iGEM 2021 — Team iBowu-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:iBowu-China/Notebook

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

# Implementation

* Project

![](https://static.igem.org/mediawiki/2019/b/bc/T--iBowu-China--banPo.jpg)

# Introduction

This year, our works in the implementation focus on three of our products--Glycyrβ Database, immobilized enzyme systems and glycyrrhetinic acid. For each product, we will identify the design, safety and users perception.

## Main Outcomes

##### Substance

Interview 1

##### Improving conditions

Interview 2

We then recognized an absence in both detection and treatment of Soft Rot Disease. Therefore, we understood the importance of a bio-control solution that is not only effective but also environmentally-friendly. Based on knowledge of the field of synthetic biology, we started to come up with different approaches to soft rot detection and treatment.

![](https://static.igem.org/mediawiki/2020/c/cb/T--iBowu-China--keystone1.png)

### Interview（Potato Center Asia-Pacific Headquarter）

We met with senior staffs of the potato center Asia-Pacific Headquarter to learn about the current detection and treatments of potato diseases. We then visited their workplace, which accounted for a large proportion of the current potato-agricultural development. We presented our initial concept to the experts (including the chairman of the potato center, Mr. Lu) and audited a group meeting on agricultural pharmacology. Through the discussions with them, we became more aware of the consequences of Soft Rot disease and the importance of creating an effective bio-control solution.

[![](https://static.igem.org/mediawiki/2020/c/cb/T--iBowu-China--keystone1.png)](./images/image-2.jpg)

[![](https://static.igem.org/mediawiki/2020/c/cb/T--iBowu-China--keystone1.png)](./images/image-2.jpg)

[![](https://static.igem.org/mediawiki/2020/c/cb/T--iBowu-China--keystone1.png)](./images/image-2.jpg)

### Experts

Our team had the pleasure of meeting with Dr. Flemming Bensenbacher on 5th July 2019. Dr. Bensenbacer is an academician of the Royal Danish academy of Sciences and President of Carlsberg Foundation. He shared his journey in scientific research with us and the problems he encountered and tackled. We presented our project to him and hosted an interview with questions relating to the application of synthetic biology. This opportunity was crucial to us as we gained from it a valuable depth of knowledge in the potential future pathways of our product development.

[](video/mda-kc21c5bv2fig3ieu.mp4)

![](https://static.igem.org/mediawiki/2020/c/cb/T--iBowu-China--keystone1.png)

## Project Analysis

We Reserch deeply to give the best quality services.

##### Here our project analysis result.

* It was popularised in the with the release of sheets containing Lorem Ipsum
* Page Maker including versions of Lorem Ipsum.
* It is a long established fact that a reader will be distracted by the readable
* content of a page when looking at its layout. The point of using Lorem Ipsum
* is that it has ult model text, and a search for 'lorem ipsum' will uncover many
  web

[Ins @ ibowuchina2020](https://www.instagram.com/ibowuchina2020/)
Facebook: iBowu China
Twitter @ IbowuC
[Ximalaya: iBowu\_China2020](https://www.ximalaya.com/zhubo/234355068/)
Email: iBowu-China2021@outlook.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:iBowu-China/Engineering

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

# Engineering Success

## Engineering Success for Silver Medal

We were privileged to test out our designed synthetic biology parts in our lab for the 2021 iGEM season. We will show here a new part we designed and present the results of our experiments which indicated the part works as we expected.

#### New part 6xHis-Beta-glucuronidase: (link in parts registry) [BBa\_K3716012 (basic)](http://parts.igem.org/Part%3ABBa_K3716012) and [BBa\_K3716022 (composite)](http://parts.igem.org/Part%3ABBa_K3716022)

##### Introduction

This biological part is the CDS sequence of β-glucuronidase, a protein that can hydrolyze the glycyrrhizic acid into glycyrrhetinic acid by cutting off the glucuronide groups. This part can be used in E. coli BL21(DE3) to express the enzyme β-glucuronidase. This specific part is the sequence found in special types of E. coli strains and then its sequence was optimized for expression in E. coli BL21(DE3).

![](https://static.igem.org/mediawiki/2021/0/04/T--iBowu-China--parts-2.png)

Figure. The plasmid constructions used for the expression of our new part 6xHis-Beta-glucuronidase. This part encodes an enzyme β-glucuronidase that can hydrolyze the glycyrrhizic acid into glycyrrhetinic acid.

##### Related parts

The enzyme can come from a variety of sources, including humans and bacteria. We also obtained the coding sequence of the enzyme from two other species from previous reports:
Aspergillus oryzae
(link in the parts registry: [BBa\_K3716013](http://parts.igem.org/Part%3ABBa_K3716013))
and Thermotoga maritima
(link in the parts registry: [BBa\_K3716013](http://parts.igem.org/Part%3ABBa_K3716013)).

In the plasmid construction, we used commonly used T7 promoters for the expression of this sequence. T7 promoter can be recognized by T7 RNA polymerase that is highly effective in transcribing the downstream genes. T7 promoter and each of the above coding sequences were designed, built and tested in combination. They are also uploaded into the parts registry for documentations, including

T7-LacO-gusA(E. coli),
(link: [BBa\_K3716022](http://parts.igem.org/Part%3ABBa_K3716022))

T7-LacO-gusA(Aspergillus\_oryzae),
(link: [BBa\_K3716026](http://parts.igem.org/Part%3ABBa_K3716026))

T7-LacO-gusA(Thermotoga\_maritima).
(link: [BBa\_K3716027](http://parts.igem.org/Part%3ABBa_K3716027))

In order to investigate the catalytic power of the enzyme β-glucuronidase under different expression concentrations,
we designed three other promoters according to previous research, namely C4, H9 and G6.
They can be recognized by T7 RNA polymerase as well, but the strength is different from T7 promoters.
Therefore users can select a proper variant of the promoter to meet the need For different expression strengths.
This series of promoters were built and tested in combination with the enzyme from E. coli source. The documentations were uploaded to the relevant part registry pages as the following:

C4-LacO-gusA(E. coli),
(link: [BBa\_K3716023](http://parts.igem.org/Part%3ABBa_K3716023))

H9-LacO-gusA(E. coli),
(link: [BBa\_K3716024](http://parts.igem.org/Part%3ABBa_K3716024))

G6-LacO-gusA(E. coli),
(link: [BBa\_K3716025](http://parts.igem.org/Part%3ABBa_K3716025))

##### Protocol

1. Transform the plasmids into E. coli BL21(DE3)

2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37 degree Celsius in a shaker overnight.

3. Fluorescence. Acquire 100 ul bacteria culture and centrifuge at 10000g for 1 min. Aspirate the supernatant and resuspend with 100 ul ddH2O to get rid of the fluorescence background from LB medium. Add 100 µl sample solution into a sterile 96-well plate. Measure fluorescence with a microplate reader and then also measure OD600 for normalization.

4. SDS-PAGE.

4.1 Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis.

4.2 Add marker in the first lane on the left at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.

5. Enzyme Activity

5.1 Take 100 ul of bacterial solution, centrifuge at 10000g for 1min at room temperature and discard the supernatant. Resuspend with 100ul ddH2O, and add into this solution 100ul of standard test solution I (containing Phenolphthalein-β-D-glucuronide, and the test box was purchased from Nanjing Jiancheng Biotech company). Incubate the mixture at 37C for 1 hour.

5.2 Observe the solution. pink or purple color indicates positive enzyme activity. Quantitatively the activity can be measured by OD540 reading on a microplate reader, divided by its OD600 reading for normalization of concentration.

##### Results

First we needed to experimentally confirm that our gene circuit would work as we had expected. We used sfGFP (Part:K515105) gene to substitute the β-glucuronidase gene in the above plasmid construction. The reconstructed plasmid was then cloned in DH5a and then extracted to be transformed into BL21(DE3). With the induction Iptg added, we can clearly measure strong signal of green fluorescence light. The intensity is about 100 fold stronger than the background, which is obtained by measuring the same bacteria culture but without Iptg induction. This result confirm that with Iptg induction, our gene circuit works normally and can be used for expression of bG enzyme.

![](https://static.igem.org/mediawiki/2021/d/d9/T--iBowu-China--parts-6.png)

Figure. Expression of GFP by the designed gene circuit with and without Iptg induction.

To express the enzyme β-glucuronidase, the plasmid was cloned in DH5a and then extracted to transform BL21(DE3). Iptg was added at 1mM at OD600=0. The medium was incubated at 37 degrees Celsius with shaking overnight and then taken to SDS-PAGE experiment to check for the expression of the enzyme. This enzyme was expected to have a size about 67 kDa, and the SDS-PAGE result showed the successful expression of the protein at the expected size band (marked by a red arrow on the figure). We included a control group of vacant-pet28a, of which the only difference is there the β-glucuronidase sequence is removed. Control groups where there is no Iptg induction for the same plasmid and control groups of vacant-pet28a (labeled Pet) showed no intrinsic protein exists in the expected bands.

![](https://static.igem.org/mediawiki/2021/1/14/T--iBowu-China--parts-4.png)

Measurement of enzyme activities can be easily achieved by using β-glucuronidase to hydrolyze specific substance in a buffer and measure the hydrolysis product. Here we used Phenolphthalein-β-D-glucuronide as the substrate and after hydrolysis reactions with β-glucuronidase, free phenolphthalein will be released into the buffer. The system would show a pink to purple color after adding NaOH solution at a prepared standard concentration. The activity can also be quantitatively measured by measuring the light emission at 540 nm, which is the most intensive emission of colorant.

In the measurement, we added a control group labeled vacant-pet, where the plasmid and the gene circuit is the same, but the β-glucuronidase is removed. The control group went through exactly the same process of incubation and induction with Iptg to provide a background reading.

We also compare between the β-glucuronidase sequence from E. coli source (T7-bG-pet), Aspergillus oryzae source (bG14-pet), the Thermotoga maritima source (bG15-pet) and also a control group (vacant-pet). We found the enzyme encoded by the E. coli source (T7-bG-pet) has a significantly higher enzyme activity compared to others; and the enzyme activity from Aspergillus oryzae source and Thermotoga maritima source is not stronger than the background. Previous research works reported positive enzyme activities of the β-glucuronidase enzyme from these two sources, but here in our experiment conditions these two enzymes do not display discernible performance.

![](https://static.igem.org/mediawiki/2021/c/c7/T--iBowu-China--parts-5-1.png)

[Ins @ ibowuchina2020](https://www.instagram.com/ibowuchina2020/)
Facebook: iBowu China
Twitter @ IbowuC
[Ximalaya: iBowu\_China2020](https://www.ximalaya.com/zhubo/234355068/)
Email: iBowu-China2021@outlook.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:iBowu-China/Experiments

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

# Experiments

### Molecular Cloning 【1】PCR Primers were designed by snapgene software and synthesized at BGI Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification. 【2】Gel extraction DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250). 【3】Gibson Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201). 【4】Endonuclease digestion and ligation The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions. 【5】Transformation Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501). 【6】Plasmid extraction Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250). 【7】Sequencing Sanger sequencing was done by GENEWIZ Company and snapgene software is used to align the sequences. Expression and Measurements 1. Transform the plasmids into E. coli BL21(DE3) 2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37 degree Celsius in a shaker overnight. 3. Fluorescence. Acquire 100 ul bacteria culture and centrifuge at 10000g for 1 min. Aspirate the supernatant and resuspend with 100 ul ddH2O to get rid of the fluorescence background from LB medium. Add 100 µl sample solution into a sterile 96-well plate. Measure fluorescence with a microplate reader and then also measure OD600 for normalization. 4. SDS-PAGE. 4.1 Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis. 4.2 Add marker in the first lane on the left at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures. 5. Enzyme Activity 5.1 Take 100 ul of bacterial solution, centrifuge at 10000g for 1min at room temperature and discard the supernatant. Resuspend with 100ul ddH2O, and add into this solution 100ul of standard test solution I (containing Phenolphthalein-β-D-glucuronide, and the test box was purchased from Nanjing Jiancheng Biotech company). Incubate the mixture at 37C for 1 hour. 5.2 Observe the solution. pink or purple color indicates positive enzyme activity. Quantitatively the activity can be measured by OD540 reading on a microplate reader, divided by its OD600 reading for normalization of concentration.

[Ins @ ibowuchina2020](https://www.instagram.com/ibowuchina2020/)
Facebook: iBowu China
Twitter @ IbowuC
[Ximalaya: iBowu\_China2020](https://www.ximalaya.com/zhubo/234355068/)
Email: iBowu-China2021@outlook.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:iBowu-China/Results

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

[![](https://static.igem.org/mediawiki/2021/6/67/T--iBowu-China--logo-nav-3.jpg)](https://2021.igem.org/Team%3AiBowu-China)

* [Home](https://2021.igem.org/Team%3AiBowu-China)
* Project
  + [Description](https://2021.igem.org/Team%3AiBowu-China/Description)
  + [Experiments](https://2021.igem.org/Team%3AiBowu-China/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AiBowu-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AiBowu-China/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AiBowu-China/Results)
  + [Model](https://2021.igem.org/Team%3AiBowu-China/Model)
* [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Parts](https://2021.igem.org/Team%3AiBowu-China/Parts)
  + [Engineering](https://2021.igem.org/Team%3AiBowu-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AiBowu-China/Contribution)
* [Safety](https://2021.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Integrated HP](https://2021.igem.org/Team%3AiBowu-China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AiBowu-China/Communication)
* [Team](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Members](https://2021.igem.org/Team%3AiBowu-China/Team)
  + [Attributions](https://2021.igem.org/Team%3AiBowu-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AiBowu-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AiBowu-China/Partnership)

# Proof of Concept

In order to confirm whether β-glucuronidase can hydrolyze the reactant substance to drop a glucuronic acid, we selected an E. coli strain, E.coli BL21 (DE3), which is commonly used to express proteins for experiments and measurements. We chose to engineer the plasmid pet28a(+) that is known to be compatible with this strain for expression of β-glucuronidase protein by inserting the corresponding Gus sequence. To Distinguish from the previous works, we call our sequence the bG sequence.
The construction of the plasmid is shown in the schematic drawing on the right.

![](https://static.igem.org/mediawiki/2021/a/af/T--iBowu-China--parts_1.png)

**Figure 1.**  Construction of plasmid T7-bG-pet28a(+) for expression of the enzyme in E. coli BL21(DE3).

In order to measure the production of glycyrrhetinic acid, HPLC is the most commonly used method in the reference articles. It can quantitatively measure the amount of the reduction of GL (glycyrrhizic acid) and the production of GA (glycyrrhetinic acid), but the apparatus is highly costly for our lab to own. We have consulted a few iGEM teams in our HP and colab activities, and also consulted a few commercial companies about HPLC measurements, and from the responses, we learned this method still costs a lot even for a single measurement run because the special container is not recyclable. At the same time, HPLC measurements may take a long time, even up to 3 weeks, due to limited access and complex sample preparations. Therefore HPLC method is not a proper method in the first stage of experiments for optimization of the enzyme expression.

In our interviews, we learned there is a special test toolkit for β-glucuronidase. The reactant substance prepared in the toolkit contains Phenolphthalein β-D-glucuronide. After being hydrolyzed with the presence of β-glucuronidase, the product releases free Phenolphthalein in the buffer. By adding NaOH into the buffer after reaction, Phenolphthalein will show pink to purple color. The amount of Phenolphthalein released is proportional to the light intensity at OD540 according to the toolkit documentation, and the measurement can directly indicate the level of (the total) enzyme activity of β-glucuronidase. The method can not tell the amount of β-glucuronidase produced in each cell. In the following figure, we show the chemical theory for the production of GA by β-glucuronidase hydrolysis and the principle of enzyme activity measurement with the toolkit.

![](https://static.igem.org/mediawiki/2021/7/70/T--iBowu-China--POF-2.png)

According to our literature searches and Human Practices interviews with Prof. Lv Bo, we selected three different kinds of β-glucuronidase which is likely to have high catalytic effectiveness, namely the β-glucuronidase from E. coli source, from Aspergillus oryzae and from Thermotoga maritima.

All three enzymes were expressed in E. coli BL21(DE3), and we carried out SDS-PAGE experiments and enzyme activity experiments to evaluate their effectiveness. The results show only the β-glucuronidase enzyme from E. coli source has been successfully expressed, while β-glucuronidase (Aspergillus oryzae) and β-glucuronidase (Thermotoga maritima) expression had to be further explored. Similarly in the enzyme activity measurement, the β-glucuronidase enzyme from E. coli source has the highest enzyme activity. Therefore, we carried out further experiments based on this enzyme.

The failure to express β-glucuronidase (Aspergillus oryzae) and β-glucuronidase (Thermotoga maritima) might be the result of the following reasons.

1. The protein structure of the two enzymes are different from that of the E. coli source and therefore the translation and folding of this protein might not be compatible with the specific plasmid and E. coli strain we currently use.

2. The expression condition might need to be further optimized for these two enzyme such as the timing for induction, the working concentration of IPTG and/or the strain we are using.

Because we had only limited time in an iGEM season, we first focused on the experiments with the successfully expressed species, and if we had a chance in the future, we would carry out more experiments to explore the expression conditions at various induction conditions such as adding IPTG at OD600=0.6~0.8, or changing the IPTG concentration from 0.5mM to 2mM, or varying the incubation temperature from 16 degree Celsius to 37 degree Celsius, or using different expression strains like E. coli Rosetta or E. coli Plyss.

![](https://static.igem.org/mediawiki/2021/5/57/T--iBowu-China--2021-bG-SDS.jpg)

![](https://static.igem.org/mediawiki/2021/e/e3/T--iBowu-China--POF-3.png)

In our Human Practices interviews, we learned that there are a few more E. coli strains frequently and routinely used for protein expression and production in the factories. Under certain circumstances, they could be advantageous compared to the conventional BL21(DE3). We then tested the enzyme activities of β-glucuronidase expressed in different E. coli expression strains and found in our case the enzyme activities do not show a significant difference. For subsequent experiments, we decided to keep using E. coli BL21(DE3) strain with the enzyme from E. coli source.

![](https://static.igem.org/mediawiki/2021/0/07/T--iBowu-China--2021-POF-4.jpg)

The appropriate enzyme expression strength may contribute to the catalytic efficiency in the unit volume bacteria culture. With this consideration in mind, we plan to control the catalytic power of the bacteria culture by optimizing the expression of β-glucuronidase in E. coli. In the expression systems of E. coli, T7 promoter is a most commonly used promoter originating from the T7 phage. Researchers have obtained a series of modified T7 promoters with different expression strength through engineered mutations (Ref [C4-1], [C4-2]). We selected to test C4, H9 and G6 promoters on a reporter gene sfGFP. To our surprise, the fluorescence intensities we measured does not show a significant difference between the mutated promoter and the original T7.

![](https://static.igem.org/mediawiki/2021/4/4f/T--iBowu-China--POF-5.png)

Figure 6. sfGFP light intensity expressed by different promoters.

Considering that the intensity of sfGFP does not necessarily represent the expression of β-glucuronidase enzyme, We used different promoters to connect the β-glucuronidase enzyme from E. coli source to detect the enzyme activity under these conditions. β-glucuronidase enzymes from Aspergillus oryzae and from Thermotoga maritima were tested again. The results indicated the promoters indeed do not bear a significant difference. Therefore, the T7 promoter can still be used as the first choice for promoters. Promoters with different strengths will be obtained through mutation or directed evolution in the future.

Of course, we believe that the induction temperature and induction method may also affect the final catalytic ability of the bacterial solution, while E. coli may not be the best species. Other prokaryotes, yeasts, molds, etc. are also candidate chassis organisms. However, due to time limits, these experiments can only be carried out in the future.

![](https://static.igem.org/mediawiki/2021/b/ba/T--iBowu-China--POF-6.png)

Figure 7. β-glucuronidase enzyme activity from different promoters. bG14 is a short label for Aspergillus oryzae and bG15 is a short lable for Thermotoga maritima.

In addition, an expression system with induction can better control the expression of enzyme at the right timing. But we learned in Human Practices interviews that iPTG, as an inducer, introduces high costs in industrial production. Therefore, constitutive expression may be more suitable for industrial production, and constitutive expression system does not need to consider the choice of induced reagents, inducing reagent concentration, and other variables, and thus the system is conducive to the reduction of inter-batch difference and to increase stability. So we plan to create a system of constitutive expression system for the enzyme.

In the BL21 strain, both the T7 RNA polymerase and the β-glucuronidase enzyme on the pet28a vector contain lacO sequences in their upstream and therefore any expression needs to be induced. In the future, we plan to remove the LacO sequence before the T7 RNA polymerase gene in the BL21 genome through gene editing, as well as the LacO sequence in the pet28a vector to obtain a system for the expression of β-glucuronidase enzymes in a constitutive way.

However, due to the time limit, it is not realistic for us to realize genome editing during the 2021 iGEM season, and we are not currently doing this experiment. Alternatively, as a proof of concept, we designed a gene circuit in DH5a bacteria, using the E. coli constitutive promoter J23101 in upstream of the β-glucuronidase enzyme coding sequence. We tested for enzyme activity of the bacteria without any addition of the inducer IPTG.

![](https://static.igem.org/mediawiki/2021/6/65/T--iBowu-China--2021-POF-7.jpg)

Figure 8. Constitutive expression system tested for β-glucuronidase enzyme activity (A) The current design（J23101-bG）（B）The successful expression indicated by the pink color due to release of Phenolphthalein. Control group was exactly the same expression system without the addition of the testing substrate Phenolphthalein-β-D-glucuronide. （C）Quantitative measurement

Of course, in the above experiments, we used Phenolphthalein-β-D-glucuronide instead of glycyrrhizic acid to conduct experiments to reduce the time cost. Finally, we also used HPLC to verify some of the above experimental results. HPLC results show that β-glucuronidase enzyme can indeed convert glycyrrhizic acid. However, the experiment did not detect a signal of glycyrrhetinic acid after the reaction. Except for the possibility that better HPLC measurement conditions might help to detect the glycyrrhetinic acid signal, there is still the possibility that some new product was formed but escaped from the detection.
It is believed that after continuous optimization, this method can be used to make glycyrrhetinic acid cheaper and more environmentally friendly in the industry for use in medical, food, and other industries.

![](https://static.igem.org/mediawiki/2021/6/6f/T--iBowu-China--POF-8.png)

Figure 9. HPLC results show that after overnight incubation, glycyrrhizic acid can be decomposed in the bacteria culture containing β-glucuronidase enzyme. The experiments failed to detect a significant signal of glycyrrhetinic acid after the reaction.

### References

---

[1]. Jones, J., Vernacchio, V., Lachance, D. et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci Rep 5, 11301 (2015). https://doi.org/10.1038/srep11301

[2]. Adams, A. M., Kaplan, N. A., Wei, Z. et al. In vivo production of psilocybin in E. coli. Metabolic Engineering, vol 56 p111-119, (2019). https://doi.org/10.1016/j.ymben.2019.09.009.

[3]. Afkhami-Poostchi A., Mshreghi, M., Iranshahi M., et al. Use of a genetically engineered E. coli overexpressing β-glucuronidase accompanied by glycyrrhizic acid, a natural and anti-inflammatory agent, for directed treatment of colon carcinoma in a mouse model. International Journal of Pharmaceutics, Vol 579, 119159 (2020). https://doi.org/10.1016/j.ijpharm.2020.119159.

[4]. Wang, X., Feng, X., Lv, B., et al. Enhanced yeast surface display of β-glucuronidase using dual anchor motifs for high-temperature glycyrrhizin hydrolysis. AIChE Journal. Vol 65, e16629 (2019).

[5]. Wang, Z., Pei, J., Li, H., and Shao, W.
Expression, Characterization and Application of Thermostable β-glucuronidase from Thermotoga maritima. Chin J Biotech Vol 24(8), 1407-1412 (2008).

[6]. Wang, Guojing. Enzymatic Production of Glycyrrhetinic Acid and the Separation Process Design. Master Thesis, Beijing Institute of Technology. (2016)

[7]. Brittona, J., Majumdar, S., and Weiss, G. A.
Continuous Flow Biocatalysis.
Chem Soc Rev. Vol 47(15): 5891–5918. (2018) doi:10.1039/c7cs00906b

![](https://static.igem.org/mediawiki/2020/4/45/T--iBowu-China--ibowu.png)
![](https://static.igem.org/mediawiki/2020/6/60/T--iBowu-China--snapgene.png)
![](https://static.igem.org/mediawiki/2020/c/c9/T--iBowu-China--BGIlogo.jpeg)

[Ins @ ibowuchina2020](https://www.instagram.com/ibowuchina2020/)
Facebook: iBowu China
Twitter @ IbowuC
[Ximalaya: iBowu\_China2020](https://www.ximalaya.com/zhubo/234355068/)
Email: iBowu-China2021@outlook.com