iGEM 2019 — Team NCKU_Tainan

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NCKU Tainan/Notebook

[![Logo](https://static.igem.org/mediawiki/2019/thumb/d/da/T--NCKU_Tainan--Logo.png/800px-T--NCKU_Tainan--Logo.png)
![logo-mobile](https://static.igem.org/mediawiki/2019/thumb/0/08/T--NCKU_Tainan--Logo_mobile.png/320px-T--NCKU_Tainan--Logo_mobile.png)](https://2019.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2019.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2019.igem.org/Team%3ANCKU_Tainan/Design)
  [Measurement](https://2019.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2019.igem.org/Team%3ANCKU_Tainan/Model)
  [Hardware](https://2019.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Entrepreneurship](https://2019.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Safety](https://2019.igem.org/Team%3ANCKU_Tainan/Safety)
* Results

  [Results](https://2019.igem.org/Team%3ANCKU_Tainan/Results)
  [Demonstrate](https://2019.igem.org/Team%3ANCKU_Tainan/Demonstrate)
  [Future](https://2019.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Human Practice](https://2019.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3ANCKU_Tainan/Public_Engagement)
  [Collaboration](https://2019.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [OMG Game Room](https://2019.igem.org/Team%3ANCKU_Tainan/Software)
* Parts

  [Parts](https://2019.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2019.igem.org/Team%3ANCKU_Tainan/Improve)
* Team

  [Team](https://2019.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2019.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2019.igem.org/Team%3ANCKU_Tainan/Notebook)
  [Protocols](https://2019.igem.org/Team%3ANCKU_Tainan/Protocols)
* Awards

  [Awards](https://2019.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2019.igem.org/Team%3ANCKU_Tainan/Criteria)

>

Collab

# Notebook

![...](https://static.igem.org/mediawiki/2019/e/ed/T--NCKU_Tainan--Notebook_Carousel_2.jpg)

![...](https://static.igem.org/mediawiki/2019/1/1b/T--NCKU_Tainan--Notebook_Carousel_1.jpeg)

![...](https://static.igem.org/mediawiki/2019/3/31/T--NCKU_Tainan--Notebook_Carousel_3.jpg)

![...](https://static.igem.org/mediawiki/2019/6/64/T--NCKU_Tainan--Notebook_Carousel_4.jpg)

[Previous](#carousel-team)
[Next](#carousel-team)

### Check this page out to see what we did! Enjoy!

### Week 1 (6/2 - 6/8)

## Wet

---

1. Fragment preparation: cmR from pkD3 for *can* gene knockout and *dap* gene knockout.
2. Learning to prepare high CFU heat shock competent cell.
3. Basic lab skills.
4. PCR to get *tyrP*, *yebF*, CBMB fragment.

## Dry

---

1. Design and fabricate the first version of microfluidic chip.
2. Design Wiki page structure.
3. Modeling research and brainstorming about possible modeling topic.

## HP

---

1. Write news script for NCKU News Agency.
2. Review Tainan First Senior High School’s feedback.

### Week 2 (6/9 - 6/15)

## Wet

---

1. First attempt *can* gene knockout and *dap* gene knockout by electroporation in *E. coli* MG1655 and *E. coli* Nissle.
2. First attempt to ligate each PCR product into pSB1C3.

## HP

---

1. Contact Genemont and Geneferm companies to entreprise visit.

### Week 3 (6/16 - 6/24)

## Wet

---

1. *E. coli* MG1655 Δ*can*::cmR, *E. coli* Nissle Δ*can*::cmR, *E. coli* MG1655 Δ*dap*::cmR, *E. coli* Nissle Δ*dap*::cmR genotype and phenotype check.
2. Vector: pSB3K3 transformation and extraction.
3. Colony PCR to check colony of bacteriocin constructs and *tyrP* construct.
4. Use commercial cloning kit to subclone each PCR product into pCR-Blunt II-TOPO and pJET1.2 cloning vector.

## HP

---

1. Contact tFDA, NHI.
2. Design and choose our souvenirs.

### Week 4 (6/23 - 6/29)

## Wet

---

1. Kill-switch comparison.
2. Remove pkD46 from *E. coli* MG1655 Δ*can*::cmR and *E. coli* Nissle Δ*can*::cmR.
3. Double digest vector pSB3K3 and Topo-*tyrP* and further ligation.
4. Use pJET1.2 to subclone *yebF*-CBMB.

## Dry

---

1. Design the second version of microfluidic chip.
2. Test and calibrate the detector.

## HP

---

1. Contact with Taiwan Chronic Kidney Disease foundation to join their event on August.
2. iGEM Collaboration.

### Week 5 (6/30 - 7/6)

## Wet

---

1. IDT fragment preparation and first PCR.
2. First attempt to construct *E. coli* Nissle Δ*can*::FRT.
3. Sequencing Topo-*tyrP*, to confirm our *tyrP* is correct.
4. Ligation and Transformation for pSB3K3-*tyrP* into *E. coli* DH5α.
5. Ligation and Transformation for pSB3K3-*yebF*-CBMBs into *E. coli* DH5α.

## Dry

---

1. Design Wiki layout.
2. Design spinner and control rotation speed through Arduino.
3. Brainstorming IoT implementations for hardware.
4. Bacteriocin model experiment

## HP

---

1. Oh My Gut logo design.
2. Video for wiki.
3. Genmont Enterprise Visit

### Week 6 (7/7 - 7/13)

## Wet

---

1. Vector: pSB1C3 transformation and extraction.
2. Second and third overhang extension PCR, double digestion and ligation for pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, pSB1C3-*pchR*-native RBS-GFP construct.
3. Extraction for pSB3K3-*tyrP*, and check by Colony PCR and Double Digestion
4. Transformation for pFNR-GFP for part characterized.
5. SDS-PAGE for pSB3K3-*tyrP* protein expression.
6. Ligation and Transformation for pSB3K3-*yebF*-CBMBs into *E. coli* DH5α.

## Dry

---

1. Identify cause of spinner vibration.
2. *p*-cresol sensing model experiment.

## HP

---

1. Radio interview confirmation.
2. Discuss events (escape room or workshop).

### Week 7 (7/14 - 7/20)

## Wet

---

1. Transform pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, pSB1C3-*pchR*-native RBS-GFP to *E. coli* top10, checked by colony PCR.
2. pFNR-GFP streak on plate, and put them in 37 degree incubator and anaerobic incubator.
3. SDS-PAGE for pSB3K3-*tyrP* to check protein expression.
4. Transformation of pSB3K3-*tyrP* into *E. coli* Nissle and *E. coli* BL21.
5. Amplify each IDT synthesis fragment.
6. Double digest pSB3K3-*yebF*-CBMBs to confirm successful of cloning.

## Dry

---

1. Microfluidic chip test failed. Redesign microfluidic chip and find other ways to separate blood.
2. IoT connection ongoing.
3. Snake game programming.
4. Seeking possibility of CKD microbiome modelling (meeting with Prof. Liu and asking for NGS data).

## HP

---

1. Arrange HP timelines.

### Week 8 (7/21 - 7/27)

## Wet

---

1. *E. coli* top10 pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, and pSB1C3-*pchR*-native RBS-GFP plasmid extraction and structure checking by digestion.
2. First attempt SDS-PAGE and western blot for *E. coli* top10 pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, and pSB1C3-*pchR*-native RBS-GFP.
3. SDS-PAGE for *E. coli* Nissle, DH5α, BL21 pSB3K3-*tyrP*.
4. PCR to add his-tag on pSB3K3-*tyrP* for Western Blot.
5. First attempt Western Blot for pSB3K3-*tyrP* in *E. coli* BL21 and Nissle.
6. Sequencing for pSB3K3-*yebF*-GS-CBMBs and pSB3K3-*yebF*-TB-CBMBs.
7. Run SDS-PAGE for bacteriocin constructs.

## Dry

---

1. Team page coding.
2. Tweaking sensing model.
3. Arduino code debug.

## HP

---

1. Mentor iGEM Korea\_HS.

### Week 9 (7/28 - 8/3)

## Wet

---

1. Transform pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL to *E. coli* Nissle, checked by colony PCR and digestion.
2. First functional test for pSB1C3-*pchR*-native RBS-GFP.
3. SDS-PAGE for pFNR-GFP in *E. coli* Nissle, samples include different time (every 8hr).
4. Transformation of pSB3K3-*tyrP* and TAL with B0034,TAL with NRBS in to same *E. coli* Nissle.
5. PCR to check if transformation of pSB3K3-*tyrP* and TAL with B0034 success or not.
6. Run SDS-PAGE and Western blot for bacteriocin constructs.

## Dry

---

1. sfGFP *E. coli* test for detector.
2. Protocols & Main page in progress.
3. Meeting with Prof. Wu.
4. Populating Wiki

## HP

---

1. National Education Radio Kaohsiung interview.

### Week 10 (8/4 - 8/10)

## Wet

---

1. Second functional test for pSB1C3-*pchR*-native RBS-GFP.
2. SDS-PAGE and western blot for *E. coli* top10 and *E. coli* Nissle pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, pSB1C3-*pchR*-GFP.
3. SDS-PAGE for pFNR-GFP in *E. coli* Nissle, samples include different time (4hr, 8hr, 12hr).

## Dry

---

1. Start acrylic disk fabrication.
2. Research about promoter activity.

## HP

---

1. iGEM Taiwan Conference.

### Week 11 ( 8/11 - 8/17)

## Wet

---

1. Second overhang extension PCR for FNR-B0034-TAL due to mutation and third overhang extension PCR for K880005-*yebF*-CBMB, K880005-*yebF*-GS-CBMBs, and K880005-*yebF*-TB-CBMBs.
2. Vector: pSB1C3 extraction.
3. Double digest pSB1C3, B0034-TAL, K880005-*yebF*-CBMB, K880005-*yebF*-GS-CBMBs, and K880005-*yebF*-TB-CBMBs. Also, ligation.
4. First TAL and *tyrP* functional assay.
5. SDS-PAGE for pSB3K3-*tyrP*.
6. Western Blot for TAL with NRBS/B0034.
7. Prepare M9 Medium for Second TAL and *tyrP* assay.

## Dry

---

1. IPTG & Arabinose test.
2. Model *p*-cresol Growth.

## HP

---

1. Go to Yunlin for volunteer with Taiwan Chronic Kidney Disease Foundation.
2. Geneferm entreprise visit.
3. NAR(National Applied Research) Lab Visit.
4. iGEM collaboration with iGEM UNSW.

### Week 12 (8/18 - 8/24)

## Wet

---

1. Transform pSB1C3- FNR-B0034-TAL, pSB1C3-K880005-*yebF*-CBMB, pSB1C3-K880005-*yebF*-GS-CBMBs, pSB1C3-K880005-*yebF*-TB-CBMBs to *E. coli* Top10. Checked by colony PCR and double digest. The right size plasmids were sent for sequencing.
2. Mutation found in B0034-TAL original plasmid IDT sent.
3. Construction of pSB1C3-*pchR*-B0034-GFP and transformed to *E. coli* DH5α.
4. Construction of pSB4A3-*tyrP* and transformed to *E. coli* Nissle.
5. pFNR-GFP fluorescent intensity test.

## Dry

---

1. Experiment of *p*-cresol growth rate.

## HP

---

1. Escape room preparation.
2. CKD Survey.
3. Education visit to Indonesia Kemurnian II High School.

### Week 13 (8/25 - 8/31)

## Wet

---

1. Spot on Lawn assay for pSB1C3-K880005-*yebF*-GS-CBMBs.
2. Mutation found on pSB1C3-K880005-*yebF*-GS-CBMBs, and pSB1C3-K880005-*yebF*-TB-CBMBs.
3. Use M9 Medium for Second TAL and *tyrP* assay.

## Dry

---

1. Proceed acrylic disk fabrication.

![blood](https://static.igem.org/mediawiki/2019/thumb/6/6b/T--NCKU_Tainan--Notebook_blood_separation.jpeg/800px-T--NCKU_Tainan--Notebook_blood_separation.jpeg)

## HP

---

1. Write news script for NCKU News Agency.
2. Review Tainan First Senior High School’s feedback.

### Week 14 (9/1 - 9/7)

## Wet

---

1. Construction of pBBR1 MCS4-*pchR*-native RBS-GFP and transformation to *E. coli* top10.
2. Construction of pSB1C3-J23100-NRBS-TAL and pSB1C3-J23100-B0034-TAL to remove Fnr promoter.

## Dry

---

1. Acrylic disk fabrication and test its functionality.
2. Test dectector’s sensitivity.
3. Ver3. PDMS chip fabrication.
4. Model co-culture of *p*-cresol and *clostridium*

## HP

---

1. Participate in NCKU Undergraduate Research.
2. Visit Intellectual Property Office.
3. Escape room preparation

### Week 15 (9/8 - 9/14)

## Wet

---

1. Construction of pBBR1 MCS4-*pchR*-native RBS-GFP and transformation into *E. coli* top10.
2. Second attempt to construct *E. coli* Nissle Δ*can*::FRT.
3. Construction of pSB1C3-J23100-NRBS-TAL, pSB1C3-J23100-B0034-TAL and transformation into *E. coli* Nissle.

## Dry

---

1. Design and manufacture hardware base.
2. Wiki content submission.
3. Visit Senior of Business Administration to have a business plan and business model review.

## HP

---

1. Synbiotech entreprise visit.

### Week 16 (9/15 - 9/21)

## Wet

---

1. Construction of pBBR1 MCS4-*pchR*-native RBS-GFP and transformation into *E. coli* top10.
2. Preparation of Pseudomonas fluorescens for transformation.
3. Test phenotype of *E. coli* Nissle Δ*can*::FRT by streaking on plates.
4. Construction of pSB1C3-J23100-NRBS-TAL and transformation into *E. coli* Nissle.
5. Digestion to check constructs of pSB1C3-J23100-NRBS-TAL and pSB1C3-J23100-B0034-TAL.
6. SDS-PAGE and Western Blot to confirm TAL and *tyrP* protein expression.

## Dry

---

1. HP wiki page design.
2. Casing of device design.
3. Working on DFBAlab.

## HP

---

1. Visit NCKU Technology Transfer & Business Incubation Center.
2. Visit Professor Jimmy from Business Administration to discuss about entrepreneurship.

### Week 17 (9/22 - 9/28)

## Wet

---

1. Construction of pBBR1 MCS4-*pchR*-native RBS-GFP is completed and are successful.
2. Preparation of Pseudomonas fluorescens for transformation.
3. Transformation of pBBR1 MCS-4 plasmid into P.fluorescens.
4. Test phenotype of *E. coli* Nissle Δ*can*::FRT by streaking on plates.
5. First functional test for new TAL and *tyrP* construct in LB Medium.
6. Second functional test for new TAL and *tyrP* construct in LB Medium with different tyrosine concentration.

## Dry

---

1. Modeling *E. coli* Nissle pathway.
2. Public and Engagement wiki page design.
3. Finish device enclosure.

## HP

---

1. Escape room.
2. Review business plan.

### Week 18 (9/29 - 10/5)

## Wet

---

1. Test phenotype of *E. coli* Nissle Δ*can*::FRT by streaking on plates.
2. Preparation of Pseudomonas aeruginosa for transformation.
3. Test antibiotic resistance of Pseudomonas fluorescens 55.

## Dry

---

1. Finish all wiki page design.
2. Finish SolidWorks illustration.
3. Finish *E. coli* Nissle & *Clostridium difficile* metabolic network.
4. Filming demonstration video.

## HP

---

1. Collaboration with Washington iGEM.
2. Visit Winston Medical Corporation.
3. Finalized business plan.

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[![](https://static.igem.org/mediawiki/2019/thumb/f/f1/T--NCKU_Tainan--Diamodtext_icon.png/499px-T--NCKU_Tainan--Diamodtext_icon.png)](http://www.diamondcapital.com.tw/)

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e-mail : igem.ncku.tainan@gmail.com

phone : 06-2757575


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NCKU Tainan/Experiments

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[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3ANCKU_Tainan)
[HOME](https://2019.igem.org/Team%3ANCKU_Tainan)

TEAM

[Team Members](https://2019.igem.org/Team%3ANCKU_Tainan/Team)
[Collaborations](https://2019.igem.org/Team%3ANCKU_Tainan/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ANCKU_Tainan/Description)
[Design](https://2019.igem.org/Team%3ANCKU_Tainan/Design)
[Experiments](https://2019.igem.org/Team%3ANCKU_Tainan/Experiments)
[Notebook](https://2019.igem.org/Team%3ANCKU_Tainan/Notebook)
[Contribution](https://2019.igem.org/Team%3ANCKU_Tainan/Contribution)
[Results](https://2019.igem.org/Team%3ANCKU_Tainan/Results)
[Demonstrate](https://2019.igem.org/Team%3ANCKU_Tainan/Demonstrate)
[Improve](https://2019.igem.org/Team%3ANCKU_Tainan/Improve)
[Attributions](https://2019.igem.org/Team%3ANCKU_Tainan/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ANCKU_Tainan/Parts)
[Basic Parts](https://2019.igem.org/Team%3ANCKU_Tainan/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ANCKU_Tainan/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ANCKU_Tainan/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ANCKU_Tainan/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ANCKU_Tainan/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ANCKU_Tainan/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ANCKU_Tainan/Hardware)
[Measurement](https://2019.igem.org/Team%3ANCKU_Tainan/Measurement)
[Model](https://2019.igem.org/Team%3ANCKU_Tainan/Model)
[Plant](https://2019.igem.org/Team%3ANCKU_Tainan/Plant)
[Software](https://2019.igem.org/Team%3ANCKU_Tainan/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=NCKU_Tainan)

# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NCKU Tainan/Results

[![Logo](https://static.igem.org/mediawiki/2019/thumb/d/da/T--NCKU_Tainan--Logo.png/800px-T--NCKU_Tainan--Logo.png)
![logo-mobile](https://static.igem.org/mediawiki/2019/thumb/0/08/T--NCKU_Tainan--Logo_mobile.png/320px-T--NCKU_Tainan--Logo_mobile.png)](https://2019.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2019.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2019.igem.org/Team%3ANCKU_Tainan/Design)
  [Measurement](https://2019.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2019.igem.org/Team%3ANCKU_Tainan/Model)
  [Hardware](https://2019.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Entrepreneurship](https://2019.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Safety](https://2019.igem.org/Team%3ANCKU_Tainan/Safety)
* Results

  [Results](https://2019.igem.org/Team%3ANCKU_Tainan/Results)
  [Demonstrate](https://2019.igem.org/Team%3ANCKU_Tainan/Demonstrate)
  [Future](https://2019.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Human Practice](https://2019.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3ANCKU_Tainan/Public_Engagement)
  [Collaboration](https://2019.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [OMG Game Room](https://2019.igem.org/Team%3ANCKU_Tainan/Software)
* Parts

  [Parts](https://2019.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2019.igem.org/Team%3ANCKU_Tainan/Improve)
* Team

  [Team](https://2019.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2019.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2019.igem.org/Team%3ANCKU_Tainan/Notebook)
  [Protocols](https://2019.igem.org/Team%3ANCKU_Tainan/Protocols)
* Awards

  [Awards](https://2019.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2019.igem.org/Team%3ANCKU_Tainan/Criteria)

>

* [*p*-Cresol Reducing](#Subtitle1)
* [*p*-Cresol Sensing](#Subtitle2)
* [Biosafety](#Subtitle3)
* [Characterization](#Subtitle4)

# Results

## Overview

1. Construct each part and test the function of TAL, *tyrP*, **pchR** and bacteriocin.
2. Using spot-on-lawn assay to test the bacteriocin bactericidal activity.
3. Use the previously developed method ([see Protocols](https://2019.igem.org/Team%3ANCKU_Tainan/Protocols)) to measure the *p*-Coumaric acid production of TAL.
4. Knockout *dapA* and *can* gene and phenotype check.
5. Characterize the anaerobic sensing promoter *Pfnr* ([BBa\_K1123000](http://parts.igem.org/Part%3ABBa_K1123000)) and improve TAL with native RBS Biobrick ([BBa\_I742148](http://parts.igem.org/Part%3ABBa_I742148))

---

## *p*-Cresol Reducing

### Bacteriocin

**Achievements:**

1. Successfully proven CBM-B bacteriocin is able to inhibit *Clostridium* growth

In the human gut microbiome, *Clostridium* related species have been reported to have the highest conversion capability of fermenting tyrosine into *p*-Cresol[[1](#ref1)]. To target the root of *p*-Cresol accumulation, reducing the population of *Clostridium* is needed. We decide to use *C. difficile* as a model of *p*-Cresol producing bacteria because it is a popular research target due to its notorious infectious ability.

Luckily, iGEM NCKU Tainan 2019 was kindly supported by one of our PIs, Professor Huang, an assistant professor from the Microbiology and Immunology Department, who is currently devoting himself to the field of developing a novel therapeutic approach for *C. difficile* infection. He kindly provided us with a plasmid containing a bacteriocin gene (CBM-B), that was proven in his lab to have bactericidal activity against certain strains of *Clostridium*, including *C. difficile*.

As proof of the concept that bacteriocin is able to inhibit *Clostridium* growth, we did a spot-on-lawn assay using purified bacteriocin protein provided by advisors to observe the inhibition zone formation. As shown in Fig. 1 below, a clear inhibition zone formed in the middle of the BHI plate streak with *C. difficile* R20291 strain.

[![](https://static.igem.org/mediawiki/2019/2/2e/T--NCKU_Tainan--spot-on-lawn.png)](https://static.igem.org/mediawiki/2019/2/2e/T--NCKU_Tainan--spot-on-lawn.png)

Fig. 1. Spot-on-lawn test using 5 μl purified bacteriocin, inhibition zone formation in the middle of the plate can clearly be seen.

To further incorporate bacteriocin into our project, we wanted to integrate the bacteriocin into a live therapeutic drug instead of having patients consuming purified bacteriocins. Thus, we decided to fuse our CBM-B with a secretion tag, YebF. YebF is a secretory protein with unknown function in *E. coli*[[2](#ref2)]. We first amplified the *yebF* fragments from the genome of *E. coli* MG1655 and the CBM-B fragment from the plasmid kindly provided by Professor Huang. He confirmed that the CBM-B gene encodes a bacteriocin protein that will perform self-cleavage, and only the C-terminus fragment has bactericidal properties. So, we designed two versions of CBM-B construct by separately amplifying the full version of CBM-B gene and also the sCBM-B (short-length CBM-B) that encodes the C-terminus protein region only. Concerned that the fusion of sCBM-B with *yebF* may affect its bactericidal function, two linker sequences was introduced between *yebF* and sCBM-B using overhang primers. We also used PCR to amplify [K880005](http://parts.igem.org/Part%3ABBa_K880005) from iGEM distribution kit, and used overextension PCR to piece these fragments together, before ligating them into pSB3K3 plasmid.

[![](https://static.igem.org/mediawiki/2019/3/31/T--NCKU_Tainan--CBMB-Amplification.png)](https://static.igem.org/mediawiki/2019/3/31/T--NCKU_Tainan--CBMB-Amplification.png)

Fig. 2. (a) Schematics of PCR strategy for each reaction. (b) 1.5% Agarose Gel figure show PCR result. M: Marker; Lane 1: Promoter+RBS; Lane 2: Promoter+RBS; Lane 3: CBM-B; Lane 4: GS linker-sCBM-B; Lane 5: TB linker-sCBM-B; Lane 6: *yebF*-GS overhang; Lane 7: *yebF*-TB overhang; Lane 8: *yebF*-CBM-B overhang.

[![](https://static.igem.org/mediawiki/2019/4/41/T--NCKU_Tainan--CBMB-Overhang.png)](https://static.igem.org/mediawiki/2019/4/41/T--NCKU_Tainan--CBMB-Overhang.png)

Fig. 3. (a) Schematic of PCR strategy for each reaction. (b) 1.5% Agarose Gel figure show PCR result. M: Marker; Lane 1: PCR product of BBa\_K2997000; Lane 2: PCR product of BBa\_K2997005; Lane 3: PCR product of BBa\_K2997006; Lane 4: PCR product of BBa\_K2997007.

However, after several attempts of constructing the *yebF*-bacteriocin plasmid, mutations kept appearing in the coding region. We will further complete *yebF*-bacteriocin construct in the future. We hypothesize that adding the yebF secretion tag into the construct is toxic to the bacteria.

[![](https://static.igem.org/mediawiki/2019/d/de/T--NCKU_Tainan--CBMB-Mutation.png)](https://static.igem.org/mediawiki/2019/d/de/T--NCKU_Tainan--CBMB-Mutation.png)

Fig. 4. Alignment results showing 1 bp deletion causing frameshifting in the coding region of *yebF*-GSlinker-sCBM-B construct. Red triangular indicates the deletion base pair.

### Tyrosine ammonia-lyase & tyrosine transporter

**Achievements:**

1. Construction of [BBa\_K2997000](http://parts.igem.org/Part%3ABBa_K2997000), [BBa\_K2997009](http://parts.igem.org/Part%3ABBa_K2997009) and [BBa\_K2997010](http://parts.igem.org/Part%3ABBa_K2997010)
2. Run SDS-PAGE to confirm expression in *E. coli* Nissle
3. Conduct functional test to measure the activity of TAL and *tyrP*

We constructed tyrosine Ammonia-Lyase with a native RBS and B0034 RBS, ([BBa\_K2997009](http://parts.igem.org/Part%3ABBa_K2997009) and [BBa\_K2997010](http://parts.igem.org/Part%3ABBa_K2997010)), and transformed both plasmids into *E. coli* Nissle 1917 and confirmed it by double digestion. The results are as follows:

[![](https://static.igem.org/mediawiki/2019/7/75/T--NCKU_Tainan--Result-C3Tori_double_digest_2.jpg)](https://static.igem.org/mediawiki/2019/7/75/T--NCKU_Tainan--Result-C3Tori_double_digest_2.jpg)

Fig. 5. Confirmation of BBa\_K2997009 by double digestion, the arrow indicates TAL with NRBS (~1600 bp).M: Marker; Lane 1: pSB1C3-BBa\_K2997009; Lane 2: BBa\_K2997009.

[![](https://static.igem.org/mediawiki/2019/e/e5/T--NCKU_Tainan--Result-C3T34_double_digest_2.jpg)](https://static.igem.org/mediawiki/2019/e/e5/T--NCKU_Tainan--Result-C3T34_double_digest_2.jpg)

Fig. 6. Confirmation of BBa\_K2997010 by double digestion, arrow indicates TAL with B0034 (~1600 bp). M: Marker; Lane 1: pSB1C3-BBa\_K2997010; Lane 2: BBa\_K2997010.

Meanwhile, we amplified the tyrosine transporter and its promoter ([BBa\_K2997000](http://parts.igem.org/Part%3ABBa_K2997000)) from *E. coli* MG1655 and cloning into pSB4A3. We then transformed the plasmid into DH5α and *E. coli* Nissle 1917.

[![](https://static.igem.org/mediawiki/2019/a/a7/T--NCKU_Tainan--Result-TyrP-double_digest_2.jpg)](https://static.igem.org/mediawiki/2019/a/a7/T--NCKU_Tainan--Result-TyrP-double_digest_2.jpg)

Fig. 7. Confirmation of BBa\_K2997000 by double digestion. M: Marker; Lane 1: BBa\_K2997000, arrow indicates *PtyrP*-*tyrP* insert at 1307 bp; Lane 2: pSB4A3-J04450 (negative control).

RT-PCR experiment was performed to confirm the transcription of constructed TAL Biobrick. As shown in Fig. 8, cDNA for both bacteria carrying TAL constructs are being detected by PCR, confirming that the TAL gene is actually being transcribed in *E. coli* Nissle.

[![](https://static.igem.org/mediawiki/2019/6/62/T--NCKU_Tainan--RT-PCR.png)](https://static.igem.org/mediawiki/2019/6/62/T--NCKU_Tainan--RT-PCR.png)

| Lane | Template | Primers | Lane | Template | Primers |
| --- | --- | --- | --- | --- | --- |
| 1 | PCR positive control |  | 8 | BBa\_K2997009 cDNA | 3+4 |
| 2 | BBa\_K2997009 cDNA | 1+2 | 9 | BBa\_K2997010 cDNA | 3+4 |
| 3 | BBa\_K2997009 RNA | 1+2 | 10 | BBa\_K2997009 RNA | 3+4 |
| 4 | BBa\_K2997009 plasmid | 1+2 | 11 | BBa\_K2997010 RNA | 3+4 |
| 5 | BBa\_K2997010 cDNA | 1’+2 | 12 | BBa\_K2997009 plasmid | 3+4 |
| 6 | BBa\_K2997010 RNA | 1’+2 | 13 | BBa\_K2997010 plasmid | 3+4 |
| 7 | BBa\_K2997010 plasmid | 1’+2 |  |  |  |

Fig. 8. Reverse Transcription (RT)-PCR Results to confirm that our construct is being transcribed. (a) Schematics show the location of amplified regions and primers. (b) 1.5 % Agarose gel shows PCR results. All products have expected sizes of 250 bp as shown in (a). Templates and primers used in this experiment are listed in the table. (cDNA: Total cDNA; RNA: Total RNA; plasmid: pSB1C3 containing respective Biobrick.)

Then, we carried out SDS-PAGE to check the protein expression of TAL, both with TyrP and without TyrP. The expected protein size of TAL is 54 kDa and the expected protein size of TyrP is 43 kDa. As seen in the results below, however, there’s no distinguishable band around both sizes.

[![](https://static.igem.org/mediawiki/2019/0/02/T--NCKU_Tainan--Result-SDSPAGE_2.jpg)](https://static.igem.org/mediawiki/2019/0/02/T--NCKU_Tainan--Result-SDSPAGE_2.jpg)

Fig. 9. 12% SDS-PAGE of *E. coli* Nissle 1917 with different plasmids. M: Marker; Lane 1: Wild Type; Lane 2: pSB1C3; Lane 3: BBa\_K2997009 ; Lane 4: BBa\_K2997010; Lane 5: Dual plasmid containing BBa\_K2997009 and BBa\_K2997000; Lane 6: Dual plasmid containing BBa\_K2997010 and BBa\_K2997000; Lane 7: Positive control (CD630\_33920)

Finally, to confirm the protein activity of TAL and TyrP, we performed a functional test using [*n*-octanol extraction method](https://2019.igem.org/Team%3ANCKU_Tainan/Design#Subtitle1), which was previously proposed by [iGEM Uppsala 2013](https://2013.igem.org/Team%3AUppsala) and has been verified by HPLC. The *p*-Coumaric acid concentration was measured through the absorbance value at 310 nm wavelength under Nanodrop UV-Vis wavelength. The standard curve of *p*-Coumaric acid was drawn in Fig. 10 to determine the relationship between *p*-Coumaric acid concentrations and its 310 nm arbitrary unit (a.u.). Our samples with TAL constructs were then mapped onto the standard curve, to know how much *p*-Coumaric acid is being produced.

[![](https://static.igem.org/mediawiki/2019/1/1f/T--NCKU_Tainan--Standard_Curve_48hr.png)](https://static.igem.org/mediawiki/2019/1/1f/T--NCKU_Tainan--Standard_Curve_48hr.png)

Fig. 10. The standard curve of *p*-Coumaric acid concentration in correlation with absorbance at 310 nm, which is provided by genetic *E. coli* Nissle in LB broth after 48 hours.

We compared the TAL constructs containing the native and B0034 ribosome binding sites, (BBa\_K2997009 and BBa\_K2997010) to determine if *p*-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig. 11, BBa\_K2997010 is able to produce a higher amount of *p*-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into *p*-Coumaric acid can increase by 1.73-fold.

We can further improve the conversion of tyrosine into *p*-Coumaric acid by adding a tyrosine transporter (BBa\_K2997000). As seen in Fig. 11, when tyrosine transporter is added, the production of *p*-Coumaric acid is significantly higher. When tyrosine transporter (BBa\_K2997000) is introduced into *E. coli* Nissle with TAL constructs containing native RBS (BBa\_K2997009) and B0034 RBS (BBa\_K2997010), conversion of tyrosine into *p*-Coumaric acid is increased by 1.44-fold and 1.31-fold respectively.

[![](https://static.igem.org/mediawiki/2019/d/d3/T--NCKU_Tainan--Results_tyrosine_to_pCA%2C_RBS_improve.png)](https://static.igem.org/mediawiki/2019/d/d3/T--NCKU_Tainan--Results_tyrosine_to_pCA%2C_RBS_improve.png)

Fig. 11. *p*-Coumaric acid/OD600 levels of *E. coli* Nissle with TAL and *tyrP* in LB with 1 mM tyrosine.

To further prove that the TAL enzyme can specifically use tyrosine as its substrate, we cultured *E. coli* Nissle with dual plasmids containing TAL with B0034 RBS (BBa\_K2997010) and tyrosine transporter (BBa\_K2997009) in LB medium with different concentrations of tyrosine with hopes to see a dose-dependent effect. As seen in Fig. 12, although no significance in *p*-Coumaric acid production in culture supplemented with 0.5 mM and 1.0 mM tyrosine was detected, there was an increasing trend. Furthermore, there was a significant increase when comparing culture supplemented with 1.0 mM and 2.0 mM tyrosine. We speculate that there was indeed a dose-dependent effect, but due to the sensitivity limitations of *n*-octanol extraction method, it was not apparent.

[![](https://static.igem.org/mediawiki/2019/6/61/T--NCKU_Tainan--Results_pCA_dose_responding.png)](https://static.igem.org/mediawiki/2019/6/61/T--NCKU_Tainan--Results_pCA_dose_responding.png)

Fig. 12. *p*-Coumaric acid/OD600 production levels from *E. coli* Nissle with dual plasmids of *tyrP* and TAL with B0034 RBS in LB with different concentrations of tyrosine.

---

## *p*-Cresol Sensing

### *pchR*

**Achievements:**

1. Construction of *pchR*-GFP [BBa\_K2997008](http://parts.igem.org/Part%3ABBa_K2997008) and confirm by double digestion.

The *pchR*-GFP ([BBa\_K2997008](http://parts.igem.org/Part%3ABBa_K2997008)) was constructed from IDT DNA synthesis in the form of three gBlock gene fragments via overhang PCR. Then, we performed digestion on the finished part and ligated it into the pSB1C3 vector. Subsequently, we transformed the *pchR*-GFP construct ([BBa\_K2997008](http://parts.igem.org/Part%3ABBa_K2997008)) into *E. coli* Top 10 using electroporation. After allowing the transformed *E. coli* Top 10 to grow on LB with Chloramphenicol plates, colony PCR and sequencing was carried out to determine successful clones. The colony PCR results indicated that the *E. coli* has pSB1C3 with *pchR*-GFP. However, after several attempts to construct *pchR*-GFP, all the sequencing resulted in mutations.

[![](https://static.igem.org/mediawiki/parts/b/bd/PchR_sequencing_result3.jpg)](https://static.igem.org/mediawiki/parts/b/bd/PchR_sequencing_result3.jpg)

Fig. 13. Sequencing results of the pSB1C3 with *pchR*-GFP construct.

These results led us to hypothesize that the *pchR* protein is toxic to our *E. coli*. Moreover, according to a research paper[[3](#ref3)] we have found, it is suggested in the study that *p*-Cresol is toxic to Gram-negative bacteria, which includes *E. coli*. Hence, this plasmid construct has been dropped.

Therefore, the construction of *pchR*-GFP was carried out again by re-doing overlap PCR, where we combine all the three IDT gene fragments into the full-length *pchR*-GFP construct. For this time, the finished full-length *pchR*-GFP part was then inserted into the vector pBBR1 MCS-4 (contains ampicillin selection marker).

[![](https://static.igem.org/mediawiki/2019/a/af/T--NCKU_Tainan--pCHR_double_digest_2.jpg)](https://static.igem.org/mediawiki/2019/a/af/T--NCKU_Tainan--pCHR_double_digest_2.jpg)

Fig. 14. Gel electrophoresis result of pBBR1 MCS-4 with full-length *pchR*-GFP. Lane 1: Marker; Lane 2: Pre-ligation of vector pBBR1 MCS-4 (4950 bp) and insert *pchR*-GFP (3138 bp); Lane 3: Post-ligation of pBBR1 MCS-4 with full *pchR*-GFP (8098 bp).

After that, we proceeded into transforming the construct into *Pseudomonas* *fluorescens* 55 and *Pseudomonas* *aeruginosa* PAD1 via electroporation.

However, due to several complications regarding the selection marker being used (Ampicillin), we were not able to obtain definitive results indicating that we have positive colonies. This is due to the fact that *P. fluorescens* 55 and *P. aeruginosa* PAD1 are naturally resistant to ampicillin and the vector we used contains ampicillin selection marker. Hence, positive colonies cannot be differentiated from negative colonies. Besides that, our colony PCR results on the single colonies were also proven to be negative.

Therefore, with time being one of the main factors, we decided to drop any further construction and functional experiments surrounding the *p*-Cresol sensing part (*pchR*-GFP).

---

## Biosafety

**Achievements:**

1. Successful knockout of *can* gene and *dapA* gene

In order to make our live therapeutic safe for human consumption, we added a kill switch into our *E. coli* Nissle in the form of gene knockouts. We used Lambda Red Recombineering system, which is based on homologous recombination, to perform gene knockouts. By using lambda red genes and hijacking *E. coli* Nissle’s own recombineering system, we are able to replace the target DNA region with a DNA fragment of our choosing. [Electroporation](https://2019.igem.org/Team%3ANCKU_Tainan/Protocols) was then used to deliver DNA fragments and plasmids into the bacteria.

We amplified the Chloramphenicol Resistance (CmR) Cassette from pKD3 plasmid using Amplification PCR, and used that fragment to replace the target gene. The resistance cassette is flanked by FRT sites, which then allows the removal of the cassettes with the help of pCP20, an FLP helper plasmid. Before we electroporated the CmR cassette into *E. coli* Nissle, we electroporated in pKD46, a recombinase helper plasmid.

We experimented with two different gene knockouts, *can* gene knockout and *dapA* gene knockout, and compared its effectiveness.

### *dapA* Gene Knock Out

*dapA* encodes for 4-hydroxy-tetrahydrodipicolinate synthase, an enzyme necessary for cell wall synthesis[[4](#ref4)]. By knocking out this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) for cell wall assembly and growth, making it a potential kill switch as the bacteria will not survive without suppliance of exogenous DAP.

We confirmed that we have successfully replaced the *dapA* gene with CmR cassette using colony PCR. The size of CmR cassette inserted between the homology arms is approximately 1600 base pairs.

[![](https://static.igem.org/mediawiki/2019/1/17/T--NCKU_Tainan--Dap_PCR_2.jpg)](https://static.igem.org/mediawiki/2019/1/17/T--NCKU_Tainan--Dap_PCR_2.jpg)

Fig. 15. (a) Schematics showing *dapA* knockout strategy (b) Confirmation of *dapA* knockout in *E. coli* Nissle via colony PCR. M: Marker; Lane 1: Wild Type (no bands); Lane 2: Δ*dapA*::CmR 1 (1.6 kb); Lane 2: Δ*dapA*::CmR 2 (1.6 kb).

To further confirm our mutant *E. coli* works as a kill switch, we did a phenotype test by streaking out bacteria on different plates.

[![](https://static.igem.org/mediawiki/2019/c/ce/T--NCKU_Tainan--dapa-plate.png)](https://static.igem.org/mediawiki/2019/c/ce/T--NCKU_Tainan--dapa-plate.png)

Fig. 16. Confirmation of *dapA* knockout in *E. coli* Nissle. *E. coli* Nissle and other strains were streaked onto agar plates containing (A) just Lysogeny Broth (LB); (B) 0.1 M DAP; (C) Chloramphenicol (Cm) and 0.1 M DAP for phenotyping.

As seen in Fig. 16, without exogenous DAP, Δ*dapA*::CmR is unable to grow. And since it *can* survive on Cm plates, we *can* confirm that it contains the CmR cassette.

However, we realized that we would have to think of a way to provide exogenous DAP in our capsule to prevent our *E. coli* from dying before it reaches the gut. After deliberating the cost-performance ratio, we decided it wasn’t a good fit for what we had in mind. Thus, we did not continue on with replacing the CmR cassette with FRT sites.

### *can* Gene Knock Out

The *can* gene of *E. coli* encodes for carbonic anhydrase (CA), an enzyme that assists rapid interconversion of CO2 and water into carbonic acid, protons and bicarbonate ions. *E. coli* requires a constant supply of bicarbonate as a metabolic substrate during normal growth[[5](#ref5)]. So, if this gene is knocked out, *E. coli* is unable to turn the CO2 into bicarbonate fast enough before the CO2 diffuses out and causes cell death[[6](#ref6)]. Inside the gut, where we want the *E. coli* to survive, the CO2 concentration is high enough (ranging from 5% to 29%) to allow the spontaneous conversion of CO2 into bicarbonate ions. However, when *E. coli* exits the human body, the lowered concentration of CO2 will result in its death.

We confirmed that we have successfully replaced the *can* gene with CmR cassette using colony PCR. The size of CmR cassette inserted between the homology arms is approximately 1400 base pairs. The size of FRT sites inserted between the homology arms is approximately 500 base pairs.

[![](https://static.igem.org/mediawiki/2019/8/8b/T--NCKU_Tainan--Can_PCR_2.jpg)](https://static.igem.org/mediawiki/2019/8/8b/T--NCKU_Tainan--Can_PCR_2.jpg)

Fig. 17. Confirmation of *can* knockout in *E. coli* Nissle via colony PCR. M: Marker; Lane 1: Wild Type (~1.1 kb), Lane 2: Δcan::CmR 1 (~1.4 kb), Lane 3: Δcan::CmR 2 (~1.4 kb), Lane 4: Δcan::FRT 1 (~500 bp), Lane 5: Δcan::FRT 2 (~500 bp)

To further confirm our mutant *E. coli* works as a kill switch, we did a phenotype test by streaking our bacteria on different plates and placing them in different conditions.

[![](https://static.igem.org/mediawiki/2019/e/ec/T--NCKU_Tainan--can-plate.png)](https://static.igem.org/mediawiki/2019/e/ec/T--NCKU_Tainan--can-plate.png)

Fig. 18. Confirmation of *can* knockout in *E. coli* Nissle. *E. coli* Nissle and other strains were streaked onto agar plates and placed in (A) 0.04% CO2; (B) 5% CO2 conditions for phenotyping.

As shown in Fig. 18, Δcan::CmR and Δcan::FRT requires a higher CO2 level to survive. In doing so, we have proved that we have successfully knocked out the *can* gene.

Thereby, we will use the can gene knockout as our kill switch.

---

## Characterization

This year, we characterized FNR promoter ([BBa\_K1123000](http://parts.igem.org/Part%3ABBa_K1123000)) which is an anaerobic promoter. Originally, we used this promoter to drive the expression of TAL constructs. However, finding that this promoter has a higher expression level of GFP under aerobic conditions, we did not use this promoter for our project. Instead, we used the strong constitutive promoter J23100 as our promoter.

We first cultured DH5α pSB1C3-*Pfnr*-GFP under aerobic and anaerobic conditions. After 10 hours of incubation, we fixed the *E. coli* cells in 2% agarose gel and placed it on a glass slide, then place the glass slide under a microscope for image capturing.

[![](https://static.igem.org/mediawiki/2019/b/b7/T--NCKU_Tainan--FNR_microscopy.png)](https://static.igem.org/mediawiki/2019/b/b7/T--NCKU_Tainan--FNR_microscopy.png)

Fig. 19. Differential interference contrast (DIC) microscope image and fluorescence microscope image for a single *E. coli* cell.

We then compared the brightness of a single *E. coli* cell under a fluorescence microscope using ImageJ Intensity Processing. The results are shown below.

[![](https://static.igem.org/mediawiki/2019/2/23/T--NCKU_Tainan--pFNR-GFP_new.png)](https://static.igem.org/mediawiki/2019/2/23/T--NCKU_Tainan--pFNR-GFP_new.png)

Fig. 20. Quantification of single *E. coli* cell (n=3) fluorescence intensity using ImageJ Intensity Processing after 10 hours of incubation in both aerobic and anaerobic conditions.

As seen in Fig. 20, the fluorescence signal in single *E. coli* cell after 10 hours of incubation is significantly higher in aerobic conditions than in anaerobic conditions. Literature has reported that GFP requires oxygen molecules to fold properly[[7](#ref7)] before it can emit fluorescence signal. We cannot exclude the possibility that in anaerobic culture, GFP protein is not folding properly and thus affecting the measurement result. However, we can still conclude this promoter is not anaerobic specific. It is recommended that future iGEMers should use other reporter genes that will not be affected by oxygen like luciferase to further characterize this Biobrick.

## References

1. Saito, Y., Sato, T., Nomoto, K., & Tsuji, H. (2018). Identification of phenol- and *p*-Cresol-producing intestinal bacteria by using media supplemented with tyrosine and its metabolites. *FEMS Microbiology Ecology*, 94(9).
2. Zhang, G., Brokx, S., & Weiner, J. H. (2005). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in *Escherichia coli*. *Nature Biotechnology*, 24(1), 100–104.
3. Passmore, I. J., Letertre, M., Preston, M. D., Bianconi, I., Harrison, M. A., Nasher, F., … Dawson, L. F. (2018). Para-cresol production by *Clostridium difficile* affects microbial diversity and membrane integrity of Gram-negative bacteria. *PLoS pathogens*, 14(9), e1007191.
4. InterPro EMBL-EBI. “4-Hydroxy-Tetrahydrodipicolinate Synthase, DapA (IPR005263) < InterPro < EMBL-EBI.” Ebi.Ac.Uk, 2019, <www.ebi.ac.uk/interpro/entry/IPR005263>. Accessed 5 July 2019.
5. Merlin, C., Masters, M., McAteer, S., & Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to *Escherichia coli*? *Journal of Bacteriology*, 185(21), 6415–6424.
6. Hashimoto, M., & Kato, J.-I. (2003). Indispensability of the *Escherichia coli* Carbonic Anhydrases YadF and CynT in Cell Proliferation at a Low CO2 Partial Pressure. *Bioscience, Biotechnology, and Biochemistry*, 67(4), 919–922.
7. Coralli, C., Maja Cemazar, Chryso Kanthou, Tozer, G. M., & Dachs, G. U. (2001). Limitations of the Reporter Green Fluorescent Protein under Simulated Tumor Conditions. *Cancer Research*, 61(12), 4784–4790.

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