iGEM 2023 — Team gxu-china

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* [**Engineering**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
* [**Results**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
* [**Experiments**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
* [**Notebook**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

[click to download](https://static.igem.wiki/teams/4678/wiki/nootbook.pdf)

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![](https://static.igem.wiki/teams/4678/wiki/wetlab-header.png)

* [**Engineering**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
* [**Results**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
* [**Experiments**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
* [**Notebook**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

![](https://static.igem.wiki/teams/4678/wiki/engineer.png)
INTRODUCTION
DESIGN
BUILD
TEST
LEARN
REDESIGN

x

### RESEARCH

Curcumin, a natural compound extracted from the ginger plant, is one of the edible natural pigments and a food additive approved by the Codex Alimentarius Commission of the Food and Agriculture Organization of the United Nations.
In addition, it has attracted much attention worldwide for its antioxidant, anticancer and anti-fungal properties. The common method used to extract curcumin from plants often involves the pressing and dissolving method, which employs various organic solvents and likely pollutes the environment.
Also, this method consumes a lot of energy, and the yield is far from meeting the market demand. So we chose to create the engineering E. coli to generate curcumin.

### DESIGN

In plants, 4-coumarate coenzyme A ligase (4cl) converts ferulic acid to feruloyl coenzyme A while Acetyl coenzyme A carboxylase (acc) can transform acetyl coenzyme A into malonyl coenzyme A during metabolism. Dcs catalyzes ferulic acid coenzyme A and malonyl CoA to β-ketoyl-CoA, and ferulic acid coenzyme A and β-ketoyl CoA synthesize curcumin by curs.
Therefore, if we insert the genes encoding the four key enzymes 4cl, acc, dcs, and curs into E. coli BL21(DE3) and take the advantage of some Endo diacyl coenzyme A which is produced by E. coli during its own metabolism, we can realize the synthesis of curcumin in E. coli by supplying ferulic acid as a substrate.

![](https://static.igem.wiki/teams/4678/wiki/kgg.png)

### BUILD

We extracted RNA from Arabidopsis thaliana and performed reverse transcription to generate cDNA. cDNA was used as a template to clone acc and 4cl genes, which were fused later as a fusion gene in the expression vector pETEXba by a linker, and pETEXba-acc-4cl plasmid was constructed successfully.
In the same way, we cloned the dcs gene from turmeric and curs from fresh ginger, and then fused the two genes into pGEX-MCM vector to construct a pGEX-dcs-curs plasmid.
We finally constructed the engineered Escherichia coli capable of synthesizing curcumin by transforming the above two plasmids into the chassis organism BL21 (DE3).

![](https://static.igem.wiki/teams/4678/wiki/engerp3.png)

### TEST

We validated the construction of the pGEXMCM-dcs-curs plasmid through antibiotic sensitivity experiments, plasmid size verification, and PCR amplification. Similarly, we validated the construction of the pET28EXba-acc-4cl plasmid through antibiotic sensitivity experiments, plasmid size verification, and PCR amplification. Our next step will be to use engineered bacteria for fermentation to produce curcumin. Prior to this, we explored the experimental conditions for detecting curcumin. Using analytical pure turmeric as the standard, turmeric stem extract was used as the experimental group. We obtained the standard sample with a maximum absorption wavelength of 420 nm, and the experimental group with a maximum absorption wavelength of 417 nm, successfully verifying the presence of curcumin in the extract of turmeric stem block. Subsequently,We investigated the mass spectrum conditions and obtained the mass spectrum information of curcumin.We opened the raw data by the Qualitative Analysis analysis software that comes with the instrument's computer. The molecular weight(M) of curcumin is 368.39, and the molecular formula(Z) is C21H20O6.
The mass spectrometry was detected using positive ion mode. The detected mass-to-charge ratio of curcumin is added to the molecular weight of a hydrogen atom, which means that the mass spectrum characteristic of curcumin is mainly [M+H]+.Therefore, the m/z of this curcumin is 369.133. Basing on the molecular weight value of 369.133, we found the detected curcumin in the data and saved the spectrum. The peak graph with this value is the curcumin we detected.

![](https://static.igem.wiki/teams/4678/wiki/yu18.png)

![](https://static.igem.wiki/teams/4678/wiki/yu19.png)

![](https://static.igem.wiki/teams/4678/wiki/1111.jpg)

![](https://static.igem.wiki/teams/4678/wiki/1112.jpg)

### LEARN

At the beginning, we attempted to clone the target genes using primers with restriction endonucleases but it failed. Afterwards, we used primers without restriction endonucleases to succeed to clone four targets' genes acc, 4cl, dcs and curs.
In the process of constructing the vector, we designed the bridging primers and cloned the fused gene fragment using dcs (removing the termination codon) and curs fusion methods.
We also used fusion expression to connect acc (without removing the termination codon) and 4cl to pETEXba at once. However, we found that the cloned fragments had many heterobands and concentrations were too low to be constructed into the vector. Subsequently, the vector was constructed using a separate connection method.
Later, we reviewed the literature and found that curcumin inhibits the growth of Escherichia coli. In order to solve the problem of insufficient growth of bacteria which results in the decrease of curcumin production, we designed a population sensing system: when the number of bacteria reaches a certain level, the curcumin synthesis pathway will be activated; when the number of bacteria decreases to a certain level, the curcumin synthesis pathway will be shut down, so that the number of bacteria will gradually increase. This strategy may realize the autonomous and dynamic regulation of metabolic flux.

### REDESIGN

![](https://static.igem.wiki/teams/4678/wiki/engerp4.png)

We used the comqxpa quorum sensing system of Bacillus subtilis, which consists of four genes: comx, comq, comp, and coma. Comx encodes a precursor peptide (pre comx) with 55 amino acid residues, which is then modified by the comq gene's encoding product, the isoprene transferase comq, to produce an active isoprene like peptide pheromone comx composed of 10 amino acid residues at its c-terminal.
As the population density of bacteria increases, the pheromone molecule comx released into the extracellular space continues to accumulate. When the pheromone molecule comx reaches a certain threshold, it binds to the transmembrane protein histidine kinase comp and activates the enzyme, promoting itself autophosphorylation.
The phosphorylated comp then transfers the phosphate group to the COMA regulatory protein and activates it. The activated coma protein then binds to the downstream target gene dcs::curs promoter Psrfa and activates the expression of downstream target genes. This receptive system can dynamically regulate the curcumin synthesis pathway and reduce the toxicity of curcumin to chassis organisms.
The curcumin produced by our constructed engineering bacteria can be used for cultural relic restoration, vegetation dyeing, and pigment production.
The engineering bacteria have been systematically optimized to provide a theoretical basis for future generations. Biosynthesis of curcumin is time-saving and efficient, with minimal environmental pollution.

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

![](https://static.igem.wiki/teams/4678/wiki/wetlab-header.png)

* [**Engineering**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
* [**Results**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
* [**Experiments**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
* [**Material**](#mat)
* [**Protocol**](#pro)
* [**Notebook**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

### Material

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Bacteria strain

1. Escherichia coli BL21(DE3): obtained from Dr. Li lab.
2. Escherichia coli DH5α, obtained from Dr. Li lab.
3. BL21/pET28EXba-acc, constructed by this experiment.
4. BL21/pET28EXba-acc-4cl, constructed by this experiment.
5. BL21/pGEXMCM-dcs-curs, constructed by this experiment.

![](https://static.igem.wiki/teams/4678/wiki/emoji-8.png)

# Genes

1. acc, sequence obtained from National Center for Biotechnology Information (NCBI).
2. 4cl, sequence obtained from NCBI.
3. dcs, sequence obtained from NCBI.
4. curs, sequence obtained from sequence obtained from NCBI.

![](https://static.igem.wiki/teams/4678/wiki/emoji-9.png)

# Plasmids

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Resistance | Size | Source |
| pET28EXba | Kanamycin | 6526 bp | Dr.Li lab |
| pGEXMCM | Ampicilin | 6190 bp | Dr.Li lab |
| pET28EXba-acc | Kanamycin | 6916 bp | This work |
| pET28EXba-acc-4cl | Kanamycin | 8602 bp | This work |
| pGEXMCM-dcs-curs | Ampicilin | 6634 bp | This work |

![](https://static.igem.wiki/teams/4678/wiki/emoji-10.png)

# Primers

|  |  |  |
| --- | --- | --- |
| Name | Sequence | Enzyme |
| dcs-F | ATATGAATTCATGGAAGCGAACGGCTACCG | EcoRⅠ |
| dcs-R | ATTGTCTAGAGCCAGATCCGTTCAGTCTGCAACTATGGA |  |
| curs-F | ACTGAACGGATCTGGCATGGGTAGCCTGCAGGCGATGC |  |
| curs-R | TGCGGCCGCTCGAGTCTACGGTATTGGTAGACTGCGTAGC | XbaⅠ |
| 4cl-F | ATGCTCTAGAATGGCGCCACAAGAACAAGC | XbaⅠ |
| 4cl-R | GCGCCTCGAGTCACAATCCATTTGCTAGTT | XbaⅠ |
| acc-F | CGGCCATATGATGGACGCTTCTATGATTAC | NdeⅠ |
| acc-R | ATCGCTCGAGACGAGATCTAGACTAAACCGTTGCGTTTGTCAGC | XhoⅠ/XbaⅠ |
| PGEXMCM-F | GCCTTAAGGAATACTGTTTCCTGTGTGAAATTG | EcoRⅠ |
| PGEXMCM-R | ATTAAGATCTCTCGAGCGGCCGCATCGTGACTGACTG | XbaⅠ |

![](https://static.igem.wiki/teams/4678/wiki/emoji-11.png)

# Enzymes and antibiotics

1. XhoⅠ, purchased from TAKARA Company (CHN).
2. NdeⅠ, purchased from TAKARA Company (CHN).
3. XbaⅠ, purchased from TAKARA Company (CHN).
4. EcoRⅠ, purchased from TAKARA Company (CHN).
5. Ampicilin (Amp), purchased from solarbio Company (CHN).
6. Kanamycin (Kan), purchased from solarbio Company (CHN).

![](https://static.igem.wiki/teams/4678/wiki/emoji-12.png)

# Reagent

1. Tris-base, purchased from sigma Company (GER).
2. Glycerinum, purchased from sigma Company (GER).
3. NaOH, purchased from Macklin Company (CHN).
4. CaCl2, purchased from APS Company (USA).
5. MgCl2, purchased from APS Company (USA).
6. NaCl, purchased from aladdin Company (CHN).
7. Agar powder, purchased from solarbio Company (CHN).
8. Extraction kit, purchased from Omega Company (USA).
9. Agarose: purchased from Sangon Biotech Company.
10. EDTA, purchased from Sangon Biotech Company (CHN).
11. SDS, purchased from Sangon Biotech Company (CHN).
12. Absolute ethyl alcohol, purchased from Sangon Biotech Company (CHN).
13. 75% ethanol, purchased from Sangon Biotech Company (CHN).
14. Ferulic acid, purchased from MACKLIN Company (CHN).
15. 6×Loading Buffer, purchased from TAKARA Company (CHN).
16. Marker, purchased from TAKARA Company (CHN).

### Protocol

![](https://static.igem.wiki/teams/4678/wiki/emoji-1.png)

# Preparation of chemicals

1. Alkali lysate I, weighed 3.03 g Tris-base, 2.92 g EDTA, 9.9 g glucose into 1 L H2O, adjusted pH to 8.0, 121 ℃ sterilized for 10 mins then stored at 4 ℃.
2. Alkali lysate II, mixed 0.4mol/L NaOH and 2% SDS in equal volume, while 0.4 mol/L NaOH and 2% were prepared by sterile water respectively and stored at the room temperature.
3. Alkali lysate III, weighed 194.42 g potassium acetate into 800mL steriled water, then 115 mL of acetic acid was added, and finally the volume was fixed to 1 L, stored at 4 ℃.
4. 50% glycerol, equal volume of 100% glycerol and sterile water were mixed and stored at 4℃.
5. 2×GC buffer, added BSA 40 mg, Betaine 24.6 g, Tris-HCl 4 mL at pH 8.8, 1 mol/L KCl 4 mL, 800 μL of 1 mol/L Mg2SO4 at pH 8.8, 12 mL of 0.5 mol/L (NH4)2SO4 at pH 8.8, 24 mL 50% glycerin, 4mL DMSO, 4 mL N, N, N-dimethylformamide, Triton X-100 400 μL, and finally the volume was fixed to 200 mL with sterilized water.
6. 0.8 mol/L Isopropyl-β-D -thiogalactopyranoside (IPTG)：weighed IPTG 2 g, added H2O to 10 mL, then filtered and sterilized and stored at -20 °C.

![](https://static.igem.wiki/teams/4678/wiki/emoji-2.png)

# Preparation of LB medium

1. Weighed NaCl 10 g, Tryptone 10 g, Yeast extract 5 g then the volume was fixed to 1 L with ddH2O and adjusted pH to 7.0 with NaOH, sterilized at 121 ℃ for 20 mins, and stored at the room temperature.
2. For solid LB medium, 1.5 g Agar powder was added every 100 mL on the basis of (1).

![](https://static.igem.wiki/teams/4678/wiki/emoji-3.png)

# Preparation of medium containing antibiotics

1. For the liquid medium, the corresponding antibiotics were added at a ratio of 1/1000 before using.
2. For the solid medium, it was heated and melted before using. When the temperature was reduced to about 50-60 ℃, the corresponding antibiotics were added in the proportion of 1/1000, which could be used to make the plate.

![](https://static.igem.wiki/teams/4678/wiki/emoji-4.png)

# Lysis of Protocol-II polysaccharide polyphenol plant tissues

1. Placed fresh leaves in a mortar and pestle pre-cooled with liquid nitrogen, and the tissue was ground with a mortar and pestle, with the addition of liquid nitrogen until it was ground to powder, and the sample (50-100 mg) was added to a 1.5sterilized tube containing 450 μL of Butter RL.
2. Centrifuged the lysate at 12000 rpm for 5 min at 4 ℃.
3. Pipetted the supernatant carefully into a new 1.5sterilized tube.

![](https://static.igem.wiki/teams/4678/wiki/emoji-5.png)

# Extraction of total RNA

1. Added 1/2 volume of anhydrous ethanol to the supernatant or mixtured from the sample lysis step and mixed with a pipette gun.
2. Immediately transferred the entire mixture to the RNA SC (2 mL tube).
3. Centrifuged at 12000 rpm for 1 min to discard the filtrate and placed the RNA Spin Column back into the 2 mL Collection tube.
4. Added 500 μL of Buffer RW to the RNA Spin Column, centrifuged at 12000 rpm for 30 s and discarded the filtrate.
5. Added 600 μL of Buffer RWB to the RNA Spin Column, centrifuge at 12000 rpm for 30 s, discarded the filtrate.
6. Used DNase I to digest.

![](https://static.igem.wiki/teams/4678/wiki/emoji-6.png)

# RT-PCR

Mixed the extracted RNA in the following proportions.

![](https://static.igem.wiki/teams/4678/wiki/pro-1.png)

Placed the mixed system in the PCR instrument and ran it (Procedure was to first incubate at 25 ℃ Celsius for 10 mins, then at 50 ℃ Celsius for 30 mins, finally at 85 ℃ Celsius for 5seconds).

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Polymerase Chain Reaction (PCR)

1. Preparation 1-reaction PCR mix into the PCR tube by adding together 8.5 µL deionized water, 12.5 µL 2×GC buffer, 2 µL dNTP mix,1 μL templated DNA, 1 μL primer pairs and 0.125 µL rTaq DNA polymerase.
2. Placed the PCR tubes into the PCR apparatus and run it (Protocol can be divided into five centrifuge tubes, pre-denaturation → denaturation → anneal→ extension → final extension). After the PCR program entered denaturation, it run 30 times successively through denaturation, annealed and extension, and finally entered complete extension. After completing extension, waited for the temperature to drop to 16 ℃ to end the pro-gram.
① Pre-denaturation, run at 95 ℃ for 5-10 mins to completely denature the primes, to ensure that the primers were single stranded. Meanwhile, some secondary structures of the template DNA aredestroyed, and the double stranded of the template DNA was fully dissociated.
② Anneal, reduced the solution temperature to the appropriate temperature (determined by annealing temperature pf the primer) and centrifuged it for 20 s to make the template DNA and primer complemented each other according to the principle of base pairing principle.
③ Extension, when the reaction temperature of the solution rose to 72 ℃, the rTaq DNA polymerase took the single strand DNA as the template and centrifuged the complementary DNA in the direction of 5'- 3' by using four kinds of deoxynucleotide triphosphate (dNTP) in the reaction mixture under the guidance of primers. The extension time of the microcenter point ends at the length of the fragment The PCR instrument used in this experiment was extended by 1k bp/min.
④ Final extension, maintained it at 72 ℃ for 8mins to make PCR reaction more completed to improve amplification yield.
⑤ Waited for the temperature to drop to 16 ℃ and the PCR procedure was completed.

![](https://static.igem.wiki/teams/4678/wiki/emoji-8.png)

# GXL system

![](https://static.igem.wiki/teams/4678/wiki/pro-2.png)

![](https://static.igem.wiki/teams/4678/wiki/pro-3.png)

![](https://static.igem.wiki/teams/4678/wiki/emoji-9.png)

# DNA/RNA extraction using commercial kits

All reagent below referent was offered by Extract kit.
1. Added twice the volume of Binding Buffer to the sample, stood at the room temperature for 1 min. Transferred to adsorption column, then centrifuged at a speed of 13000 rcf/min for 1 min and discarded the liquid supernatant of collect column (If the sample was attained from the gel extraction, the volume of Binding Buffer was exceeded the gel).
2. Added 300 μL Binding Buffer to adsorption column, then centrifuged at a speed of 10000rcf/min for 1min and discarded the liquid supernatant of collect column.
3. Added 700 μL Washing Buffer to adsorption column, stood at the room temperature for 2 mins, then centrifuged at a speed of 10000 rcf/min for 1min and discarded the liquid supernatant of collect column (Repeated this step twice).
4. Added nothing to adsorption column, stood at the room temperature for about 5mins, then discarded collect column and transferred adsorption column to a new centrifuge tube.
5. Added 30~50 μL 55 ℃ ddH2O, stood at the room temperature for 1 min and then centrifuged at a speed of 13000 rcf/min for 1 min.
6. Transferred the water in the centrifuge tube to adsorption column and then centrifuged one more time.
7. Discarded the adsorption column and restored the sample at -20 ℃.

![](https://static.igem.wiki/teams/4678/wiki/emoji-10.png)

# Activation of glycerol bacteria stock and Cultivation of single colonies

1. Activation of glycerol bacteria stock, 5 mL of liquid medium, 5 μL of corresponding antibiotic and 100 μL of glycerol bacteria were added to sterile 30 mL cylindrical glass bottles, and incubated overnight in shaker at 37 ℃, 160 rpm/min.
2. Cultivation of single colonies picked out the single colonies on the plate by a 10 μL tip, and then the tip was put into a 30 mL cylindrical glass bottle with 5 mL liquid medium and 5 μL of corresponding antibiotics. Finally, the 30 mL cylindrical glass bottle was incubated overnight in shaker at 37 ℃, 160 rpm/min.

![](https://static.igem.wiki/teams/4678/wiki/emoji-11.png)

# Plasmid extraction of E. coli

1. Inoculated E.coli strains containing the plasmid in 5 mL of LB liquid medium with selective antibiotics and then cultured in a shaking table at 37 ℃, 160 rpm for 12-16 h.
2. Transferred the bacterial solution to the micro centrifuge tube, centrifuged it at a speed of 8000 rpm/min at 4 ℃ for 1 min, discarded the liquid supernatant, and under the same conditions until all the bacterial solutions were transferred.
3. Added 100 μL alkali lysate I to the bacteria and suspended fully.
4. Added 200 μL alkali lysate II, slowly inverted mixed, and flicked the bottom, until the solution became clear, then added 150 μL alkali lysate III, reversed and mixed micro centrifuge system, stood at 4 ℃ for 5 mins, then 4 ℃ centrifuged at a speed of 12000 rpm/min for 5 mins.
5. Transferred the liquid supernatant to a clean centrifugal tube, added anhydrous ethanol of 2 times volume of the liquid supernatant, mixed and precipitated at 4 ℃ for 5mins, then centrifuged at a speed of 12000 rpm/min for 5 mins, and discarded the liquid supernatant.
6. Added 500 μL 75% ethanol solution to the precipitation, discarded the liquid supernatant after 4 ℃ centrifuged at a speed of 12000 rpm/min for 2 mins, and placed at the room temperature to volatilize the remaining liquid.
7. Added an appropriate amount of sterile water at about 60 ℃, and the shaker oscillated evenly, and stored the plasmid solution at -20 ℃.

![](https://static.igem.wiki/teams/4678/wiki/emoji-12.png)

# Enzyme digestion

1. Single Enzyme digestion system of plasmid, 80% plasmid, 10% buffer, 5% enzyme, 5% ddH2O.
2. Double Enzyme digestion system of plasmid, 80% plasmid, 10% buffer, 5% enzyme A, 5% enzyme B.
3. Single enzyme digestion system of gene fragment, 80% plasmid, 10% buffer, 2% enzyme, 8% ddH2O.
4. Double enzyme digestion system of gene fragment, 80% gene fragment, 16% buffer, 2% enzyme A, 2% enzyme B.

![](https://static.igem.wiki/teams/4678/wiki/emoji-1.png)

# DNA gel electrophoresis

1. Sample Preparation, added electrophoresis Loading buffer, Gel Loading Dye, Purple (6×) to nucleic acid sample, that was, added 1 part buffer to parts of sample.
2. Agarose gel Preparation, weighed 0.8 g agarose, added 100 mL of TAE(1×) buffer，prepared 0.8% agarose, heated it in a microwave oven for several minutes to completely melted the agarose, mixed it well, poured it into a gel making mold, inserted a comb with a suitable aperture, and after the gel solidified, pulled out the comb and put it into an electrophoretic tank containing TAE(1×) buffer.
3. Electrophoresis, completely immersed the agarose gel in the buffer, added mark and the sample into the loading well, and performed electrophoresis at 120 V for 30 mins.
4. Imaging dyed the electrophoretic gel in EB for 2 mins, then performed UV imaging in the GelDoc Go Imaging System and took photos for recording.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Gel extraction

1. Sample Preparation added electrophoretic Loading buffer, Gel Loading Dye, Purple (6×) into the products of endonuclease digestion, that was, added 1 part buffer to 5 parts of sample.
2. Agarose gel Preparation, weighed 0.8 g agarose, added 100 mL of TAE(1×) buffer，prepared 0.8% agarose, heated it in a microwave oven for several minutes to completely melted the agarose, mixed it well, poured it into a gel making mold, inserted a comb with a suitable aperture, and after the gel solidifies, pulled out the comb and put it into an electrophoretic tank containing TAE(1×) buffer.
3. Electrophoresis, completely immersed the agarose gel in the buffer, added mark and the sample into the loading well, and performed electrophoresis at 100 V for 45 mins.
4. Stain placed the gel in 10 μg/mL EB dye solution for 2-4 mins at room temperature.
5. Observation and cutting, observed the electrophoresis tape and its position on the ultraviolet fluoroscope, and cut the fragments in the required range.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Enzyme-linked Ligation

1. Enzyme-linked system 1 (total volume 10 μL), 20% plasmid, 60% gene fragment, 10% buffer, 10% T4 DNA ligase.
2. Enzyme-linked system 2 (total volume 10 μL), 40% plasmid, 40% gene fragment, 10% buffer, 10% T4 DNA ligase.
3. Reaction condition, stood at 16 ℃ for 4hours or overnight.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Preparation of chemically competent cells of Escherichia coli (E. coli)

1. A tube of 100 μL DH5α was inoculated in 5mL LB liquid medium and incubated overnight in shaker at 37 ℃, 160 rpm/min.
2. Inoculated the overnight culture in 100 mL of LB medium at 1% ratio and incubated at 37 ℃ for 2-3 h (Cultured to OD600=0.5-0.6).
3. Cooled the bacteria culture in the mixture of ice and water for 5-10 mins in advance. Then transferred the treated bacteria culture to a centrifugal tube and placed it on ice for 10 mins to cool the bacteria liquid.
4. Transferred the cold bacterial liquid into 4 ℃ centrifuged at a speed of 6000 rpm/min for 5 mins. Discarded the liquid supernatant and collected the bacterium.
5. Added 60 mL cold 0.1 mol/L CaCl2 solution to re-suspend the bacterium, and then cooled it in an ice bath for 45 mins.
6. Transferred the bacterial liquid into a 4℃ centrifuged at a speed of 6000rpm/min for 5mins to discard the liquid supernatant and collected the bacterium.
7. Added 1 to 2 mL cold 15% glycerol solution (containing 0.1 mol/L CaCl2 solution) to re-suspend the bacterium and divided them into 300 μL of each micro centrifuge tube and then stored at -80 ℃.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Transformation of DH5α chemically competent cells

1. Added an appropriate amount of DNA sample or ligated reaction product to 100 μL competent cells, mixed slightly, and took an ice bath for 30 mins.
2. The micro centrifuge system was placed in a water bath at 42 ℃ for 90 s incubation.
3. After incubation, placed the competent cells quickly in the mixture of ice and water for cooling 2 mins, and then added into 800 μL LB liquid medium for shaking culture at 37 ℃ for 45-60 mins.
4. Took out, centrifuged at a speed of 8000 rpm/min for 1 min, removed the liquid supernatant, and left 100 μL.
5. Coated on LB solid plate containing corresponding antibiotics and cultured upside down overnight in 37 ℃ incubator to observe the growth of transformation.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Induced expression of protein

1. Inoculated the glycerol bacteria in 5 mL LB liquid medium containing 1/1000 antibiotics and incubated in a shaking at 37 ℃ and 160 rpm to OD600=0.6.
2. Added 1/1000 of 0.8 mol/L IPTG to the bacteria solution and continued to be cultured in the shaking at 37 ℃ and 160 rpm for 4h to induce the expression of protein.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# SDS-PAGE

1. Taked 20 μL of the induced bacterial solution and centrifuged at 8000 rpm for 1 min. Removed the supernatant and added 20 μL H2O for resuspension.
2. Added equal volume of SDS and DTT mixture (mixed volume ratio of SDS: DTT=4:1), incubated in a 100 ℃ metal bath for 10 mins, and then apply the sample.
3. Used SDS-PAGE gel to detect the purity and molecular weight of proteins. The formula of SDS-PAGE gel is as follows:

![](https://static.igem.wiki/teams/4678/wiki/pro-4.png)

![](https://static.igem.wiki/teams/4678/wiki/pro-5.png)

{% endblock %}

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

![](https://static.igem.wiki/teams/4678/wiki/wetlab-header.png)

* [**Engineering**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
* [**Results**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
* [**Experiments**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
* [**Notebook**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

We selected four coding genes acc (encoding acetyl CoA carboxylase, ACC), 4cl (encoding 4-alginate CoA ligase, 4Cl), dcs (encoding diketone CoA synthase, DCS), and curs (encoding curcumin synthesis, CURS) as candidate genes that play important roles in the curcumin synthesis pathway. In plants, 4Cl converts ferulic acid to ferulic CoA, ACC converts acetyl CoA to malonyl CoA, DCS catalyzes the binding of ferulic CoA and malonyl CoA to produce ferulic diketone CoA, CURS catalyzes the conversion of ferulic diketone CoA to β- Ketonic acid condenses with another molecule of ferulic coenzyme A to form curcumin (as shown in **Fig 1**). We chose pETEXba and pGEXMCM as carriers and cloned four catalytic enzymes into BL21 (DE3) substrate organisms to achieve the goal of high curcumin production (as shown in **Fig 2**).****

![](https://static.igem.wiki/teams/4678/wiki/resultgraph1.png)

**Fig 1**: The biosynthetic pathway of curcumin.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph2.png)

**Fig 2** The recombinant plasmid and engineering bacteria constructed in this project.

### **Construction of acc-4cl expression system**

![](https://static.igem.wiki/teams/4678/wiki/emoji-1.png)

# Design

Cloned acc into the expression vector pET28EXba containing a constitutive, shown in **Fig 3.1.**

![](https://static.igem.wiki/teams/4678/wiki/resultgraph3.png)

**Fig 3.1** The construction of P-acc.

![](https://static.igem.wiki/teams/4678/wiki/emoji-1.png)

# Build

We extracted RNA from Arabidopsis thaliana (as shown in **Fig 3.2A**) and obtained the entire genome of Arabidopsis thaliana through reverse transcription. Using the entire Arabidopsis genome as a template and amplified acc by polymerase chain reaction (PCR) as shown in **Fig 3.2B**.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph4.png)

**Fig 3.2 (A)**. Lane 1-3: RNA extracted from Arabidopsis thaliana. **(B)** Lane 1: DNA Marker, Lane 2: acc gene fragment, and the band size was as expected.

Extracted pET28EXba carrier using alkaline lysis method (as shown in **Fig3.3A**). According to the restriction endonuclease sites, the corresponding restriction endonucleases were used to digest acc and pET28EXba. The digested pET28EXba vector is shown in **Fig 3.3B**. After digestion, we used T4 DNA ligase to connect acc and pET28EXba, and then transferred them to DH5α.

![](https://static.igem.wiki/teams/4678/wiki/newfig3-3.jpg)

**Fig 3.3 (A)**. Lane 1: DNA Marker, Lane 2: pET28EXba. **(B)** Lane 1: DNA Marker, Lane 2: pET28EXba vector digested with NdeⅠ and XhoⅠ, and the band size was as expected.

![](https://static.igem.wiki/teams/4678/wiki/emoji-1.png)

# Test

Through antibiotic sensitivity experiments (as shown in **Fig 3.4A**), plasmid size, and validation of target genes (as shown in **Fig 3.4B**), pET28EXba-acc was successfully obtained.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph6.png)

**Fig 3.4** Validation of pET28EXba-acc. **(A)**. The result of resistance screening experiment, the left: DH5α/ pET28EXba-acc ,the right: DH5α. **(B)**. Validation of the plasmid size and PCR amplification of the candidate genes. Lane 1: DNA Marker, Lane 2: pET28EXba, Lane 3: pET28EXba-acc, Lane 4: acc gene fragment, used pET28EXba-acc, as the template and the band size was as expected.

### **Construction of pET28EXba-acc-4cl expression system**

![](https://static.igem.wiki/teams/4678/wiki/emoji-2.png)

# Design

Cloned 4cl into the expression vector pET28EXba-acc containing a constitutive, shown in **Fig 4.1.**

![](https://static.igem.wiki/teams/4678/wiki/resultgraph7.png)

**Fig 4.1** The construction of P-acc-4cl.

![](https://static.igem.wiki/teams/4678/wiki/emoji-3.png)

# Build

Using the entire Arabidopsis genome as a template and amplified 4cl by PCR as shown in **Fig 4.2A**. Extracted pET28EXba-acc from DH5α (as shown in **Fig4.2B**). According to the restriction endonuclease sites, the corresponding restriction endonucleases were used to digest acc and pET28EXba-acc. After digestion, we used T4 DNA ligase to connect 4cl and pET28EXba-acc, and then transferred them to DH5α.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph8.png)

**Fig 4.2** Lane 1: DNA Marker, Lane 2: 4cl gene fragment, amplified by PCR, and the band size was as expected. **(B)** Lane 1: DNA Marker, Lane 2: pET28EXba-acc.

![](https://static.igem.wiki/teams/4678/wiki/emoji-4.png)

# Test

Through antibiotic sensitivity experiments (as shown in **Fig 4.3A**), plasmid size, and validation of target genes (as shown in **Fig 4.3B**), pET28EXba-acc-4cl was successfully obtained.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph9png.png)

**Fig 4.3** Validation of pET28EXba-acc-4cl. **(A)**. The result of resistance screening experiment, the left: DH5α, the right: DH5α/pET28EXba-acc-4cl. **(B)**. Validation of the plasmid size and PCR amplification of the candidate genes.
Lane 1: DNA Marker, Lane 2: pET28EXba-acc, Lane 3: pET28EXba-acc-4cl, Lane 4: acc gene fragment, Lane 5: 4cl gene fragment, Lane 4 and Lane 5 all amplified used pET28EXba-acc, as the template and the band size was as expected.

### **Construction of pGEXMCM-dcs-curs expression system**

![](https://static.igem.wiki/teams/4678/wiki/emoji-5.png)

# Design

Cloned dcs-curs into the expression vector pGEXMCM, shown in **Fig 5.1.**

![](https://static.igem.wiki/teams/4678/wiki/resultgraph10.png)

**Fig 5.1** The construction of P-dcs-curs.

![](https://static.igem.wiki/teams/4678/wiki/emoji-6.png)

# Build

We purchase and plant turmeric and ginger, extract their RNA (as shown in **Fig5.2A**), and obtain the entire genome of turmeric through reverse transcription. Using the turmeric genome as a template, the dcs gene was amplified using primers with linkers added.
The curs gene was amplified using primers with linkers added to the ginger genome as a template, and then the dcs and curs fragments were fused into the dcs-curs through linkers to form a connection (as shown in **Fig 5.2B**). Using corresponding restriction endonucleases to digest dcs-curs and pGEXMCM, the digested pGEXMCM carrier is shown in **Fig 5.2C**. After digestion, we used T4 DNA ligase to connect dcs-curs and pGEXMCM, and then transferred them to DH5α.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph11.png)

**Fig 5.2 (A)**. Lane 1-3: RNA extracted from turmeric. **(B)** Lane 1-3: RNA extracted from ginger. **(C)** Lane 1: DNA Marker, Lane 2: dcs gene fragment, Lane 3: zurs gene fragment, Lane 4: fusion fragment dcs-curs, and the band size was as expected.

![](https://static.igem.wiki/teams/4678/wiki/emoji-7.png)

# Test

Through antibiotic sensitivity experiments (as shown in **Fig 5.3A**), plasmid size, and validation of target genes (as shown in **Fig 5.3B**), pGEXMCM dcs curs were successfully obtained.

![](https://static.igem.wiki/teams/4678/wiki/resultgraph12.png)

**Fig 5.3** Validation of pGEXMCM -dcs-curs. **(A)**. The result of resistance screening experiment, the left: DH5α, the right: DH5α/pGEXMCM-dcs-curs. **(B)**. Validation of the plasmid size and PCR amplification of the candidate genes.
Lane 1: DNA Marker, Lane 2: pGEXMCM, Lane 3: pGEXMCM-dcs-curs, Lane 4: dcs-curs gene fragment, amplified used pGEXMCM-dcs-curs, as the template and the band size was as expected.

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