iGEM 2021 — Team WHU-China

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# Team:WHU-China/Notebook

[![](https://static.igem.org/mediawiki/2021/8/87/T--WHU-China--logo.png)](https://2021.igem.org/Team%3AWHU-China)

* Project
  + [Description](https://2021.igem.org/Team%3AWHU-China/Description)
  + [Design](https://2021.igem.org/Team%3AWHU-China/Design)
  + [Contribution](https://2021.igem.org/Team%3AWHU-China/Contribution)
  + [Prospect](https://2021.igem.org/Team%3AWHU-China/Prospect)
  + [Safety](https://2021.igem.org/Team%3AWHU-China/Safety)
  + [Sustainable](https://2021.igem.org/Team%3AWHU-China/Sustainable)
* Wet Lab
  + [Parts](https://2021.igem.org/Team%3AWHU-China/Parts)
  + [Proof Of Concept](https://2021.igem.org/Team%3AWHU-China/Proof_Of_Concept)
  + [Measurement](https://2021.igem.org/Team%3AWHU-China/Measurement)
  + [Engineering](https://2021.igem.org/Team%3AWHU-China/Engineering)
  + [Results](https://2021.igem.org/Team%3AWHU-China/Results)
  + [Notebook](https://2021.igem.org/Team%3AWHU-China/Notebook)
  + [Protocols](https://2021.igem.org/Team%3AWHU-China/Protocols)
* [Model](https://2021.igem.org/Team%3AWHU-China/Model)
* [Hardware](https://2021.igem.org/Team%3AWHU-China/Hardware)
* HP
  + [Integrated
    Human
    Practices](https://2021.igem.org/Team%3AWHU-China/Human_Practices)
  + [Interview](https://2021.igem.org/Team%3AWHU-China/Interview)
  + [Education](https://2021.igem.org/Team%3AWHU-China/Education)
  + [Inclusivity](https://2021.igem.org/Team%3AWHU-China/Inclusivity)
  + [Communication](https://2021.igem.org/Team%3AWHU-China/Communication)
* Application
  + [Entrepreneurship](https://2021.igem.org/Team%3AWHU-China/Entrepreneurship)
  + [Implementation](https://2021.igem.org/Team%3AWHU-China/Implementation)
* About
  + [Attributions](https://2021.igem.org/Team%3AWHU-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AWHU-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AWHU-China/Partnership)
  + [Team](https://2021.igem.org/Team%3AWHU-China/Team)
* Awards
  + [Awards](https://2021.igem.org/Team%3AWHU-China/Awards)
  + [Excellence in

    another area](https://2021.igem.org/Team%3AWHU-China/Excellence_In_Another_Area)
  + [Criteria](https://2021.igem.org/Team%3AWHU-China/Criteria)

Wet Lab > Notebook

![](https://static.igem.org/mediawiki/2021/8/85/T--WHU-China--note_first.png)
Notebook

### Week 1 (5.24-5.30)

## Wet

---

1. We tested the condition of inducing the GFP to express.
2. Experiment preparation and extraction of pET26b+.
3. Search the literature and identify the target gene for knockout
   experiment.
4. Obtained relevant gene sequence information and designed expression
   vector.

## HP

---

1. Interview professors in related fields, introduce our project to them
   and ask them to make suggestions to help us refine the design and
   improve the feasibility of the project.
2. In conjunction with world skin care days, skin health-related tweets
   are tweeted within our official account.

### Week 2 (5.31-6.6)

## Wet

---

1. We tested the condition of inducing the GFP to express, and asked
   NTHU for advice about how to diffuse the oleic acid.
2. Extraction of vector pUC-PctA and construction of vector
   pET26b+-PctA.
3. Contact the professor's lab for material support and technical
   guidance.Experiment preparation.
4. Designed and synthesized the primer of fadD and fadE.

## HP

---

1. Etodole surrounding community, to popularize the knowledge of skin
   health as well as acne for the group of elementary school students.

### Week 3 (6.7-6.13)

## Wet

---

1. We devoted to find out the suitable way measuring the fluorescence
   intensity and make it.
2. First attempt to transform pET26b+-PctA into *E. coli* Dh5alpha.
3. Conduct the first round of hisBCD gene knockout.
4. Construction of the recombinant vectors of FadD and FadE.

## HP

---

1. Participation in the CCiC HP discussion meeting.

### Week 4 (6.14-6.20)

## Wet

---

1. We explore how the concentration of oleic acid inluence the express
   of GFP.
2. Extraction and PCR identification of pET26b+ and pUC67.
3. Conduct the second round of hisBCD gene knockout.
4. Construction of the recombinant vectors of FadD and FadE.

### Week 5 (6.21-6.27)

## Wet

---

1. We explore how the time of induction influence the express of GFP.
2. Second attempt to transform pET26b+-PctA into *E. coli* Dh5alpha.
3. Conduct the second round of hisBCD gene knockout4.
4. Construction of the recombinant vectors of FadD and FadE.

## Dry

---

1. Discussed how to use modeling method in our project and brought up
   several potential modeling ideas

## HP

---

1. Exchange the progress and experience of model establishment with
   HUST2-China.

### Week 6 (6.28-7.4)

## Wet

---

1. Searched for the articles that concerned FadR and directed evolution
   on the internet;
2. Extraction of pET26b+ and pUC67
3. Analyze the reasons for the experiment failure. Search the literature
   and preliminarily determine the feasibility of using CRISPR-Cas9 as
   gene editing tools in *E. coli*.
4. Try to explore the best expression conditions of the target
   protein(FadD,FadE)

## Dry

---

1. Designed physical models in conjunction with directed evolution
   projects

## HP

---

1. Contact relevant pharmaceutical enterprises to prepare for the
   commercialization of the project.

### Week 7 (7.5-7.11)

## Wet

---

1. Reorganized all the mathods used in the articles we have read, and
   start searching for the advantages and disadvantages of each method;
2. Extraction of pET26b+ and pUC67 and enzyme cut.
3. Search the literature and choose glyA as target knockout.
4. Try to explore the best expression conditions of the target
   protein(FadD,FadE)

## Dry

---

1. Determined to use Microfluididic chips to build a high throughput
   directional evolution platform

## HP

---

1. Participate in the "Luojiazhihang" activity organized by Wuhan
   University and practice for the commercialization and real world
   application of our project.

### Week 8 (7.12-7.18)

## Wet

---

1. Through discussion, decided on the method best suited for our
   project, and started searching for protocols in the internet and
   prepare for the experiment;
2. Construction of pET26b+-PctA.
3. Conduct the first round of glyA gene knockout based on DH5α strain.
4. Try to explore the best expression conditions of the target
   protein(FadD,FadE)

## Dry

---

1. Used L-edit to design chip structure

### Week 9 (7.19-7.25)

## Wet

---

1. Designed plasmid and primers, and ordered on the internet.
2. Conduct the second round of glyA gene knockout based on DH5α strain.
3. Induced protein expression and detected the ability of engineered
   bacteria β- oxidative metabolism.

## Dry

---

1. Lithographic printed mask and first edition chip

## HP

---

1. Participate in iGEM central China exchange meeting and carry out
   project exchange and cooperation with iGEM teams in Central China.

### Week 10 (7.26-8.1)

## Wet

---

1. Tried different conditions of epPCR, in search for the best
   condition.
2. Successful identification of pET26b+-PctA in DH5alpha.
3. Conduct the second round of glyA gene knockout based on DH5α strain
   and analyse the sequencing results. There may be something wrong
   with the primers, so we redesigned the primers and decided to try
   again.We also chose MG1655 as a new chassis for gene knockout
   experiment in case of the influence of DH5α itself.
4. Induced protein expression and detected the ability of engineered
   bacteria β- oxidative metabolism.

## Dry

---

1. Pre-tested the first version of the chip

## HP

---

1. Cooperate with the high school team (XHD-Wuhan-Pro), help them
   improve their project and visit the laboratory.

### Week 11 (8.2-8.8)

## Wet

---

1. Extracted genomic DNA, and used the genomic DNA as the template of
   PCR and epPCR to obtained FadR.
2. Extraction of pET26b+-PctA from DH5alpha and transformation of it
   into *E. coli* BL21(DE3)
3. Contact with the professor's lab to attain MG1655 strain.Extract
   pKD46 and pKD13.
4. Purified the target protein with nickel column and then detected its
   expression with SDS-PAGE electrophoresis.

## Dry

---

1. Constructed growth simulator model to predict the coculture of
   engineering bacteria and *P. acnes* by differential equations.
2. Redesigned the second version of the chip

### Week 12 (8.9-8.15)

## Wet

---

1. Performed PCR product purification, enzyme cut, gel electrophoresis
   and gel purification of FadR, but found that the purified product
   has such a low concentration that it is hard to continue in the next
   step.
2. Failed identification of pET-26b+-PctA in BL21(DE3) and second
   transformation of pET26b+-PctA.
3. We extracted the pKD13 plasmid from the lab-supplied strain, but
   encountered some problems in culturing the strain, which did not
   seem to grow on kanamycin resistant plates. In the process of
   communication with the laboratory,we learned that the graduate
   students were also troubled by this problem, but the dilemma is that
   we couldn't obtain plasmids from other sources for the time being.

## Dry

---

1. Used simulated data to solve the parameters in growth simulator and
   made sure the momdel was usable.
2. Brought up the FBA model.
3. Remade the second version chip and tested the performance

## HP

---

1. Cooperate with CAU-China and HUST-China to conduct online publicity
   and complete public education.
2. Interview Dr. Min Wang, a plastic surgeon of Central South Hospital,
   to understand the cutting-edge acne treatment strategies.

### Week 13 (8.16-8.22)

## Wet

---

1. Changed some experimental conditions and tried again to get FadR
   digested with enzyme.
2. Successful sequencing for pET26b+-PctA in BL21(DE3) and SDS-PAGE for
   *E. coli* BL21(DE3) pET26b+-PctA
3. Culture strains containing pKD13 from previous lab and contact with
   other labs for pKD13.

## Dry

---

1. Made beads loading,bacteria loading,and water in oil droplets in the
   second version of the chip
2. Found data and solved the FBA model

## HP

---

1. Interview Prof. Zhixiongxie and ask for the problems encountered in
   the experiment for a second visit.

### Week 14 (8.23-8.29)

## Wet

---

1. Tried to get FadR digested with enzyme again, this time using *E. coli*
   DH5α as a template directly, without the step of genomic DNA
   extraction, and tried new gel extration kit, which yield a better
   rate of recovery.
2. SDS-PAGE orthogonal experiment of induced expression of pET26b+-PctA
   and optimization of experiment conditions

## Dry

---

1. Searched online FBA tools

## HP

---

1. Participate in CCiC activities.
2. Interview Renfu pharmaceutical and other related pharmaceutical
   enterprises.

### Week 15 (8.30-9.5)

## Wet

---

1. Tried to get pBAD33 digested with enzyme, joined DNA fragments and
   transformed into DH5α., when the product was linked into the
   plasmid, no transformant were found.
2. SDS-PAGE orthogonal experiment of induced expression of pET26b+-PctA
   and optimization of experiment conditions
3. Conduct the third round of glyA gene knockout based on MG1655 strain.
   4. Access to information and analyze the reason why the molecular
   weight of protein is lower than expected.

## Dry

---

1. Observed the capture efficiency was observed under a microscope
2. Used Escher tool to build up a new FBA model

## HP

---

1. Interview the counters of skin care products to get to know the sales
   of skin care products.
2. Carry out science popularition on synthetic biology and acne
   treatment for students in the middle school attached to Wuhan
   University.

### Week 16 (9.6-9.12)

## Wet

---

1. Performed epPCR and PCR again using the bacteria as the template to
   get FadR for many times, but after electrophoresis, there are only
   diffusion band.
2. Optimization of experimental conditions of SDS-PAGE and induction of
   pET-26b+-PctA.
3. Conduct the third round of glyA gene knockout based on MG1655 strain.
4. Changed the engineering strain - the *E. coli* DH5α strain was replaced
   with BL21 strain.

## Dry

---

1. Designed and printed in 3-D for microscope viewing
2. Assigned tasks to sort out modeling results and write articles

## HP

---

1. Interview Dr. Ding Hong, a professor in the field of acne treatment
   again, and ask about what kind of product form is more suitable for
   our project.

### Week 17 (9.13-9.19)

## Wet

---

1. Wondered whether the diffusion band is due to degration of the primer
   or due to contemination of the PCR mastermix, another mastermix and
   another primer were ordered.
2. Salt fractionation purification of PctA and SDS-PAGE of unpurified
   and purified PctA
3. Conduct the fourth round of glyA gene knockout based on DH5α strain.
4. Induced the target protein in *E. coli* BL21 strain and then purified
   the target protein with nickel colum,detected its expression with
   SDS-PAGE electrophoresis

## HP

---

1. Participate in the project defense of "Luojiazhihang" and
   successfully conclude the project.
2. Popularize synthetic biology and iGEM to undergraduates in the
   college of life sciences of Wuhan University.

### Week 18 (9.20-9.26)

## Wet

---

1. Performed both kinds of PCR again, using the new agents we have
   ordered, still using the genomic DNA as the template, this time,
   there are correspounding band after the gel electrophoresis.
2. Ni-IMAC of PctA and SDS-PAGE of unpurified and purified PctA.
3. Conduct the fourth round of glyA gene knockout based on DH5α strain.
   Design the primers and sequence genes. Analyse the data.

## Dry

---

1. Designed the third version chip for better performance in water in
   oil droplets

### Week 19 (9.27-10.3)

## Wet

---

1. Due to the lack of time, the product wasn't linked into the plasmid,
   but rather, it was sequenced to see if there were indeed a higher
   mutation rate after the epPCR, compared to the normal PCR.
2. Western blot of unpurified and purified PctA and design of
   pET26b+-OmpA-PctA with optimized signal sequence

## Dry

---

1. Revised our modeling articles and translated them into English.

## HP

---

1. Participate in ICII and publicize iGEM for the whole school.

Contact Us

[![](https://static.igem.org/mediawiki/2021/9/9f/T--WHU-China--web.png)](http://www.bio.whu.edu.cn/en.htm)
![](https://static.igem.org/mediawiki/2021/4/43/T--WHU-China--wx.png)

[![](https://static.igem.org/mediawiki/2021/c/c3/T--WHU-China--weibo.png)](https://weibo.com/u/7100890882)
[![](https://static.igem.org/mediawiki/2021/7/7b/T--WHU-China--bilibili.png)](https://space.bilibili.com/13333167)

Acknowledgement

[![](https://static.igem.org/mediawiki/2021/8/86/T--WHU-China--wuda.png)](https://www.whu.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/4/48/T--WHU-China--wuda_cs.png)](http://cs.whu.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/2/22/T--WHU-China--wuda_hy.png)](http://hyxt.whu.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/c/c4/T--WHU-China--wuda_shu.png)](http://maths.whu.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/c/ce/T--WHU-China--wuda_sk.png)](http://www.bio.whu.edu.cn/)

[![](https://static.igem.org/mediawiki/2021/9/97/T--WHU-China--wuda_yao.png)](http://www.pharm.whu.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/7/76/T--WHU-China--jiyi.png)](http://wbm.whu.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/7/7e/T--WHU-China--yasheng.png)](https://www.ascentagepharma.com/?lang=zh-hant)
[![](https://static.igem.org/mediawiki/2021/1/1a/T--WHU-China--renfu.png)](http://www.humanwell.com.cn/)

![](https://static.igem.org/mediawiki/2021/9/90/T--WHU-China--zhi_ding.png)

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

# Team:WHU-China/Engineering

[![](https://static.igem.org/mediawiki/2021/8/87/T--WHU-China--logo.png)](https://2021.igem.org/Team%3AWHU-China)

* Project
  + [Description](https://2021.igem.org/Team%3AWHU-China/Description)
  + [Design](https://2021.igem.org/Team%3AWHU-China/Design)
  + [Contribution](https://2021.igem.org/Team%3AWHU-China/Contribution)
  + [Prospect](https://2021.igem.org/Team%3AWHU-China/Prospect)
  + [Safety](https://2021.igem.org/Team%3AWHU-China/Safety)
  + [Sustainable](https://2021.igem.org/Team%3AWHU-China/Sustainable)
* Wet Lab
  + [Parts](https://2021.igem.org/Team%3AWHU-China/Parts)
  + [Proof Of Concept](https://2021.igem.org/Team%3AWHU-China/Proof_Of_Concept)
  + [Measurement](https://2021.igem.org/Team%3AWHU-China/Measurement)
  + [Engineering](https://2021.igem.org/Team%3AWHU-China/Engineering)
  + [Results](https://2021.igem.org/Team%3AWHU-China/Results)
  + [Notebook](https://2021.igem.org/Team%3AWHU-China/Notebook)
  + [Protocols](https://2021.igem.org/Team%3AWHU-China/Protocols)
* [Model](https://2021.igem.org/Team%3AWHU-China/Model)
* [Hardware](https://2021.igem.org/Team%3AWHU-China/Hardware)
* HP
  + [Integrated
    Human
    Practices](https://2021.igem.org/Team%3AWHU-China/Human_Practices)
  + [Interview](https://2021.igem.org/Team%3AWHU-China/Interview)
  + [Education](https://2021.igem.org/Team%3AWHU-China/Education)
  + [Inclusivity](https://2021.igem.org/Team%3AWHU-China/Inclusivity)
  + [Communication](https://2021.igem.org/Team%3AWHU-China/Communication)
* Application
  + [Entrepreneurship](https://2021.igem.org/Team%3AWHU-China/Entrepreneurship)
  + [Implementation](https://2021.igem.org/Team%3AWHU-China/Implementation)
* About
  + [Attributions](https://2021.igem.org/Team%3AWHU-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AWHU-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AWHU-China/Partnership)
  + [Team](https://2021.igem.org/Team%3AWHU-China/Team)
* Awards
  + [Awards](https://2021.igem.org/Team%3AWHU-China/Awards)
  + [Excellence in

    another area](https://2021.igem.org/Team%3AWHU-China/Excellence_In_Another_Area)
  + [Criteria](https://2021.igem.org/Team%3AWHU-China/Criteria)

Wet Lab > Engineering

![](https://static.igem.org/mediawiki/2021/7/76/T--WHU-China--engi_first.png)
Engineering

[Overview](#title1)

[DBTL cycle of fatty acid consumption module](#title2)

[DBTL cycle of the signal peptide for PctA](#title3)

[DBTL cycle of fatty acids sensing module](#title4)

[DBTL cycle of gene knockout module](#title5)

[DBTL cycle of Hardware](#title6)

---

## Overview

The idea of engineering and DBTL(Design-Build-Test-Learn cycle) was always kept in
our minds since it’s one of the major ideas of synthetic biology. With comprehensive efforts in the
field of wet lab, model, hardware as well as human practice, we endeavored to learn from what we
obtained for improving our methodologies to demonstrate our designs.

In all, we did generate expected results to a certain degree. DBTL cycles of our
four parts designed are described in detail in the following contents.

---

## DBTL cycle of fatty acid consumption module

In our design, consuming excess sebum on the skin surface to prevent it from
blocking pores is one of the two important ways for our engineered bacteria to treat acne.

In the early stage of acne, the skin secretes a large amount of sebum which blocks
the pores. The blocked pores form an anaerobic environment, which provides a more suitable
environment for *Propionibacterium acnes* and proliferates *Propionibacterium acnes* in large numbers.

Over-proliferated *Propionibacterium acnes* will decompose sebum and produce a large
number of free fatty acids. These fatty acids penetrate into skin capillaries through pores, causing
local stress response,swelling, redness and acne, resulting in skin tissue damages. In view of this
pathogenic mechanism, we proposed to consume the excessive sebum on the skin surface in order to
block the excessive proliferation of *Propionibacterium acnes* and the immune response triggered by
fatty acids.

In order to make our engineered bacteria consume the excess sebum on the skin
surface more efficiently, we put our efforts on improving the β-oxidation ability of engineered
bacteria (i.e. improving the decomposition ability of fatty acids). As shown in the following
figure, fatty acids need to be activated to produce Fatty acyl-CoA before β-oxidation. Then, Fatty
acyl-CoA enters the β-oxidation pathway. After oxidation, hydration, oxidation and cleavage, the
fatty acyl-CoA breaks to form acetyl-CoA and fatty acyl-CoA with two carbon atoms less. When we
inquired about the literature and the work of iGEM teams in previous years, we learned that
overexpression of two enzymes in the β-oxidation pathway, fatty acyl-CoA synthetase (FadD) and fatty
acyl-CoA dehydrogenase (FadE), in engineered bacteria can significantly improve the oxidation
ability of engineered bacteria.

![](https://static.igem.org/mediawiki/2021/7/77/T--WHU-China--engi_1.png)

Figure 1. Fatty Acid β-oxidation pathway

Therefore, we tried to repeat this result, that is, overexpressing these two enzymes
in our engineered.bacteria to improve their β-oxidation ability, so as to achieve the purpose of
rapid consumption of excess sebum on the skin surface by engineered bacteria.

In order to overexpress FadD and FadE protein in engineered bacteria, we designed
the following two plasmids. We first designed primers, amplified FadD and FadE genes from the genome
of E. coli K12 strain by PCR, and added corresponding enzyme digestion sites on both sides of the
gene; At the same time, we added six his tags at the N-terminal of the protein for subsequent
purification and detection. After that, we connected our target fragment with the vector by enzyme
digestion and enzyme linkage.

![](https://static.igem.org/mediawiki/2021/f/f1/T--WHU-China--engi_2.png)
![](https://static.igem.org/mediawiki/2021/2/20/T--WHU-China--engi_3.png)

Figure 2. The maps of pBAD33-FadE and pDSW208-FadD

After the construction of the vector, we transferred the recombinant plasmid into *E. coli* DH5α
Strain and induced expression. After a simple protein expression test, we began the functional
test. We mixed the two-hour protein induced bacterial solution with equal volume of LB medium
dissolved with fatty acid sodium and continued to culture at 37 ℃ for 4h. Then we detected the
residual amount of fatty acids in the culture medium However, in this experiment, we did not get
the expected results. The fatty acid decomposition ability of engineered bacteria which
overexpresses FadD and FadE was not significantly higher than that of negative control.

![](https://static.igem.org/mediawiki/2021/d/dc/T--WHU-China--engi_4.png)

Figure 3. The decomposition ability of engineering bacteria

In order to explore the reasons for the failure of our experiment, we tested the
protein expression more carefully. Our protein was purified by nickel column and then detected by
SDS-PAGE . The detection results show that the molecular weight of our target protein is about 20kDa
less than our expected molecular weight - which explains why our constructed system does not achieve
our expected results. Because the engineered bacteria we use this time is *Escherichia coli* DH5α. The
internal protease system of the strain is not knocked out, so the overexpressed protein is easy to
be degraded. For this reason, we replaced our host strain with *E. coli* BL21 strain for following
experiments.

---

## DBTL cycle of the signal peptide for PctA

The successful expression and secretion of PctA by our engineered bacteria is of
vital importance to our project. In the expected situation, our engineering should transport PctA
across plasma membrane and then outer membrane, letting it perform its duty in the surrounding
environment.

The original PctA itself is a secretory protein purified from the supernatant of the
culture. So in the first version of our design, we keep the original Sec signal peptide in the full
sequence of PctA gene [1]. We adapted this signal peptide because the Sec system is also found in
*Escherichia coli* . According to our prediction, the bacteria will first produce a 11 kDa precursor
protein, then transport it into the periplasmic space and at the same time cut off the signal
peptide after the Ala-Met-Ala sequence. Finally, a 7 kDa mature PctA protein will diffuse across
outer membrane into the environment.

![](https://static.igem.org/mediawiki/2021/e/e9/T--WHU-China--engi_5.png)

Figure 4. The full sequence of PctA gene

We adapt the original Sec signal peptide, and made no adjustment except adding a His
tag. We put the sequence in plasmid pET26b(+) with T7 promotor to test the expression, secretion and
the function of PctA.

In order to test the expression and secretion of PctA, we induced the engineered
bacteria in different temperature and IPTG concentration, then analyze protein composition with
SDS-PAGE (later tricine-SDS-PAGE) and Western Blot. Most of PctA form inclusion body and end up in
precipitation; limited amounts of PctA in solution can be tested by WB; no PctA can be found in
culture supernatant both by SDS page and Western Blot, even after the supernatant is concentrated
for 40 times. This could result from the overpowerful of T7 promotor, or the original Sec signal
peptide doesn’t fit in *Escherichia coli* expression system. PctA with Sec peptide is likely to end up
in plasma or periplasmic space, which is different from our prediction.

![](https://static.igem.org/mediawiki/2021/0/05/T--WHU-China--engi_6.png)

Figure 5. The SDS page of induced engineered bacteria. PctA is successfully induced,
but most end up in precipitation. A: the solution of bacteria lysis; B: the precipitation of
bacteria lysis.

On the basis of former experiment, we begin the second turn of DBTL cycle. We look
through papers and find a new way of transporting PctA. A new signal peptide, OmpA was chosen,
because it is widely used in *E. coli* and at least one experiment can prove that protein with OmpA
was
successfully transported across outer membrane and entered the culture supernatant [2].

![](https://static.igem.org/mediawiki/2021/0/05/T--WHU-China--engi_7.png)

Figure 6. The redesigned OmpA-PctA recombined protein. OmpA is added to replace Sec
signal peptide.

Due to the lack of time, we could not practice our new design to see if it works
out. However, we are planning to finish this experiment in the future.

---

## DBTL cycle of fatty acids sensing module

In the initial design of the project, there was actually no such module. At first,
we hoped that the bacteriocin against *Propionibacterium acnes* could be expressed directly in a
constitutive manner to achieve the optimal bactericidal effect. However, after consulting literature
and communicating with medical college professors, we found that *Propionibacterium acnes* also
existed on the skin surface of healthy people and played a certain role. It can produce protease and
lipase to release arginine from the skin and degrade triglycerides to survive in the anoxic
sebaceous gland. The released fatty acid also increases the adhesion between microflora [3]. In
order to maintain a balanced, symbiotic relationship between the skin and skin microbiota, we
designed this module and selected the fatty acid-sensitive promoter, in the hope of providing a
relatively accurate control over the yield of bacteriocin.

However, we found that the existing fatty acids-sensitive promoters have a bad
performance, taking a long time to better start the expression with high leakage expression.
Therefore, we first used the existing fatty acid sensitive promoters and made artificial
improvements on the RBS. The plasmids we designed is shown in the figure below.

![](https://static.igem.org/mediawiki/2021/2/24/T--WHU-China--engi_8.png)
![](https://static.igem.org/mediawiki/2021/7/7b/T--WHU-China--engi_9.png)

Figure 7. The maps of pDSW208-99-1 and pDSW208-99-2. (a)
pDSW208-99-2(pFadD\_Lac+B0030+GFP) contains RBS without modification, and can test promoter
function. (b) pDSW208-99-2(pFadD\_Lac+ BBa\_K3763000+GFP) Contains RBS with modification, and
can test promoter function.

After consulting a large number of RBS data, we found that most RBS with a higher binding
efficiency had the repetitive sequence of "aggg", or "aaa" after the start codon. B0030 was the
one with the highest expression efficiency among the four existing RBS, with the sequence of
"aaa" but no sequence of "aggg". Therefore, we modified the sequence to change "aagagg" to
"aggagg", so that it could simultaneously have the characteristics of two sequences with high
binding efficiency. In later experiments, we wanted to compare the performance of the two
sequences.

![](https://static.igem.org/mediawiki/2021/e/e0/T--WHU-China--engi_10.png)

Figure 8. The change in sequence

After the successful transformation of the plasmid, we used different concentrations of fatty
acids for induction for different times. The expression level of eGFP was determined by
fluorescence detected with a microplate reader, reflecting the performance of the promoter. The
results are as follows:

![](https://static.igem.org/mediawiki/2021/9/9b/T--WHU-China--engi_11.png)

Figure 9. Promoter and eGFP

![](https://static.igem.org/mediawiki/2021/b/b9/T--WHU-China--engi_12.png)

Figure 10. BBa\_K3763002(pFadD\_Lac-RBS BBa\_B0030-GFP-rrnB T1 and T2 terminator,
pDSW208-99-2) contains RBS without modification

![](https://static.igem.org/mediawiki/2021/b/bf/T--WHU-China--engi_13.png)

Figure 11. BBa\_K3763003(pFadD\_Lac-RBS BBa\_K3763000-GFP-rrnB T1 and T2 terminator,
pDSW208-99-2) Contains RBS with modification

According to the results, we can find that the performance of our fatty
acids-sensitive promoter is improved to a certain extent. Taking 0.125× fatty acid concentration as
an example, the fluorescence intensity of BBa\_K3763003 is over 11000 after 6 hours, while the
promoter of BBa\_K3763002 is less than 10000 at three fatty acid concentrations. Although we can see
that the optimized promoter still has a high expression leakage and low expression in a short time,
the experimental results have been able to prove the function of the fatty acids-sensitive promoter,
and partially prove the effectiveness and rationality of our design. For future optimization, after
the first DBTL cycle, we have conducted a new round of design and experimental work.

We wanted to regulate the exact expression of bacteriocins from multiple
perspectives. After the optimization of RBS, we turned our target to the improvement of FadR, and
designed a directed evolution method to optimize the function of the repressor FadR corresponding to
this promoter.

FadR is one of the regulatory enzymes in fatty acid biosynthesis in *E. coli*., which
acts as the repressor in the fatty acid metabolism system. In the absence of fatty acids, FadR will
attach to the gene fragment, and block the entire gene expression.

![](https://static.igem.org/mediawiki/2021/b/bd/T--WHU-China--design_3.gif)

Figure 12. pFadD\_Lac promoter and FadR

Directed evolution (DE) is a method used in protein engineering that harnesses the
power of selection to evolve molecules, typically proteins or nucleic acids, from large,
stochastically permuted pools or combinatorial libraries, to obtain a desired properties. It mainly
comprises the following steps:

(1) gene diversification by random mutagenesis and/or gene recombination to generate
a diverse library of variants;

(2) screening/selection to obtain variants with improved phenotypes.

(3)The improved variant will serve as a new starting point for the next round of
gene diversification. [4]

The experimental process we designed is shown in the figure:

![](https://static.igem.org/mediawiki/2021/8/82/T--WHU-China--engi_15.png)

Figure 13. The process of directed evolution

We obtain the sequence of FadR directly by cell disruption of *E. coli* DH5α. After
obtaining FadR, we further combined it with araBAD promoter. The following is our design of plasmid:

![](https://static.igem.org/mediawiki/2021/c/c3/T--WHU-China--engi_16.png)

Figure 14. The map of pBAD33-FadR

After that, we designed to transfer the obtained plasmid into DH5α which already
contained a plasmid with a fatty acids-sensitive promoter and GFP, induce it with different
concentrations of fatty acids, and measure the fluorescence intensity.

At the same time, we obtained a mutation library of FadR by error-prone PCR, and
performed the same operations as for FadR that were not subjected to error-prone PCR.

The gene sequencing results indicated that we successfully get FadR gene and
introduce random nucleotide mutations into the parent sequence qualitatively because there are
overlapping peaks.

a)

![](https://static.igem.org/mediawiki/2021/5/5f/T--WHU-China--engi_17.png)

b)

![](https://static.igem.org/mediawiki/2021/5/58/T--WHU-China--engi_18.png)

Figure 15. Partial FadR sequencing results: a)results of PCR b) results of
error-prone PCR

We haven’t finished this DBTL cycle due to the lack of time, but more work will be
done in the future.

When discussing the problem of the time consumption of the directed evolution
module, the students in the hardware group designed a microfluidic chip to help us. This is
described in detail in the DBTL section of the physical model.

In summary, in this module, we have completed a complete DBTL cycle and obtained
parts with acceptable performance, and we are performing a second cycle in order to make the
engineered bacteria more sensitive to fatty acid. At the same time, we optimized our experiment by
cooperate with hardware. Although some of the results are not completely obtained due to the lack of
time, we believe that we are nearing success.

---

## DBTL cycle of gene knockout module

In our design, a defect type of strain is constructed to prevent pollution in case
of engineered bacteria escaping. In the literature review process, we found several common types of
gene defects, including nutritional defects, cell wall defects and so on.[5][6][7] We chose to
construct the deficient strain by knockout, because it is easier to ensure the normal growth of the
deficient strain by replenishing certain nutrients compared to other methods. Among the common
nutritional defects, the construction technology of amino acid deficient strains is relatively
mature, and amino acids are often used in cosmetics as beneficial ingredients, so we chose to
construct amino acid deficient strains.

*Escherichia coli* histidine operon is composed of 9 genes including
hisL、hisG、hisD、hisC、hisB、hisH、hisA、hisF and hisIE. They are responsible for the synthesis of enzymes
needed for histidine synthesis in *E. coli*. HisDCB gene in histidine operon is responsible for the
last three steps of histidine synthesis: aminotransference, dephosphorization and dehydrogenation.
They are essential components of histidine synthesis, and their absence will cause the nutritive
deficiency type of histidine, making the growth of strain dependent on histidine.[7]

pKD46 plasmid was transfected into DH5α and the expression of λ -RED system was
induced by arabinose. PCR amplification with pKD13 as template was used to obtain 1.4 kb DNA
fragment. The 5'-end primer hisBCD-knockout-F included bases homologous to the upstream sequence of
hisB gene of host DH5α chromosome and priming sequence of pKD13 plasmid. The 3'-end primer
hisBCD-knockout-R includes bases homologous to hisD downstream of host DH5α chromosome and priming
sequence of pKD13 plasmid. The PCR product was electrically transferred into DH5α receptor state
containing pKD46 plasmid, and the recombinant was screened on kanamycin resistant LB plate with
glycine.[7]

![](https://static.igem.org/mediawiki/2021/7/7e/T--WHU-China--engi_19.png)

Figure 16. The template of hisBCD knock-out (1394bp) in DH5α

However, there was no colony growth on the resistance screening plate, indicating
that no homologous recombination reaction occurred in the bacteria. We tried again but failed again
with no colony growth on the resistance screening plate.

After analysis, it is speculated that probably the base sequence of the target gene
is too long, resulting in the decrease success rate of gene knockout experiment.

Therefore, we tried to look for single-gene knockout strains that had been
successfully conducted. And the Kerio collection appeared to be a good reference.[8][9] Based on
this list, we selected glyA as the target gene. Gene glyA encodes protein serine
hydroxymethyltransferase that converts serine to glycine, transferring a methyl group to
tetrahydrofolate, thus forming 5,10-methylene-tetrahydrofolate (5,10-mTHF).

Furthermore, another problem rised our attention. Considering the fact that DH5α is
genetically modified to amplify plasmids, probably it is not suitable for gene knockout. In addition
to using the DH5α strain as the chassis, we decided to use *Escherichia coli* MG1655 strain
simultaneously for gene knockout. Based on the chromosome gene sequences of the two strains, we
redesigned the gene knockout primers. Similar to the previous experiments, the primers included a
sequence of bases upstream or downstream of gene glyA and the priming sequence of the pKD13 plasmid.

![](https://static.igem.org/mediawiki/2021/9/93/T--WHU-China--engi_20.png)

Figure 17. The template of glyA knock-out (1395bp) in MG1655

We overcame many difficulties in the experimental process and repeated four rounds
of gene knockout experiments. Unfortunately, due to bacillus contamination in the laboratory, the
first round of knockdown experiment based on DH5α strain failed. Although there was colony growth on
kanamycin resistant plate, it was most likely not DH5α strain due to the appearance of the colony.
In the second round, DH5α was used as the chassis for knockout experiment, and no colony growth was
found on kanamycin resistant plate, which may be due to problems in experimental operation, such as
low sensitive state activity or high voltage during switching, etc. In the third round, MG1655 was
used as the chassis for knockout experiment, but also ended in failure. In the fourth round of
knockout experiment, we used DH5α as the chassis. Colony growth was found on kanamycin resistance
plate, though gene sequencing results indicate that bacteria still had glyA gene. Considering that
the colony is kanamycin-resistant, it is likely to have off-target effect in the knockout process.

![](https://static.igem.org/mediawiki/2021/b/b8/T--WHU-China--engi_21.png)

Figure 18. Colony growth on kanamycin resistant plate. (a) The first round of
knockdown experiment (b) The fourth round of knockdown experiment

Although off-target effect is very common in gene knockout, we still believe that
some methods can be used to minimize the off-target rate and increase the success rate. Due to the
lack of time, we did not carry out the next round of gene knockout attempt. But we planned to
redesign the primers for gene knockout to make them more specific based on the lessons of previous
experiments. Although there is a description of glycyrrhizin nutritional deficiency caused by glyA
gene defects in Kerio Collection, we still retain the suspicion that glyA gene is so important for
bacterial growth that it is difficult to knock out. In further validation, we still hope to try to
construct *E. coli* strains with different genetic defects.

---

## DBTL cycle of Hardware

Our ultimate goal is to obtain highly expressed promoters through directed evolution, so that our bacteria have high protein expression and further achieve the effect of acne treatment. While one of the biggest challenges in directed evolution is the need for high throughput. To solve this problem-raised from our design, an automated, high throughput microfluidic chip was developed for directed evolution.

Initially, I used a square array to try to capture cells and microbeads. （the structure is shown below in Figure 19）

![](https://static.igem.org/mediawiki/2021/6/63/T--WHU-China--engi_22.png)

Figure 19. The capture efficiency of square arrays
1. 80\*80 square array; 2. Bird head channel; 3. Air chamber; 4. Microbeads

We designed a simple experiment: Vacuum the chip and solvate each array with 5 ml of bead loading buffer and use a 200 microliter pipette to transfer 200 microliter solution containing 100000 beads, in a drop-wise fashion, to the surface of each array, and finally observe it with the microscope. (For more detailed experimental procedures, please refer to [Hardware](https://2021.igem.org/Team%3AWHU-China/Hardware) 4.4)

But we cannot achieve only one microbead per array (often two or more, show in Fig.1). Moreover, great difficulties occurred in the matching between two layers of chips. Thus we redesigned the chip with smaller circular channels, hoping for better results. (The structure is shown below in Figure 20)

![](https://static.igem.org/mediawiki/2021/8/87/T--WHU-China--engi_23.png)

Figure 20. The circular array

![](https://static.igem.org/mediawiki/2021/9/95/T--WHU-China--engi_24.png)

Figure 21. Matching between two layers of chips

The results show that the improved circular array can capture one microsphere in one array, which lays a solid foundation for the subsequent directional evolution step (More detailed results can be found at [Hardware](https://2021.igem.org/Team%3AWHU-China/Hardware)).

---

## Reference

[1] Faye T, Langsrud T, Nes IF, Holo H. Biochemical and genetic characterization of
propionicin T1, a new bacteriocin from Propionibacterium thoenii. Appl Environ Microbiol. 2000
Oct;66(10):4230-6. doi: 10.1128/AEM.66.10.4230-4236.2000. PMID: 11010864; PMCID:
PMC92290.
[2]Pechsrichuang P, Songsiriritthigul C, Haltrich D, Roytrakul S, Namvijtr P, Bonaparte N, Yamabhai M. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. Springerplus. 2016 Jul 28;5(1):1200. doi: 10.1186/s40064-016-2893-y. PMID: 27516938; PMCID: PMC4963352
[3]
Byrd AL, Belkaid Y, Segre JA. The human skin microbiome. Nat Rev Microbiol. 2018 Mar; 16(3):143-155.
doi: 10.1038/nrmicro.2017.157. Epub 2018 Jan 15. PMID: 29332945.
[4] Wang Y, Xue P, Cao M, Yu T,
Lane ST, Zhao H. Directed Evolution: Methodologies and Applications. Chem Rev. 2021 Jul 23. doi:
10.1021/acs.chemrev.1c00260. Epub ahead of print. PMID: 34297541.
[5] Liu, Daniel Yuen-Teh et al.
“Global gene expression profiling of wild type and lysC knockout *Escherichia coli* W3110.” FEMS
microbiology letters vol. 276,2 (2007): 202-6.
[6] 史新昌.
含真核表达质粒的营养缺陷型大肠杆菌工程菌株作为基因治疗口服递送载体系统的研究[D].第四军医大学,2014.
[7]王芃,袁盛凌,郑继平,李淑琴,段海清,张兆山.一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法[J].微生物学通报,2004(02):95-99.
[8]
Baba, Tomoya et al. “Construction of *Escherichia coli* K-12 in-frame, single-gene knockout mutants:
the Keio collection.” Molecular systems biology vol. 2 (2006): 2006.0008.
[9]
https://cgsc.biology.yale.edu/Auxotrophs.php

Contact Us

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# Team:WHU-China/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

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

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

# Team:WHU-China/Results

[![](https://static.igem.org/mediawiki/2021/8/87/T--WHU-China--logo.png)](https://2021.igem.org/Team%3AWHU-China)

* Project
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* Wet Lab
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* Application
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    another area](https://2021.igem.org/Team%3AWHU-China/Excellence_In_Another_Area)
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Wet Lab > Results

![](https://static.igem.org/mediawiki/2021/4/41/T--WHU-China--results_zi.png)
Results

[Inhibition Module](#title1)

[Fatty Acids Sensing Module](#title2)

[Fatty Acid Consumption Module](#title3)

[Safety module](#title4)

---

## Inhibition Module

### GOAL

1. To express and purify PctA, then test its ability to inhibit the growth of *P. acnes*.

2. To prove that engineered bacteria can secret PctA into the environment, and inhibit the growth of
*P. acnes*.

### ACHIEVEMENT

1. The expression vector of PctA was successfully constructed and transfromed into BL21(DE3) strain.

2. Expressed and purified the precursor PctA in BL21(DE3) strain.

3. Tested the antibiotic activity of precursor PctA against *P. acnes*.

#### Expression of PctA

In order to get enough protein products for functional test, we chose pET26b(+) as
vector and *E. coli* BL21(DE3) as host cell. We got *E. coli* DH5ɑ with pET26b(+) from Dr.
Chen’s lab and
synthesized PctA gene (with restriction site for NdeI at 5’ and XhoI at 3’) from Tsingke
Biotechnology company. Plasmid was extracted, and digested with NdeI and XhoI. Correct bands of
pET26b(+) vector (little more than 5000 bp) and PctA gene sequence (250-500bp, 289 bp in theory) were
observed.

![](https://static.igem.org/mediawiki/2021/6/6e/T--WHU-China--results_f1.png)

Figure 1. Enzyme digestion of plasmids we need (pET26b(+) and pUC67 loaded with PctA
gene). L1: undigested pET26b(+); L2,L3: pET26b(+) digested with NdeI and XhoI; L4: undigested
pUC67; L5,L6: pUC67 digested with NdeI and XhoI.

The pET26b(+) vector and PctA gene fragment were mixed and incubated with T4 DNA
ligase to form our target plasmid, and then transform DH5α competent cell. After colony PCR and
plasmid sequencing verification, the expression vectors were transformed into BL21(DE3) and
sequenced again to make sure PctA gene sequence was correct.

![](https://static.igem.org/mediawiki/2021/1/1d/T--WHU-China--results_f2.png)

Figure 2. Results of colony PCR. L1—L5: transformants 1—5; L6: pET26b(+) plasmid. The
PCR product of successful transformants is 200-300kb larger than the product of empty
pET26b(+) vector.

The expression of PctA was induced with IPTG in a set of different conditions and
checked with SDS-PAGE. Results showed that 11kDa precursor PctA is successfully induced in the
bacteria, but no band of 7kDa mature PctA is observed in the culture media, which meant that PctA
was not successfully secreted. As shown in Fig.3 (A) and (B), after induction, the pellet of
bacteria lysate contained most of the precursor PctA, while the lysate supernatant had a much
smaller portion. This indicates that a portion of the expressed precursor PctA was localized at the
inclusion body. The molecular weight of PctA seemed to be greater than we expected, this may result
from the fact that SDS-PAGE is not suitable for isolating protein under 15kDa, and the relationship
between molecular weight and small protein’s mobility can be unreliable.

| lane | volume/ml | IPTG/µl | c(IPTG)/mM | temperature/˚c | Time/h |
| --- | --- | --- | --- | --- | --- |
| 0 | 5 | 0 | 0 | 37.0 | 0 |
| 1 | 5 | 1.5 | 0.3 | 16.0 | 1.0 |
| 2 | 5 | 1.5 | 0.3 | 30.0 | 2.0 |
| 3 | 5 | 1.5 | 0.3 | 37.0 | 3.0 |
| 4 | 5 | 2.5 | 0.5 | 16.0 | 2.0 |
| 5 | 5 | 2.5 | 0.5 | 30.0 | 3.0 |
| 6 | 5 | 2.5 | 0.5 | 37.0 | 1.0 |
| 7 | 5 | 5.0 | 1.0 | 16.0 | 3.0 |
| 8 | 5 | 5.0 | 1.0 | 30.0 | 1.0 |
| 9 | 5 | 5.0 | 1.0 | 37.0 | 2.0 |

![](https://static.igem.org/mediawiki/2021/d/d8/T--WHU-China--results_f3.png)

Figure 3. SDS-PAGE of BL21 with expression vector induced in different conditions,
lysate supernatant (A) and precipitation (B). 0: uninduced BL21(DE3); 1-9: induced group,
lane 1-9. Arrow indicates the band of precursor PctA.

#### Secretion of PctA

We tested the secretion of PctA by Western Blot for this method is highly sensitive
and thus fit for identifying protein from culture supernatant even at a low concentration. The
result is as follows.

![](https://static.igem.org/mediawiki/2021/b/be/T--WHU-China--results_f4.png)

Figure 4. The result of Western Blot. Lane 1-3: lysate pellet induced for 0, 2, 4h;
4-6: lysate supernatant induced for 0, 2, 4h; 7-9: culture medium concentrated by salt out
for 50 times, induced for 0, 2, 4h. 10:purified precursor PctA, serving as positive
contrast.

As the result shows, no band of 7kDa mature PctA was found, indicating that the
precursor PctA failed to cut off the Sec signal peptide to form the mature PctA, and consequently was
unable to get into the culture medium. This may contribute to the formation of inclusive body in
lysate pellet. We discussed about the problem of secretion at [Engineering](https://2021.igem.org/Team%3AWHU-China/Engineering).

#### The function of PctA

After a few attempts of optimizing conditions for PctA induction, we still couldn’t
observe the band of 7kDa mature PctA, but we did find that at 30℃, 0.5mM IPTG concentration, 4h we
could get most precursor PctA in the supernatant of cell lysate. So we decided to first purify the
soluble precursor PctA and test its function.

We used Ni-resin to purify the precursor PctA with His tag from lysate supernatant.
In order to get a better resolution at low molecular weight range, the product was analyzed by
Tris-Tricine SDS PAGE instead of Tris-glycine SDS PAGE. We tried protocols both according to the
instruction of the resin and a method of imidazole gradient purification, but neither method had a
satisfying result: the former one left the product impure and the latter one had too little protein
of interest in the product.

![](https://static.igem.org/mediawiki/2021/0/0d/T--WHU-China--results_f5.png)

Figure 5. The result of Ni-resin purification. Proteins were detected by Coomassie
Brilliant Blue Staining. 1-11 : Imidazole gradient elution, with concentration from 10mM,
20mM, 40mM, 60mM, 80mM, 100mM, 150mM, 200mM, 250mM, 300mM to 500mM; 12-14: purified protein
elution washed out from Ni resin by elution buffer (imidazole concentration of 250mM), the
first, second and third 1ml respectively for each group. Arrow indicates the band of
purified precursor PctA.

Then we used the resin-purified product to test the activity of precursor PctA (the
later product is too thin to carry out experiment). We mixed the product with the culture of *P.
acnes* and measured the antibiotic activity by the change of OD 600. We also conducted the
inhibition
zone experiment. The result was as follows.

![](https://static.igem.org/mediawiki/2021/b/b2/T--WHU-China--results_f6.png)

Figure 6. the result of antibiotic test of precursor PctA, measured by A600. We set
two groups of precursor PctA as parallel control. Blue: water(negative contrast 1); Orange:
elution buffer(negative contrast 2); Gray: 15μg/ml Chloromycetin solution (positive
contrast); Yellow & Bright blue: precursor PctA solution ; Green: precursor PctA solution
diluted for 2 times; Dark Blue: precursor PctA solution diluted for 10 times. It seemed that
*P. acnes* was relatively resistant to antibiocs. We had tried both 50μg/ml Kanamycin and
15μg/ml Chloromycetin, but none of them was especially effective.

![](https://static.igem.org/mediawiki/2021/0/03/T--WHU-China--results_f7.png)

Figure 7. the result of inhibition zone experiment. Papers soaked with different
solutions were tested for *P. acnes* growth inhibition. area A: 10% purified precursor PctA
solution; area B: 50mg/L Kan; area C : elution buffer; area D: water

As the results showed, the OD 600 of *P. acnes* culture didn’t have a distinct
decline
after the adding precursor PctA, and in another experiment no inhibition zone is observed around the
filter with precursor PctA. So we can conclude that precursor PctA doesn’t have antibiotic activity
against *P. acnes*.

In future experiments, we will try to express and purify mature PctA and test its
activity.

---

## Fatty Acids Sensing Module

### Goal

1. Design a system that enables the engineered bacteria to sense fatty acids, and measure this
ability.

2. Improve the sensitivity towards fatty acids and reduce gene expression leakage of our engineered
bacteria, mainly through the following two ways:

A) Retrofit ribosomal binding site.

B) Introduce FadR, a repressor, and carry out directed evolution on it.

### Achievements

1. *Escherichia coli* containing a fatty acid-sensitive promoter was successfully constructed
(the
strain needs to be further identified) and its ability to sense fatty acids was measured.

2. The RBS was successfully modified to improve the sensitivity to fatty acids of our engineered
bacteria.

3. FadR was successfully cloned in *E. coli* MG1655 and a mutation library was constructed by
error-prone PCR.

We synthesized BBa\_K3763002(pFadD\_Lac-RBS BBa\_B0030-GFP-rrnB T1 and T2 terminator)
and BBa\_K3763003(pFadD\_Lac-RBS BBa\_K3763000-GFP-rrnB T1 and T2 terminator) from Tsingke
Biotechnology company. After the successful transformation of the plasmid into Top10 *E. coli*
cells,
we used different concentrations of fatty acids for eGFP induction for different times. The
expression level of eGFP was determined by fluorescence detection with a microplate reader,
reflecting the performance of the promoter and the RBS.

![](https://static.igem.org/mediawiki/2021/9/90/T--WHU-China--results_f8.png)

Figure 8. BBa\_K3763002(pFadD\_Lac-RBS BBa\_B0030-GFP-rrnB T1 and T2 terminator,
pDSW208-99-2) contains RBS without modification

![](https://static.igem.org/mediawiki/2021/6/68/T--WHU-China--results_f9.png)

Figure 9. BBa\_K3763003(pFadD\_Lac-RBS BBa\_K3763000-GFP-rrnB T1 and T2 terminator,
pDSW208-99-2) Contains RBS with modification

According to the results, we can find that the performance of our fatty
acids-sensitive promoter is improved to a certain extent. Taking 0.125× fatty acid concentration as
an example, the fluorescence intensity of BBa\_K3763003 is over 11000 after 6 hours, while the
promoter of BBa\_K3763002 is less than 10000 at three fatty acid concentrations. However, we can see
that the optimized promoter still has a high expression leakage and low expression in a short time.
However, the experimental results have been able to prove the function of the fatty acids-sensitive
promoter, and partially prove the effectiveness and rationality of our design.

We cloned genomic DNA FadR from *E. coli* MG1655 and digested with HindIII and
XbaI.
Plasmid (pBAD33) was extracted and digested with HindIII and XbaI as well. Correct bands of pBAD33
vector (around 5000 bp) and FadR gene sequence (1038bp) were observed.

![](https://static.igem.org/mediawiki/2021/5/5d/T--WHU-China--results_f10.png)

Figure 10. Enzyme digestion of plasmids and gene we need (pBAD33 and FadR gene). L1,
L2: FadR gene digested with HindIII and XbaI; L4, L5: pBAD33 digested with HindIII and XbaI

At the same time, we obtained a mutation library of FadR (around 750bp) by
error-prone PCR.

![](https://static.igem.org/mediawiki/2021/4/43/T--WHU-China--results_f11.png)

Figure 11. Error-prone PCR (FadR gene). L1: cloned genomic DNA FadR from *E.
coli*
MG1655 by error-prone PCR

The gene sequencing results indicated that we successfully get FadR gene and
introduce random nucleotide mutations into the parent sequence qualitatively because there are
overlapping peaks.

![](https://static.igem.org/mediawiki/2021/f/fb/T--WHU-China--results_f12.png)

Figure 12. Partial FadR sequencing results: a)results of PCR b) results of
error-prone PCR

Our future work will include overexpressing FadR before and after directed evolution
in our chassis to reduce the leakage expression of our promotor. Furthermore, we will use the
specially designed hardware to perform directed evolution. This will include using the microfluidic
hardware to carry out epPCR, setting up cell free expression system to measure the characters of
different promotors, and select them using flow cytometry.

---

## Fatty Acid Consumption Module

### Goal：

1. To overexpress 2 enzymes in the β-oxidation pathway—fatty acyl-CoA synthetase (FadD) and fatty
acyl-CoA dehydrogenase (FadE) —in our engineered bacteria.

2. To equip our engineered bacteria with higher fatty acid decomposition ability by overexpress FadD
and FadE.

### Achievement：

1. Ni2+ affinity chromatography column and SDS-PAGE proved that we successfully express
the target
protein with the right molecular weight.

2. The β-oxidation ability of our engineered bacteria was improved.

### Experiment：

In order to test whether the overexpression of fatty acyl-CoA synthetase (FadD) and
fatty acyl-CoA dehydrogenase (FadE) has a significant effect on improving the fatty acid
decomposition ability of our engineered bacteria, we designed the expression vectors of FadD and
FadE (Fig1 A,B). FadD is driven by trac promoter, whereas FadE is driven by araBAD promoter. Then we
constructed the expression vectors by means of enzyme digestion and ligation and transformed them
into *E. coli* DH5α strains (Figure 1).

![](https://static.igem.org/mediawiki/2021/b/b0/T--WHU-China--results_f13.png)

Figure 13. Construction of overexpression vectors of FadD and FadE. (A-B) Design of
overexpression vectors of FadD gene and FadE gene. (C) FadD and FadE gene PCR products in
agarose gel electrophoresis.（lane 1-2 FadD PCR product) (D) PCR products and digested
vectors in agarose gel electrophoresis. (lane 1-2 FadE gene digestion products; lane3 FadE
Gene ; lane4-5 FadD gene digestion products ;lane 6 FadD gene ; lane 7-8.pBAD33 plasmid
digestion products ;lane 9. pBAD33 plasmid; lane 10-11.pDSW208 plasmid digestion productas;
lane 12.pDSW208 plasmid)

After verifying our vector construction by colony PCR and sequencing, we carried out
protein expression test. Firstly, we explored the optimal culture temperature, culture time and
inducer concentration in the process of FadD and FadE protein expression by orthogonal test. After
confirming the optimal expression conditions, we conducted the experiment following the relevant
experimental methods of 2019 BNU China—the control group and the experimental group were cultured
overnight in LB medium at 37 ℃, and then mixed with the same volume of LB medium containing 400mM
fatty acid sodium to make the final concentration of fatty acid sodium 200mM, and then induced them
by 1 mM IPTG (FadD) and 0.2% ara respectively (FadE). Samples were taken at 0 h, 2 h and 4 h. The
free fatty acid (FFA) content detection kit was used to measure the fatty acid consumption (Fig. 2).
The experimental results are shown in the figure below. T-test analysis showed that there was no
significant difference in the β-oxidation ability between our engineered bacteria and the original
bacteria.

![](https://static.igem.org/mediawiki/2021/e/ea/T--WHU-China--results_f14.png)

Figure 14. Comparison between the fatty acid consumption abilities of bacteria in
experimental group and control group. T-test analysis showed that there was no significant
difference in the β-oxidtion ability between our engineered bacteria and the original
bacteria.

In order to explore the reasons for the failure of the experiment, we verified he
protein expression more carefully. After inducing the expression of FadD and FadE proteins, they
were purified by Ni2+ affinity chromatography column. After purification, SDS-PAGE
results showed
that the molecular weight of our target band was about 20 kDa lower than our predicted value. It is
speculated that in the engineered strain we used is *E. coli* DH5α, the endogenous protease
system of
this strain has not been artificially knocked out, so the overexpressed protein is easy to be
degraded. Considering this possibility, we replaced our engineered strain with *E. coli* BL21
strain.
Then we purified the protein by Ni2+ affinity chromatography column and detected it by
SDS-PAGE. The
results showed that the bands of FadD and FadE protein were in line with our prediction.

![](https://static.igem.org/mediawiki/2021/8/8a/T--WHU-China--contribution_f4.png)

Figure 15. The result of Ni-resin purification. (A) Detection of protein expression
in *E. coli* DH5α strain. Lane 1-4. Ni-resin purification result of FadD protein. Lane
4. The
purpose bond was about 20 kDa lower than our predicted value. Lane 5-8 Ni-resin purification
result of FadE protein. Lane 8. The purpose bond was about 20 kDa lower than our predicted
value. (B) Detection of FadD protein expression in *E. coli* BL21strain . Lane 1-4.
Ni-resin
purification result of FadD protein. Lane 3 The FadD protein bond was is basically the same
as we expected. (C) Detection of FadE protein expression in *E. coli* BL21 strain. Lane
1-4.
Ni-resin purification result of FadD protein. Lane 4 The FadE protein bond was is basically
the same as we expected.

While conducting our own experiments to explore the optimal protein expression
condition, we sought the help of Ailurus biotechnology and asked them to help us design the best
expression vector of FadD protein. Their scalable pipeline enables an ultra-high throughput assay of
soluble protein yields under different vectors, with much lower costs than typical lab works.

The construction results of the best FadD protein expression vector are as follows：

![](https://static.igem.org/mediawiki/2021/d/d5/T--WHU-China--res_24.png)

---

## Safety module

### Goal

1. To obtain a glycine auxotroph.

2. To test the growth rate of auxotroph on additional nutrients and basic media.

### Experiment：

We conducted two rounds of gene knockout experiments on hisBCD and four rounds of
gene knockout experiments on glyA. Unfortunately, until the end of the experiment process, we have
not been able to obtain a successful knockout of nutritionally deficient strain by λ -Red homologous
recombination method.

There was no colony growth on the resistance screening plate in the two rounds of
hisBCD knockout, indicating that no homologous recombination reaction occurred in the bacteria. So
we decided to conduct a single gene knockout. In addition, considering that DH5α has undergone large
genetic modifications, MG1655 strain was used as the chassis for gene knockout at the same time in
order to avoid the impact of this characteristic on gene knockout.

We repeated four rounds of gene knockout experiments targeting at glyA. Colony
growth was found on kanamycin resistance plate in the first and fourth round of experiment with DH5α
as the chassis. Primers were designed for PCR amplification of the base sequence near the
pre-knockout glyA gene. PCR products were detected by agarose gel electrophoresis to estimate the
band size and sequenced to determine whether they contained glyA gene.

The results of first round experiment are as below:

![](https://static.igem.org/mediawiki/2021/d/d8/T--WHU-China--results_f16.png)

Figure 16. Gene hisBCD knockout results. A) colony growth on kanamycin resistant
plate. B) detect the molecular weight of PCR products. 1: PCR product with clone as
template; 2: PCR product with clone as template; 3: PCR product with DH5α as template. C)
gene sequencing alignment results

The results of fourth round experiment are as below:

![](https://static.igem.org/mediawiki/2021/f/f0/T--WHU-China--results_f17.png)

Figure 17. Gene glyA knockout results. A) colony growth on kanamycin resistant
plate. B) detect the molecular weight of PCR products. 1: PCR product with clone as
template; 2: PCR product with DH5α as template. C) gene sequencing alignment results. D)
gene sequencing result with glyA

Unfortunately, gene sequencing results indicated that we failed to knock glyA out.
Considering that the colony is kanamycin-resistant, it is likely to have off-target effect in the
knockout process.

Due to the lack of time, we did not carry out the next round of gene knockout
attempt. However, we still believed that off-target effects can be avoided by a suitable primer
design. In addition, further research would manage to construct different genetic defects supposing
that glyA is important for bacterial growth.

Contact Us

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Acknowledgement

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