bath, repeat once), and 500 μl of PEG/LiAc according to the
combinations of the negative control, the positive control and the experimental group.

3、Pipetting and mixing several times, 30 degrees water bath for 30 minutes (turn 6-8 times at 15 minutes to
mix).

4、Water bath at 42 degrees for 15 min (7.5 min, turn 6-8 times to mix).

5、Centrifuge at 5000 rpm for 40s and discard the supernatant. ddH2O 400μl resuspension, centrifuge for 30s
and discard the supernatant.

6、ddH2O 50μl resuspension, applied to the SD plate (also known as two lack of plate), put into 28 ℃
biochemical incubator culture 48-96h. After culture, will grow white yeast cells.

Yeast color development

1、the preparation of chromogenic medium: in a conical flask add 3.35g YNB, 0.36g D0/-Trp-Ura, 412.5ml H2O,
and then adjust the PH to 5.8, after the completion of the conical flask add 10g agar.

2、Put the configured mixture into the sterilizer, 115 ℃ sterilization for 15 min. after the end of
sterilization, lower the temperature to 55 ℃ in the ultra-clean bench to add the following reagents:

10×BU salt (55℃ preheating) 50ml

40% galactose (55℃ preheated) 25ml

40% cotton seed sugar (55°C preheated) 12.5ml

X-Gal (mother liquor concentration 20mg/ml) 2ml

3、Prepare 10×BU salt solution (100ml): add 3.71g Na2HPO4 and 3g NaH2PO4 in 100ml water, and then adjust the
PH value to 7.0, then put it into the sterilizer and sterilize it at 115℃ for 15min.

4、40% galactose (25ml): take 10g of galactose dissolved in 25ml H2O, then filtered and sterilized.

40% cotton candy (12.5ml): take 10g cotton candy dissolved in 12.5ml H2O and then filtered and sterilized.

5、X-Gal solution: take 40mg of X-Gal powder and dissolve it in 2ml DMSO solution.

6、Spot the yeast grown on the SD plate into the chromogenic medium as needed, and incubate it at 29℃ for
48-96h to develop the color and take pictures.

Overexpression primer design

1、Use SnapGene software to open the sequence of pCY-35S overexpression vector, look for two endonucleases
downstream of 35S to cut the plasmid, choose BamHI and PmlI for this experiment, and look for another 15
bases before the BamHI site to act as homology arm F, and look for another 15 bases after PmlI site to act
as homology arm R.

2、in order to facilitate the later detection of gene overexpression effect, you can add a piece of flag tag
sequence during the amplification process, and divide the amplification process into two steps.

3、Add the gene cloning primer F to the homology arm F to form the overexpression primer F (OE-F), and invert
the flag tag sequence to add the gene cloning primer R to form the overexpression primer R1 (OE-R1).

4、Add the reversed and complementary flag tag sequence to the homology arm R to form the overexpression
primer R2 (OE-R2).

The first round of PCR amplification was performed using OE-F and OE-R1, and then the second round of
amplification was performed using OE-F and OE-R2.

Transformation of Agrobacterium tumefaciens receptor cells

1、Sequencing the correct DH5α bacteriophage with genes and expression vectors were plasmid extracted to
obtain plasmid DNA.

2、go to -80 ℃ stored Agrobacterium susceptible cells (GV3101) on ice to melt.

3、Add 4-6ul plasmid DNA to each tube of GV3101, gently dial the bottom of the tube by hand to mix well, and
then stand on ice for 5min, liquid nitrogen for 5min, 37℃ water bath for 5min, and ice bath for 5min in
turn.

4、Add 300ul LB solution, placed in 28 ℃ shaker shaking culture 2-3 hours.

5、Shake the good bacterial solution sucked 150ul coated to Kana-Rif medium, 28 ℃ incubator culture 2-3 days.

6、Pick a single colony to add Kana-Rif culture medium, 28 ℃ shaking incubated for 4 hours, and finally for
colony PCR identification, there are bands can be preserved bacteria standby.

Genetic transformation

1、Soak the seeds of cucumber periwinkle milfoil in warm water at 50℃ for 30 min, dehull and sterilize the
seeds.

2、Place the seeds in 75% ethanol for surface disinfection for 45 s, and rinse with sterile water for 3 times.

3、Disinfect the seeds with 4% NaClO for 8 min and rinse with sterile water for 3 times.

4、Seeds were inoculated in MS medium (4.43 g‧L-1MS powder, 30 g‧L-1 sucrose and 3 g‧L-1 gel) and incubated in
the dark (28°C) for germination.

5、After 1 d of seed germination, the hypocotyls of cucumber seedlings are carefully removed on an ultra-clean
bench, the cotyledons are cut across the cucumber with a scalpel, the distal hypocotyls are carefully
removed, and the proximal half of the cotyledons are cut in half to serve as explants for the regeneration
of the cucumber.

6、Centrifuge the overexpressed Agrobacterium spp. from overnight incubation, resuspend it to OD 0.2-0.3 by
adding infestation solution, infest the explants in two times, each time for 90s, and place the back of the
explants upwards in the co-culture medium.

7、Three days later, the exoskeleton was inoculated vertically in the adventitious shoot induction medium with
the exoskeleton opening downward.

8、After a period of time, observe whether fluorescent buds are produced under ultraviolet light, if so, it
means that Agrobacterium infestation is successful, cut into the rooting medium to root, and then move into
the nutrient soil to cultivate into regeneration plants.

9、Take the tissue samples of overexpressed plants for sequence, expression and flavonoid content
determination.

Fluorescence quantification (qRT-PCR)

1、NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK\_LOC=BlastHome) fluorescence
quantitative primer search page can get some primers by inputting the information of the gene, and choose
the appropriate primers.

2、After the primers are synthesized, the genomic plasmid of the gene should be used for PCR amplification to
test the specificity of the primers, if the runners show only one band, it means that the primers have good
specificity.

3、Extract RNA from overexpressed plant tissue samples, reverse transcribe it into cDNA, and add 1ul cDNA, 3ul
RNAfree H2O, 0.5ul primer F, 0.5ul primer R, and 5ul qRT-PCR Mix to PCR tubes sequentially.

4、PCR amplification was performed using QuantStudio™ Real-Time PCR instrument in the following steps:

Holding Stage

Step 1: 95℃ 30 sec

Cycling Stage

Number of Cycles: 40

Step 1: 95℃ 3 sec

Step 2: 60℃ 30 sec

Melt Curve Stage

5、Finally, the data were analyzed using the 2-ΔΔCt method.

LC-MS (Liquid Chromatography-Mass Spectrometry)

(1) Chromatographic conditions

a. Chromatographic column: ZORBAX SB-C18 (4.6×150 mm, 5-Micron)

b. Mobile phase: acetonitrile: 0.1% (v/v) formic acid, elution gradient see the following table：

|
|  |

Duration(min) |

Acetonitrile(v/v) |

0.1% Carboxylic acid(v/v) |

|
|  |

0 |

5% |

95% |

|
|  |

10 |

15% |

85% |

|
|  |

25 |

22% |

78% |

|
|  |

30 |

15% |

85% |

|
|  |

40 |

5% |

95% |

c. Detection wavelength: 254 nm

d. Detection column temperature: 25 ℃

e. Flow rate: 1.0 ml/min

f. Injection volume: 10 μl

(2) Standard solution preparation: Dissolve the standard with methanol and dilute with methanol to obtain a
series of standard solutions with concentrations of 5.0, 10.0, 25.0, 50.0 and 100.0 μg/mL, respectively.

(3) Preparation of test solution: weigh the sample, add liquid nitrogen into the mortar and grind, add -20 ℃
pre-cooled 70% (v/v) ethanol solution 8 ml, 55 ℃ ultrasonic extraction for 50 min, cooled to room
temperature, take the supernatant through a 0.45 μm filter membrane to get 7 ml solution, the solution was
placed in vacuum centrifugal concentrator to concentrate to 2 ml of the test solution.

(4) The standard curve was obtained after the standard product was passed through the column, and the peak
time was clarified.

(5) The test material was passed through the column, and the concentration of the test material was
calculated according to the peak area and the standard curve.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5488/cover/{{self.title()|lower}}.png {% endblock %}
{% block page\_content %}

{{self.title()}}

[Overview](#a1)
[Successful cloning of CsTBH gene](#a2)
[Successful construction of yeast single heterozygous vectors and overexpression
vectors](#a3)
[Colony PCR of E. coli single colony](#b1)
[Yeast single hybridization test to verify the interaction between PAL and TBH](#b2)
[The phenotype of overexpression plants was successfully identified](#a4)
[Determination of total human flavonoids by liquid chromatography mass
spectrometry (LC-MS)](#b3)
[References](#a5)

Overview

In this section, we present all the results of the experiments that served as important checkpoints. These
include the validation of successful vector construction, the successful validation of PAL-TBH interactions by
yeast one-hybrid assay color development, the successful phenotypic characterization of PAL-TBH overexpression
plants, and the significant increase of flavonoid substances in PAL-TBH overexpression plants.

Successful cloning of CsTBH gene

The following are the results of our successful cloning of CsTBH gene with the help of TOPO vector. We verified
the consistency of our cloned CsTBH gene with that in the cucumber genome database
(http://cucurbitgenomics.org/) by both PCR and sequencing of a single colony of E. coli, indicating that the
cucumber material we selected in the natural state did not undergo mutation.

![](https://static.igem.wiki/teams/5488/{{self.title()|lower}}/img1.webp)

Fig.1 Sequencing results of CsTBH gene clones

![](https://static.igem.wiki/teams/5488/results/img2.webp)

Fig. 2 PCR results of single colony of E. coli with CsTBH gene clone

Successful construction of yeast single heterozygous vectors and
overexpression vectors

Colony PCR of E. coli single colony

In order to further study the gene function and explore the relationship between genes and genes, we need to
construct different vectors to carry out our research, the following are the gel electrophoresis graphs obtained
from the PCR of E. coli colonies and single colonies of overexpression of PCY, yeast monoheterozygous PJG and
PLacZi vectors constructed by us, respectively, in which the red box, yellow box and blue box represent the
length of the target fragments of PCY, PJG and PLacZi, respectively, and the length of the bands are consistent
with our expectation. PLacZi, and the lengths of the target fragments shown by the bands were all consistent
with our expectations.

![](https://static.igem.wiki/teams/5488/results/img3.webp)

Fig. 3 Growth of constructed E. coli colonies of TBH-PCY, PAL-PCY, PJG and PLacZi
vectors on culture medium

![](https://static.igem.wiki/teams/5488/results/img4-1.webp)
![](https://static.igem.wiki/teams/5488/results/img4-2.webp)
![](https://static.igem.wiki/teams/5488/results/img4.webp)

Fig.4 Single colony PCR running gel bands of constructed TBH-PCY, PAL-PCY, PJG
and PLacZi vectors for E. coli sequencing

We directly sequenced the single colonies picked from the plates coated after ligation transformation to verify
whether they were consistent with the sequence of our target gene. Below are the graphs of the sequence results
comparison.

![](https://static.igem.wiki/teams/5488/results/img5.webp)

Fig.5 Sequencing results of CsTBH-PCY vector

![](https://static.igem.wiki/teams/5488/results/img6.webp)

Fig.6 Sequencing results of CsPAL-PCY vector

![](https://static.igem.wiki/teams/5488/results/img7.webp)

Fig.7 Sequencing results of CsTBH-PJG vector

![](https://static.igem.wiki/teams/5488/results/img8.webp)

Fig.8 Sequencing results of CsPAL-LacZi vector

Yeast single hybridization test to verify the interaction between
PAL and TBH

By observing our color development results, we found that the yeast round spots in the positive control group
turned blue, the yeast round spots in the experimental group turned blue, while the yeast round spots in the
negative control group did not undergo any color change, which proved that our selected CsTBH gene would bind
with the downstream cis-acting regulatory element on the promoter of the CsPAL gene, and the two of them would
interact.

![](https://static.igem.wiki/teams/5488/results/img9.webp)

Fig. 9 Yeast color development in chromatographic plates and analysis of
interactions

The phenotype of overexpression plants was successfully identified

Observation of the overexpression plants showed that their cucumber fruits grew many small black spines
compared with the wild type, which might be the key to the production of flavonoids, and further testing is
needed to verify this.

![](https://static.igem.wiki/teams/5488/results/img10.webp)

Fig.10 Fruit phenotypes of CsTBH overexpressing transgenic plants

Determination of total human flavonoids by liquid chromatography
mass spectrometry (LC-MS)

In order to clarify whether the fruits of CsTBH overexpression plants that we cultivated using gene editing
could produce more flavonoids, by referring to the methods of some scientists [1], we quantified the flavonoid
content within the wild-type and mutant fruits by using liquid chromatography mass spectrometry (LC-MS), and the
following are the results of the assay, which are presented in the form of bar graphs.

![](https://static.igem.wiki/teams/5488/results/img11.webp)

Fig.11 Mass spectra and bar graphs of total flavonoids content in the fruits of
CsTBH overexpression plants by LC-MS

References

[1] Feng Z, Sun L, Dong M, et al. Novel players in organogenesis and flavonoid biosynthesis in cucumber
glandular trichomes. Plant Physiol. 2023;192(4):2723-2736. doi:10.1093/plphys/kiad236

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