iGEM 2020 — Team NCKU_Tainan

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NCKU Tainan/Notebook

[![Logo](https://static.igem.org/mediawiki/2020/2/27/T--NCKU_Tainan--logo_title.png)
![logo-mobile](https://static.igem.org/mediawiki/2020/2/27/T--NCKU_Tainan--logo_title.png)](https://2020.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2020.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2020.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2020.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2020.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2020.igem.org/Team%3ANCKU_Tainan/Model)
  [Safety](https://2020.igem.org/Team%3ANCKU_Tainan/Safety)
  [Poster](https://2020.igem.org/Team%3ANCKU_Tainan/Poster)
* Device

  [Hardware](https://2020.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Software](https://2020.igem.org/Team%3ANCKU_Tainan/Software)
* Results

  [Results](https://2020.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2020.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2020.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2020.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2020.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2020.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Overview](https://2020.igem.org/Team%3ANCKU_Tainan/hp_overview)
  [Entrepreneurship](https://2020.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Human Practices](https://2020.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Partnership](https://2020.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Collaborations](https://2020.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Sustainable Development](https://2020.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Inclusivity](https://2020.igem.org/Team%3ANCKU_Tainan/Inclusion)
  [Education](https://2020.igem.org/Team%3ANCKU_Tainan/Education)
* Parts

  [Parts](https://2020.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2020.igem.org/Team%3ANCKU_Tainan/Improvement)
* Team

  [Team](https://2020.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2020.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2020.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2020.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2020.igem.org/Team%3ANCKU_Tainan/Criteria)

Notebook

![](https://static.igem.org/mediawiki/2020/4/4c/T--NCKU_Tainan--notebook-cover.jpeg)

# Notebook

### Weekly journal of wet, dry, and HP

### This is not just a journal, it is the history of iGEM NCKU Tainan.

## Week 1 5/31-6/6

## Wet

---

1. Do the PCR to copy bsNOS gene from bacillus subtilis.
2. Basic lab skills.
3. First attempt to make the competent cell of DH5α and BL21.
4. Design the primer of IDT fragment.

## Dry

---

1. Test the different solution ratio of contact lenses.
2. Looking for current model simulating ocular drug delivery system.
3. Device brainstorming - looking for relevant information.
4. Design Wiki page structure.

## HP

---

1. Establish the future plan of HP events.
2. Contact with Camax Optical Corp.

## Week2 6/7-6/13

## Wet

---

1. Culture the competent cell of DH5α and BL21.
2. Clone bsNOS into DH5α.
3. Design the functional test for NOS production.

## Dry

---

1. Trial production of contact lenses by using glass plates.
2. Discussing about how to modified current model to fit our situation.
3. Discuss with a professor about the device.
4. Design Wiki layout.

## HP

---

1. Preparation of Tainan First High School education event.

## Week3 6/14-6/20

## Wet

---

1. Culture different competent cell for csgD functional test.
2. Freeze the engineered bacteria.

## Dry

---

1. Trial production of contact lenses by using the mold from CAMAX
2. Establishing our own models.
3. Design the first version of the device.
4. Design the basic page.

## HP

---

1. 6/16: Meetup with president.

## Week4 6/21-6/27

## Wet

---

1. Finalize the primer design.
2. Design for the kill switch functional test.
3. Establish the protocol of biofilm assay.

## Dry

---

1. Design our mold of the contact lens by solidwork.
2. Looking for parameters.
3. Go to Prof. Huang laboratory to do porcine eye experiment.
4. Making the basic page.

## HP

---

1. 6/24: Tainan First High School education event.

## Week5 6/28-7/4

## Wet

---

1. Establish the protocol of functional test for NOS production.
2. Transform plasmid with csgD gene into bacteria.
3. Amplify the product we got from IDT synthesized with PCR.

## Dry

---

1. Print the mold by 3D-printing.
2. Looking for parameters.
3. Test 40kHz transducer and design the prototype.
4. Modify the design of the basic page.

## HP

---

1. 6/29: Glaucoma awareness week online education event
2. Preparation of visiting Camax Optical Corp.

## Week6 7/5-7/11

## Wet

---

1. Double Digest the gene fragment.
2. Extract the pSB1C3 from the plate.

## Dry

---

1. Making the contact lenses by our mold.
2. Confirming the parameters that cannot be found in current research, and try to estimate them.
3. Arduino learning.
4. Discuss main page design.

## HP

---

1. 7/10: Visit Camax Optical Corp.

## Week7 7/12-7/18

## Wet

---

1. Culture IDT fragment into different bacteria.
2. Use the three fragment assembly to clone NOS gene, csgD, and csgA together on psb1C3.
3. Change the plasmid that with EcorV for kill switch.
4. Do the congo red asset for testing csgD and csgA gene’s function.

## Dry

---

1. Improve the contact lens
2. Confirming all the compartment’s conditions
3. The first porcine eye experiment, guided by a Dr. Hung.
4. Design the main page.

## HP

---

1. 7/15 Meetup with Ming-Dao High School.
2. Preparation of Synthetic biology in Five levels video.
3. Preparation of Read for Love Summer Camp education event.

## Week8 7/19-7/25

## Wet

---

1. Clone both csgD, csgA, and also NOS successfully into DH5α.
2. Redo the csgD functional test with different control, we use PCA24N instead of pSB1C3.
3. Run SDS PAGE for csgD.

## Dry

---

1. Assemble device of Digital Image Correlation.
2. Looking for theories that can describe our equations’ boundary conditions, and ask wet team to do necessary experiments in order to obtain some critical parameters and conditions.
3. Test arduino to read high frequency signal.
4. Design the main page.

## HP

---

1. 7/20: Take Synthetic biology in Five levels video.
2. 7/21: Release survey.
3. 7/25: Read for Love Summer Camp education event.

## Week9 7/26-8/1

## Wet

---

1. Clone both csgD, csgA and also NOS into BL21.
2. Do the congo red assay.
3. Clone hicB, FLP represently intoDH5α.

## Dry

---

1. Calibration of VIC-3D (software of strain analysis)
2. Solving the partial differentiation equations we established.
3. Design the function generator and draw the schematic circuit diagram with DXP.
4. Drawing the basic page picture.

## HP

---

1. 7/31: Discussion with NarLab.
2. 8/1: I’ve Gotta PhD announcement with CSMU.
3. Preparation of Taiwan iGEM conference.

## Week10 8/2-8/8

## Wet

---

1. Run the SDS PAGE for NOS.
2. Do the heat-inactivated test to test the efficiency of heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR with 2% arabinose.
3. Use CI857 to replace EL222 gene, change the design of growth switch.
4. Analyze the data of congo red assay.

## Dry

---

1. Familiar with the operation process of DIC.
2. Solving the partial differentiation equations we established.
3. Design and test the amplifying circuit, and continue to draw circuit diagrams.
4. Populating Wiki for basic page.

## HP

---

1. Preparation of The Something Podcast.
2. Preparation of YMCA education event.

## Week11 8/9-8/15

## Wet

---

1. Do the NOS assay to test the efficiency of NO producing.
2. Use new strain WM3064 in DAP deficient functional test.
3. Do the heat-inactivated test to test the efficiency of heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR with 2% arabinose.
4. Do the three fragment assembly with CI857, FRT, OFP and GFP.

## Dry

---

1. Perform porcine eyes experiments for five-time.
2. Solving the partial differentiation equations we established.
3. The second porcine eye experiment.
4. Make the basic page picture.

## HP

---

1. W8/10: YMCA education event.
2. 8/13: Molecularcloud live stream.
3. 8/15 Online meet up with iGEM iBowu China.

## Week12 8/16-8/22

## Wet

---

1. Update the newest result from congo red assay
2. Improve the protocol in NOS assay.
3. Do the kill switch functional test.
4. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR and tetracycline with 0.2% arabinose.
5. Freeze current bacteria.

## Dry

---

1. Data analysis and processing.
2. Solving the partial differentiation equations we established.
3. Design and test the envelope detector, and order the designed PCB board.
4. .Make the basic page picture.

## HP

---

1. 8/17, 18: Taiwan iGEM conference.

## Week13 8/23-8/29

## Wet

---

1. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR and tetracycline with 0.02% arabinose.
2. Knockout DapA gene from BL21.
3. Change growth switch design and test that it can work.
4. Test NOS kinetic.

## Dry

---

1. Perform porcine eyes experiments for five-time.
2. Analyze the datas from the DIC experiment and wet team experiment to fit our model results, and adjust the parameters and conditions.
3. Envelope Circuit Ver.2
4. Design the HP page picture.

## HP

---

1. Preparation of Undergraduate research presentation.

## Week14 8/30-9/5

## Wet

---

1. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR and tetracycline with 0.02% arabinose.
2. Do the NO assay.
3. Biofilm assay with hand.

## Dry

---

1. Data analysis and processing.
2. Analyze the datas from the DIC experiment and wet team experiment to fit our model results, and adjust the parameters and conditions.
3. Soldering the PCB board and integrate the Arduino program.
4. Design the HP page picture.

## HP

---

1. 8/31: Undergraduate research presentation.
2. 9/05: Releasing The Something Podcast.
3. 9/5,6: Online meet up iGEMeetParis.
4. Preparation of Into the darkness LARP event.

## Week15 9/6-9/12

## Wet

---

1. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR and tetracycline with 0.002% arabinose.
2. IPTG concentration and time difference.
3. Biofilm assay with book.

## Dry

---

1. Analyze the datas from the DIC experiment and wet team experiment to fit our model results, and adjust the parameters and conditions.
2. The third porcine eye experiment.
3. Make the HP page picture.

## HP

---

1. 9/8: Meet up with NCKU College X.
2. 9/12, 13: Into the darkness LARP event.

## Week16 9/12-9/20

## Wet

---

1. Test the best concentration of arabinose to induce pBAD promoter.
2. Biofilm assay with book.

## Dry

---

1. Analyze the datas from the DIC experiment and wet team experiment to fit our model results, and adjust the parameters and conditions.
2. DDesign the app and combine the whole system
3. Populating Wiki for HP page.

## HP

---

1. 9/17: NCKU Club Festival.
2. 9/20: Contact lens recycling week.

## Week17 9/21-9/27

## Wet

---

1. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR and tetracycline with 0.002% arabinose.
2. Porcine eye NO penetration.

## Dry

---

1. Comparing MATLAB simulation results with wet and dry experimental datas, adn try to adjust our models..
2. Test the entire system and make improvements.
3. Check for every pages.

## HP

---

1. Preparation of SDGs Panel discussion.
2. Preparation of collaboration with NYMU.

## Week18 9/28-10/3

## Wet

---

1. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR and tetracycline with 0.0002% arabinose.
2. Do kanamycin resistance gene deletion experiments.
3. Biofilm SDS PAGE.

## Dry

---

1. Comparing MATLAB simulation results with wet and dry experimental datas, adn try to adjust our models.
2. Device appearance design.
3. Team page coding.

## HP

---

1. 9/29: SDGs Panel discussion with Taiwan iGEM team.
2. 10/3: Collaboration with NYMU.
3. Preparation of visiting TWBIO.

## Week19 10/4-10/10

## Wet

---

1. Do heat-inactivated chlortetracycline in pTet-GFP-pBAD-TetR with 0.0001% arabinose.
2. IPTG induces NOS SDS PAGE.

## Dry

---

1. Documenting our results on the wiki page.
2. Printed and assembled device.
3. Finish all wiki page design.

## HP

---

1. 10/4: Online meet up cGEM.
2. 10/8: Visit TWBIO.
3. Preparation of discussion with AIESEC.

## Week20 10/11-10/17

## Wet

---

1. Wrinting wiki page.

## Dry

---

1. Writing software (computational method) page, documenting double laplace transform (the method we use to solve our models) as a new tool to help future iGEMers.
2. Complete device and demo.
3. Wiki content submission.

## HP

---

1. 10/11: Discussion with AIESEC.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NCKU Tainan/Engineering

[![Logo](https://static.igem.org/mediawiki/2020/2/27/T--NCKU_Tainan--logo_title.png)
![logo-mobile](https://static.igem.org/mediawiki/2020/2/27/T--NCKU_Tainan--logo_title.png)](https://2020.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2020.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2020.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2020.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2020.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2020.igem.org/Team%3ANCKU_Tainan/Model)
  [Safety](https://2020.igem.org/Team%3ANCKU_Tainan/Safety)
  [Poster](https://2020.igem.org/Team%3ANCKU_Tainan/Poster)
* Device

  [Hardware](https://2020.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Software](https://2020.igem.org/Team%3ANCKU_Tainan/Software)
* Results

  [Results](https://2020.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2020.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2020.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2020.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2020.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2020.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Overview](https://2020.igem.org/Team%3ANCKU_Tainan/hp_overview)
  [Entrepreneurship](https://2020.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Human Practices](https://2020.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Partnership](https://2020.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Collaborations](https://2020.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Sustainable Development](https://2020.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Inclusivity](https://2020.igem.org/Team%3ANCKU_Tainan/Inclusion)
  [Education](https://2020.igem.org/Team%3ANCKU_Tainan/Education)
* Parts

  [Parts](https://2020.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2020.igem.org/Team%3ANCKU_Tainan/Improvement)
* Team

  [Team](https://2020.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2020.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2020.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2020.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2020.igem.org/Team%3ANCKU_Tainan/Criteria)

[Clarence Taylor](#page-top)

* Engineering

* [- Overview](#overview)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- TA System](#tasystem)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- *EL222*](#el222)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- pCP20](#pcp20)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- *FRT* Change](#frtchange)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- Proof of Design](#prove)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

![](https://static.igem.org/mediawiki/2020/7/7f/T--NCKU_Tainan--engineer-cover.jpeg)

# Engineering Success

### Most valuable parts (MVP) of this year's hard work

## Overview

For our project, we need the bacteria to stay alive in our contact lenses until it can be used. However, bacteria can’t live for a long time in such a small space, especially with such limited nutrients. To solve this problem, we designed a growth switch to put bacteria into hibernation for storage, which we can then resuscitate after exposure to external stimulation.

[![](https://static.igem.org/mediawiki/2020/0/0f/T--NCKU_Tainan--design_HicAB123.gif)](https://static.igem.org/mediawiki/2020/0/0f/T--NCKU_Tainan--design_HicAB123.gif)

Fig. 1. The big picture of growth switch.

---

## Toxin-antitoxin system (TA system)

The *hicA-hicB* locus located on *E. coli* chromosome is a toxin-antitoxin system (TA system). The TA system is composed of linked genes, encoding a toxic protein that can inhibit cell growth and an antitoxic protein which neutralize the toxic protein[[1](#ref1)]. For our project, a kind of the TA system effect called the stress tolerance effect, is used to induce bacteria hibernation. By making use of this mechanism of the TA system, we designed a growth switch to regulate the growth of bacteria.

According to a research[[2](#ref2)], HicA is a toxin promoting mRNA degradation, leading to the hibernation of bacteria, while HicB is an antitoxin that can neutralize the effect of HicA. Hence, through regulating the expression of *hicA* and *hicB* genes, we can control the growth of bacteria, hibernating the bacteria before the contact lens are used.

---

## The blue light-sensitive system: *EL222*

Since contact lens are sealed during the process of production, it is impossible to regulate the growth switch through chemical induction; hence, we turned to external stimuli such as light, temperature, etc. At first, we used *EL222* to regulate the system[[3](#ref3)] (a blue light-sensitive protein), *pBlind* (a promoter activated by *EL222*), *FLP*, and *FRT* to change the orientation of our promoter[[4](#ref4)], which is activated by blue light (Fig. 2). In other words, we have to illuminate the contact lens with blue light for 30 minutes before use.

[![](https://static.igem.org/mediawiki/2020/a/af/T--NCKU_Tainan--engineering_fig1.png)](https://static.igem.org/mediawiki/2020/a/af/T--NCKU_Tainan--engineering_fig1.png)

Fig. 2. The first version of design: BBa\_K3490020.

This design enables us to control the bacteria growth in three stages.

First, the production stage is when we are culturing our bacteria. Without any arabinose addition during culture, the bacteria will be able to grow normally. After the production process, we will put the bacteria and arabinose into the contact lens together, so the pBAD promoter will transcribe *hicA*, causing the bacteria to hibernate. When we need to resuscitate the bacteria, *EL222* will be activated by blue light to induce *pBlind* promoter, and then *FLP* will be transcribed. *FLP* will act on the *FRT* sites to change the orientation of the *pBad* promoter, as a result, the bacteria will start to transcribe *hicB* to neutralize HicA and thus continue to grow.

We replaced *hicA* and *hicB* with *GFP* and *OFP* gene to build a test plasmid because it is much easier to observe the color change than to count the CFU (Fig. 3). If the color of fluorescence changes from green to orange, the experiment is successful to prove that the design is feasible.

However, during the experiment process, we had difficulty constructing both plasmid. With the doubt that the *EL222-FLP* system might be harmful to the growth of bacteria, we changed the design of blue-sensitive protein EL222 into thermo-sensitive protein CI857 on the plasmid pCP20 provided by our PI, Prof. Ng (Fig. 4).

[![](https://static.igem.org/mediawiki/2020/4/48/T--NCKU_Tainan--engineering_fig3.png)](https://static.igem.org/mediawiki/2020/4/48/T--NCKU_Tainan--engineering_fig3.png)

Fig. 3. The test plasmid design: BBa\_K3490021.

[![](https://static.igem.org/mediawiki/2020/3/34/T--NCKU_Tainan--engineering_fig4.png)](https://static.igem.org/mediawiki/2020/3/34/T--NCKU_Tainan--engineering_fig4.png)

Fig. 4. The heat-activated plasmid pCP20.

---

## The thermo-sensitive system: pCP20

Therefore, we employed heat-activation, as our new method to resuscitate the bacteria. The *pR* of the thermo-sensitive system can be activated through the degradation of CI857 protein when the temperature rises to 36-40 ºC[[5](#ref5)]. This time, the experiments of ligation and transformation were successful, but the color of GFP were not observed in the bacteria after transformation. Suspecting that the sequence might be mutated, we sent plasmid for DNA sequencing. As we expected, the sequencing region matched poorly to our GFP-OFP design. As shown in Fig. 4, one of the *FRT* sequences is severely mutated next to the promoter J23100(Fig. 5). This suggests that our design had violated some fundamental principle.

Since the *FLP* recombinase works by twisting the DNA to bring two *FRT* sites together and nicking them to generate insertion or deletion. We speculated that the distance between the two *FRT* sites is insufficient for the two *FRT* sites to form a loop in vivo. We then check for the optimal distance between two *FRT* sites for the *FRT-FLP* system to function. Previous report states that the optimal distance for the recombination to take place is around 200bp in vitro[[6](#ref6)], while our original design between two *FRT* sites is only 160bp, this may be the cause of malfunction.

Nevertheless, we had to come up with a new design.

[![](https://static.igem.org/mediawiki/2020/6/69/T--NCKU_Tainan--engineering_sequencing.png)](https://static.igem.org/mediawiki/2020/6/69/T--NCKU_Tainan--engineering_sequencing.png)

Fig. 5. The sequencing result of *sfGFP-FRT-J23100-FRT-OFP*, *J23100* is highlighted, and the red arrow indicates *FRT* sequence flanking *J23100*.

---

## The change of *FRT* mechanism

*FRT* sites can either cause inversion or deletion depending on the orientation of the two *FRT* sequences[[7](#ref7)]. Since the design of changing the promoter direction failed, we choose to delete the gene, which is more common in general lab settings. For the new design, we still used the plasmid pCP20 for heat-activation, but we put *hicA* gene between the *FRT* sites for deletion on the plasmid pSB1C3 (Fig. 6.A), and the plasmid pSB3K3 with *hicB* gene was also designed to continuously produce HicB (Fig. 6.B). Because pSB3K3 is a low copy number plasmid, the expression of HicB is less than HicA, therefore, the bacteria will hibernate. After heat activation, hicA gene will be deleted while *hicB* gene is still expressed, so the bacteria can resuscitate. For these three plasmids, we can regulate the growth of bacteria, meeting our need to design a growth switch.

[![](https://static.igem.org/mediawiki/2020/d/d1/T--NCKU_Tainan--engineering_30312.png)](https://static.igem.org/mediawiki/2020/d/d1/T--NCKU_Tainan--engineering_30312.png)

Fig. 6. (A) The design of BBa\_K3490030; (B) The design of BBa\_K3490031

---

## The experiment of kanamycin-resistant gene deletion

Due to time constraint, we can only demonstrate this model using pKD3 plasmid, which carries a kanamycin-resistance gene flanked between two *FRT* sites provided by our PI, Prof. Ng. If the bacteria cannot survive at LB plate with kanamycin after heat-activation, we can demonstrate that the gene between the *FRT* sites was deleted, indicating that our design is feasible. For the experiment, we co-transformed pKD3 and pCP20 into E. coli DH5α, and cultured a colony in 37℃ for 8 hr and transfer to 42℃ for 1 hr to heat-activate the bacteria, and then spread on the LB plate (dilute 105 times) and culture for 12 hr. After the colony grew, we screened the colony on a kanamycin plate to check whether the bacteria will grow or not. Results show that most of the bacteria can not survive on the Kanamycin plate but can still survive on the LB plate without kanamycin (Fig. 7A, 7B.), which means the antibiotic-resistance gene was deleted successfully. With this data, we are convinced that the design of pSB3K3 with hicA gene is feasible.

[![](https://static.igem.org/mediawiki/2020/3/3b/T--NCKU_Tainan--engineering_plate.png)](https://static.igem.org/mediawiki/2020/3/3b/T--NCKU_Tainan--engineering_plate.png)

Fig. 7. The bacteria can grow on the LB plate without antibiotics (A, the left one), but when we pick the colony from it to the kanamycin plate (B, the right one), there is almost no colony on the plate.

[![](https://static.igem.org/mediawiki/2020/e/ef/T--NCKU_Tainan--engineering_gel.png)](https://static.igem.org/mediawiki/2020/e/ef/T--NCKU_Tainan--engineering_gel.png)

Fig. 8. The plasmid pattern before and after activating *FRT* genes checked by DNA gel electrophoresis.

Lane1: The uncut plasmid with activation of the *FRT* system.

Lane2: The plasmid cut by HindIII with activation of the *FRT* system.

Lane3: The uncut plasmid without activation of the *FRT* system.

Lane4: the plasmid cut by HindII without activation of the *FRT* system.

LaneM: the marker

---

## References

1. Guglielmini J, Van Melderen L. Bacterial toxin-antitoxin systems. *Mobile Genetic Elements.* 2011;1(4):283-306.
2. Jørgensen MG, Pandey DP, Jaskolska M, Gerdes K. HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea. *Journal of Bacteriology.* 2008;191(4):1191-1199.
3. Motta-Mena LB, Reade A, Mallory MJ, et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. *Nature Chemical Biology.* 2014;10(3):196-202.
4. Branda CS, Dymecki SM. Talking about a Revolution. *Developmental Cell.* 2004;6(1):7-28.
5. Jechlinger W, Szostak MP, Witte A, Lubitz W. Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene expression in Escherichia coli. *FEMS Microbiology Letters.* 1999;173(2):347-352.
6. Ringrose L. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. *The EMBO Journal.* 1999;18(23):6630-6641.
7. ‌Park Y-N, Masison D, Eisenberg E, Greene LE. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. *Yeast.* 2011;28(9):673-681.

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# Team:NCKU Tainan/Experiments

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* Project

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Experiments

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# Experiments

### The details about our experimental protocols

### This page is about our protocols, you can find all the experimental process you need. Enjoy!

## Congo red Assay

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Centrifuge

3. ELISA reader

4. Incubator

5. 96 well plate

6. Centrifuge tube

#### Consumables:

1. Congo red

2. IPTG

3. LB

4. ddH2O

Protocol:

1. Culture bacteria NOS-DA-BL21, BW25113 csgD, BW25113 control for 2hr.

2. Add IPTG for induce for 12hr.

3. Adjust OD to 1.

4. Add congo red 3μl to 1ml bacteria.

5. Wait 2hr.

6. Centrifuge full speed 2min.

7. Remove supernatant.

8. Add 1 ml water.

9. Put 200μl into 96 well plate.

10. Air dry.

11. Add water 200μl.

12. Wait 20 min.

13. Remove water.

14. Add water 200μl.

15. Test OD 500

## Biofilm Assay

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Centrifuge

3. ELISA reader

4. Incubator

5. 96 well plate

6. Centrifuge tube

#### Consumables:

1. IPTG

2. LB

3. ddH2O

Protocol:

1. Culture bacteria NOS-DA-BL21, BW25113 csgD, BW25113 control for 2hr.

2. Add IPTG for induce for 12hr.

3. Adjust OD to 1.

4. Add congo red 3μl to 1ml bacteria.

5. Wait 2hr.

6. Centrifuge full speed 2min.

7. Remove supernatant.

## NOS Kinetic Test

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Centrifuge

3. ELISA reader

4. Incubator

5. 96 well plate

6. Centrifuge tube

#### Consumables:

1. NOS kit

2. IPTG

3. LB

4. ddH2O

Protocol:

#### Preparation:

1. Culture bacteria (NOSDA BL21) 3ml for 13 hr.

2. Adjust OD to 1.

3. Add IPTG 0.1 one hr.

4. Sonication.

5. Put 60μl of cell (NOSDA BL21) into 96well.

#### NOS assay:

1. Prepare 40μl reaction mix for each well.

| Components | Volume (μl) |
| --- | --- |
| NOS cofactor 1 10 | |
| NOS cofactor 2 20 | |
| Substrate 5 | |
| Nitrate reductase 5 | |
| Total Volume | 40 |

2. Add 40μl reaction mix for each well by 0, 5, 10, 15, 20, 30, 40, 50 min.

3. Incubate 10 mins 37oC.

4. Add 95μl NOS assay buffer to each well.

5. Add 5μl enhancer to each well.

6. Incubate room temperature 10mins.

7. Add 50μL reagent 1 and 50μL reagent 2 to each well.

8. Incubate for 10 mins.

9. Measure OD540.

## IPTG Induce NOS Test

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Pipette and pipette tips

3. Centrifuge

4. ELISA reader

5. Incubator

6. 96 well plate

7. Centrifuge tube

#### Consumables:

1. NOS kit

2. IPTG

3. LB

4. ddH2O

Protocol:

#### Preparation:

1. Culture bacteria (NOSDA BL21) 3ml for 12 hr.

2. Adjust OD to 0.5.

3. Add IPTG with different time (0.025mM, 0.05mM, 0.1mM) and concentration (0.5hr, 1hr, 1.5hr).

4. Sonication.

#### NOS assay kit:

1. Put 60μl of cell (NOSDA BL21) into 96well.

2. Prepare 40μl reaction mix for each well.

| Components | Volume (μl) |
| --- | --- |
| NOS cofactor 1 10 | |
| NOS cofactor 2 20 | |
| Substrate 5 | |
| Nitrate reductase 5 | |
| Total Volume | 40 |

3. Add 40μl reaction mix for each well by 0, 5, 10, 15, 20, 30, 40, 50 min.

4. Incubate 10 mins 37oC.

5. Add 95μl NOS assay buffer to each well.

6. Add 5μl enhancer to each well.

7. Incubate room temperature 10mins.

8. Add 50μL reagent 1 and 50μL reagent 2 to each well.

9. Incubate for 10 mins.

10. Measure OD540.

## LB Preparation

Materials:

#### Equipment:

1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium)

2. Magnetic stirrer

3. Magnetic stirring bar

4. Plastic measure jug

5. Measure cylinder

6. Spoon

7. Petri dish (for agar medium)

8. Aluminum foil

9. Electronic balance

10. Weighing paper or weighing bowl

11. Pipette and pipette tips

#### Consumables:

1. DW (distilled water)

2. Tryptone

3. Yeast extract

4. NaCl

5. NaOH (10N)

6. Agar (for LB agar)

7. Antibiotics (for selection plate)

Protocol:

1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.

| Components | Amount |
| --- | --- |
| DW 98 ml | |
| Tryptone 1 g | |
| Yeast Extract 0.5 g | |
| NaCl 1 g | |
| NaOH (10N) 20 μl | |
| Total Volume | 100 ml |
| Agar (for LB agar) 1.50% | |

2. Mix well, pour the mixture into the bottle or flask and autoclave. Notes: For agar medium, pour the mixture to the petri dish and dry the plates.

For selection plates, add antibiotics as the following before pouring the mixture to the petri dish:

- Kanamycin: 100 μl/100 ml media

- Chloramphenicol: 50 μl/100 ml media

- Ampicillin: 100 μl/100 μl media

## Competent Cell Preparation

Protocol:

#### Equipment:

1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium)

2. Magnetic stirrer

3. Magnetic stirring bar

4. Plastic measure jug

5. Measure cylinder

6. Spoon

7. Petri dish (for agar medium)

8. Aluminum foil

9. Electronic balance

10. Weighing paper or weighing bowl

11. Pipette and pipette tips

#### Consumables:

1. CaCl2

2. Glycerol

3. LB

4. ddH2O

Protocol:

#### Culture bacteria for making competent cell:

1. Add 6mL LB into the test tube.

2. Pick one colony from the plate and add it into the test tube.

3. Culture O.N. 37C, 200 rpm.

#### Test O.D. value & make competent cell:

1. Add 1mL overnight bacterial culture to 50mL LB.

2. When the O.D. is about 0.3~0.4, centrifuge 5000 rpm, 10 mins, discard the supernatant.

3. Resuspend the bacteria pellet in 25 ml ice cold 100 mM CaCl2 solution for 30 minutes on ice. Centrifuge the cell suspension at 5000 rpm in the Sorvall GSA rotor for 10 minutes. Discard the supernatant.

4. Resuspend the bacteria pellet on ice in 1ml of ice cold 100 mM CaCl2/15% glycerol (sterile) in each.

5. Dispense the bacteria in 100 mL aliquots (on ice) to 10 eppendorf.

6. Freeze cells at -80oC.

## Standard PCR

Materials:

#### Equipment:

1. PCR tubes

2. Ice bucket

3. Thermocycler

4. Pipette and pipette tips

#### Consumables:

1. MQ or ddH2O

2. 2x PCR buffer for KOD FX

3. dNTPs 2 mM

4. Forward and reverse primer (10 μM)

5. KOD FX polymerase

Protocol:

1. Prepare 40μl reaction mix for each well.

| Components | Volume (μl) |
| --- | --- |
| MQ or ddH2O 1.1 | |
| 10x PCR buffer 1 | |
| dNTP 2.5 mM 0.8 | |
| Template 1 | |
| Forward primer 10 μM 1 | |
| Reverse primer 10 μM 0.1 | |
| Taq polymerase + vent 0.1 | |
| Total Volume | 10 |

2. Gently mix the PCR reactions and centrifuge briefly.

3. Transfer the PCR tubes to a thermocycler.

| step | Temperature | Time |
| --- | --- | --- |
| Initial denaturalization 94°C | 2 mins | |
| 25 - 35 cycles 98°C (denaturation) | 30 secs | |
| 55°C (annealing) | 30 secs |
| 68°C (extension) | 2 mins (depend on sequence size 2kbp/min.) |
| Final Extension 68°C | 2 mins | |
| Hold 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ | |

## PCR Clean-up

Materials:

#### Equipment:

1. Eppendorf tubes

2. Collection tubes

3. Spin column

4. Pipette and pipette tips

5. Centrifuge

#### Consumables:

1. Isopropanol

2. Binding buffer

3. Washing buffer

4. Elution buffer

5. MQ or ddH2O

Protocol:

##### DNA binding:

1. Add MQ or ddH2O to DNA solution until the volume reaches 100 μl.

2. Add 300 μl of binding buffer to the solution.

3. Add 150 μl of isopropanol and invert the eppendorf 10 times.

4. Place a spin column in a provided collection tube. Transfer 550 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.

5. Discard flow-through and place the spin column back into the collection tube.

##### Wash

1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.

2. Discard flow-through and place the spin column back into the collection tube.

3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.

4. Discard flow-through and place the spin column back into the collection tube.

##### DNA elution

1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.

2. Collect the pure sample in the eppendorf and discard the spin column.

3. Keep the DNA solution in -20°C freezer.

## Restriction Enzyme Digestion

| Restriction Enzymes | Buffer  (Buffer will depend on restriction enzyme being used) |
| --- | --- |
| EcoRI SuR E/Cut Buffer H (10X) - Roche | |
| XbaI CutSmart Reaction Buffer (10x) - NEB or 3.1 - NEB | |
| SpeI CusSmart Reaction Buffer (10x) - NEB | |
| PstI 3.1 (10X) - NEB | |

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Ice bucket

4. Incubator (37°C)

#### Consumables:

1. Restriction enzymes

2. Buffer 10x (depends on its restriction enzymes)

3. DNA sample

4. MQ od ddH2O (for single digestion)

Protocol:

1. On the ice, add all the components.

Double Digestion:

| DNA | 15 μl |
| --- | --- |
| Buffer 10x | 3 μl |
| Restriction enzyme 1 | 1 μl |
| Restriction enzyme 2 | 1 μl |
| Total Volume | 20 μl |

Single Digestion (Structure Check):

| Plasmid DNA 2 μl | |
| --- | --- |
| Buffer 10x 1.5 μl | |
| Restriction enzyme 0.5 μl | |
| MQ or ddH2O 11 μl | |
| Total Volume | 15 μl |

2.Mix gently and incubate for 1-2 hours for double digestion or 30 mins - 1 hour for single digestion at 37°C. Note: Incubation time varies along the total volume of the reaction.

## Gel Extraction and Purification

Materials:

#### Equipment:

1. Eppendorf tubes

2. Collection tubes

3. Spin column

4. Pipette and pipette tips

5. Centrifuge

6. Scalpel

7. Dry Thermounit

#### Consumables:

1. Isopropanol

2. Binding buffer

3. Washing buffer

4. Elution buffer

Protocol:

##### Gel excision, solubilization and DNA binding:

1. Excise band with scalpel and transfer to a new eppendorf tube.

2. Weigh the gel slice in a tube (by measuring the weight difference of an empty eppendorf and the eppendorf with gel slice in the tube).

3. Add 3 volumes of binding buffer to 1 volume of gel (100 mg = 100 μl).

4. Incubate at 60°C for 2-10 mins (or until the gel has completely dissolved).

5. Add 1.5 volume of isopropanol and invert the eppendorf 10 times.

6. Place a spin column in a provided collection tube. Transfer 700 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.

7. Discard flow-through and place the spin column back into the collection tube.

##### 2. Wash:

1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.

2. Discard flow-through and place the spin column back into the collection tube.

3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.

4. Discard flow-through and place the spin column back into the collection tube.

##### 3. DNA elution:

1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.

2. Collect the pure sample in the eppendorf and discard the spin column.

Keep the DNA solution in -20°C freezer.

## Ligation

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Centrifuge

#### Consumables:

1. T4 DNA ligase (NEB)

2. T4 DNA ligase buffer (NEB)

Protocol:

1. Prepare 40μl reaction mix for each well.

| Components | Volume (μl) |
| --- | --- |
| Vector DNA 12 | |
| Insert DNA 40 | |
| Fast Ligase 2X buffer 5.8 | |
| T4 ligase 0.5 | |
| Total Volume | 58.3 |

2. Gently mix the reaction and centrifuge.

3. Incubate at 16°C overnight.

4. Keep the DNA solution in -20°C freezer.

## Colony PCR

Materials:

#### Equipment:

1. PCR tubes

2. Ice

3. Thermocycler

4. Pipette and pipette tips

#### Consumables:

1. MQ or ddH2O

2. 10x PCR buffer

3. dNTPs 2.5 mM

4. Forward and reverse primer (10 μM)

5. Taq polymerase with 3 μl vent

Protocol:

1. On ice, add all components in a PCR tube, making up to 50 µl volume reaction.

| Components | Volume (μl) |
| --- | --- |
| MQ or ddH2O 1.1 | |
| 10x PCR buffer 1 | |
| dNTP 2.5 mM 0.8 | |
| Template 1 | |
| Forward primer 10 μM 1 | |
| Reverse primer 10 μM 0.1 | |
| Taq polymerase + vent 0.1 | |
| Total Volume | 10 |

2. Gently mix the PCR reactions and centrifuge briefly.

3. Transfer the PCR tubes to a thermocycler.

| step | Temperature | Time |
| --- | --- | --- |
| Initial denaturalization 94°C | 5 mins | |
| 25 - 35 cycles 94°C (denaturation) | 30 secs | |
| 55°C (annealing) | 30 secs |
| 72°C (Extension) | 2 mins (depend on sequence size 2kbp/min.) |
| Final Extension 72°C | 5 mins | |
| Hold 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ | |

## Bacterial Glycerol Stock

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Cryovial

#### Consumables:

1. 50% glycerol (autoclaved)

2. Bacterial overnight culture with an antibiotic if necessary

Protocol:

1. Put 1 ml of bacterial overnight culture into the cryovial and add 500 μl of 50% glycerol. Mix it well.

2. Keep it in the -80°C freezer.

## SDS-PAGE

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Centrifuge

4. Vortex

5. Ice bucket

6. Empty box

7. Dry Thermounit

8. Glass plates

9. 10-well comb

10. Spacer

11. Clamp

12. Casting stand

13. Buffer tank

14. Voltage source

15. Shaker

#### Consumables:

1. H2O

2. 30% acrylamide

3. 1.5M tris pH 8.8

4. 1M tris pH 6.8

5. 10% SDS

6. 10% APS

7. TEMED

8. Prestained Protein Marker - Bioman

9. Protein dye

10. Overnight culture (sample)

11. Coomassie Brilliant Blue (CBB) staining solution

12. Destaining buffer

13. Tank buffer (1X)

14. Isopropanol

Protocol:

#### Resolving gel preparation:

1. Adjust the amount of agarose to get the desired gel concentration.

2. In a 50 ml eppendorf tube add all the components 15% gel concentration.

| Total Volume | 5 ml | 10 ml | 15 ml |
| --- | --- | --- | --- |
| H2O (ml) 1.15 | 2.3 | 3.4 | |
| 30% acrylamide mix (ml) 2.5 | 5 | 7.5 | |
| 1.5M Tris pH 8.8 (ml) 1.25 | 2.5 | 3.3 | |
| 10% SDS (ml) 0.05 | 0.1 | 0.15 | |
| 10% APS (ml) | 0.1 0.2 0.3 | | |
| TEMED (ml) | 0.002 0.004 0.006 | | |

3. Pour the mixture in between the glasses and add iso-propanol afterward.

4. Wait for 10-15 mins or until the gel solidifies.

5. Pour out the iso-propanol.

#### Stacking gel preparation:

1. In a 50 ml eppendorf tube add all the components.

| Total Volume | 1.5 ml | 3 ml | 5 ml |
| --- | --- | --- | --- |
| H2O (ml) 1.05 | 2.1 | 3.4 | |
| 30% acrylamide mix (ml) 0.25 | 0.5 | 0.83 | |
| 1M Tris pH 6.8 (ml) 0.19 | 0.38 | 0.63 | |
| 10% SDS (ml) 0.015 | 0.03 | 0.05 | |
| 10% APS (ml) | 0.03 0.06 0.05 | | |
| TEMED (ml) | 0.0015 0.003 0.005 | | |

2. Pour the mixture in between the glass plates and add the 10-well comb.

3. Wait for 10-15 mins or until the gel solidifies.

#### Sample preparation:

1. Take overnight cultures with the desired volume and centrifuge.

2. Take 12 μl of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf.

3. Add 3 μl dye into the eppendorf. Vortex and centrifuge briefly.

4. Heat the eppendorf at 100°C for 10 mins and centrifuge briefly.

5. Put samples in the ice bucket.

#### Gel running:

1. When the gel solidified, set up the running equipment and pour 1X tank buffer.

2. Take out the comb and load 3 μl of the marker into a well and load the samples into the remaining wells.

3. Run the first 30 mins at 120V then continue to run for 1 hour at 150 V. Note: voltage and time may vary.

#### Staining:

1. Take out the glass plates out of the buffer tank and split up the glass plates to take out the gel.

2. The stacking gel and put the resolving gel inside an empty box, then add the CBB staining solution until it covers the gel.

3. Put the box on a shaker and shake for 30 mins or until the gel turns blue.

#### Destaining:

1. Pour the CBB staining solution back into the bottle and add the destaining buffer into the box until it covers the gel.

2. Put the box on a shaker and shake until the protein bands are visible or until the gel turns white.

3. Pour out the destaining buffer.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NCKU Tainan/Results

[![Logo](https://static.igem.org/mediawiki/2020/2/27/T--NCKU_Tainan--logo_title.png)
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* Project

  [Description](https://2020.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2020.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2020.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2020.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2020.igem.org/Team%3ANCKU_Tainan/Model)
  [Safety](https://2020.igem.org/Team%3ANCKU_Tainan/Safety)
  [Poster](https://2020.igem.org/Team%3ANCKU_Tainan/Poster)
* Device

  [Hardware](https://2020.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Software](https://2020.igem.org/Team%3ANCKU_Tainan/Software)
* Results

  [Results](https://2020.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2020.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2020.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2020.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2020.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2020.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Overview](https://2020.igem.org/Team%3ANCKU_Tainan/hp_overview)
  [Entrepreneurship](https://2020.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Human Practices](https://2020.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Partnership](https://2020.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Collaborations](https://2020.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Sustainable Development](https://2020.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Inclusivity](https://2020.igem.org/Team%3ANCKU_Tainan/Inclusion)
  [Education](https://2020.igem.org/Team%3ANCKU_Tainan/Education)
* Parts

  [Parts](https://2020.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2020.igem.org/Team%3ANCKU_Tainan/Improvement)
* Team

  [Team](https://2020.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2020.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2020.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2020.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2020.igem.org/Team%3ANCKU_Tainan/Criteria)

[Clarence Taylor](#page-top)

* Results

* [- NO Production](#nitric)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- Biofilm Production](#biofilm)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

* [- Kill Switch](#kill)

![magnifier](https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png)

![](https://static.igem.org/mediawiki/2020/0/03/T--NCKU_Tainan--results_cover.jpeg)

# Results

### No pain, no gain

## Nitric Oxide (NO) Production

#### *Nitric Oxide Synthase* (*NOS*) express

**Goal:**

Design *NOS* construction and express *Nitric Oxide Synthase* (*NOS*) gene.

**Achievements:**

1. Construction of *NOS*
2. Run SDS-PAGE to confirm expression in *E. coli*

This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered *E. coli* that can produce Nitric Oxide (NO). We aimed to make our bacteria bind to the contact lenses while at the same time express *NOS*. Since *E. coli* doesn't produce nitric oxide, we ordered a DNA sequence containing a *lacO-T7* promoter, B0034 RBS, and *bsNOS*. We ligated them altogether and inserted them into pSB1C3 plasmid and combined it with another sequence (*pLac*, RBS, *csgA*, RBS, *csgD*, *LacI*) from IDT, then transformed into *E. coli* DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide which will be released into the eyes[[1](#ref1)].

To ensure the plasmid contains those two sequences, we conducted PCR with their primer separately. Fig. 1 shows that the plasmid contains both of the desired sequences.

[![](https://static.igem.org/mediawiki/2020/6/6a/T--NCKU_Tainan--Result-gel1.png)](https://static.igem.org/mediawiki/2020/6/6a/T--NCKU_Tainan--Result-gel1.png)

Fig. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: *NOS* (~2400 bp); Lane 2: *csgA*-*csgD* (~1500 bp).

In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria was cultured for 2 hours and induced with IPTG for 12 hours. In Fig. 2 we can see that from the first to the third lane, the NOS expression was not significantly different. Meanwhile, we can see a thicker band in the fourth lane, which means higher protein expression was achieved when the IPTG concentration is 1 mM.

[![](https://static.igem.org/mediawiki/2020/c/c8/T--NCKU_Tainan--results_sds_page.png)](https://static.igem.org/mediawiki/2020/c/c8/T--NCKU_Tainan--results_sds_page.png)

Fig. 2. SDS-PAGE of *E.coli* BL21(DE3) with different concentrations of IPTG. The bacteria is cultured for two hours and induced with IPTG for 12 hours. M: Marker; Lane 1: 0.1 mM; Lane 2: 0.05 mM; Lane 3: 0.025 mM; Lane 4: 1 mM. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).

#### *NOS* expression control

**Goal:**

Control NOS production by IPTG inducible systems to produce more nitric oxide.

**Achievements:**

1. Measure the NOS activity in different IPTG concentration at different time
2. NOS kinetic test

As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and it’s induction time can control the production of NO. Therefore, we conducted experiment by inducing *E. coli* BL21(DE3) strain with different concentration of IPTG and with different period of induction time. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can conclude that the IPTG inducible system can dynamically produce nitric oxide.

[![](https://static.igem.org/mediawiki/2020/d/d3/T--NCKU_Tainan--results-iptg2.png)](https://static.igem.org/mediawiki/2020/d/d3/T--NCKU_Tainan--results-iptg2.png)

Fig. 3. Nitric oxide production dynamic.

After confirming the production of NO can be induced by IPTG, we tested the kinetic of NOS. The bacteria were cultured for 12 hours and induced with 0.1 mM IPTG for 2 hours. We sampled different time points and we can saw the substrates ran out within 20 min, producing approximately 1.6 nmol nitric oxide, which we assumed all substrate was converted. The maximum NO concentration can be achieved as seen in Fig. 4. We then set up a proof of concept experiment where we applied homogenized bacteria and necessary substrates onto the porcine cornea to observe whether the concentration of nitric oxide can penetrate into the aqueous humor. Through Fig. 5, we can see the nitric oxide in the aqueous humor of the porcine eye that supplied with our engineered bacteria and the necessary substrates increased by 6 times compared to control.

[![](https://static.igem.org/mediawiki/2020/4/45/T--NCKU_Tainan--Result-data1.png)](https://static.igem.org/mediawiki/2020/4/45/T--NCKU_Tainan--Result-data1.png)

Fig. 4. NOS activity at different times to observe the time required for the substrate to be depleted.

[![](https://static.igem.org/mediawiki/2020/1/14/T--NCKU_Tainan--results_nos_diffusion.png)](https://static.igem.org/mediawiki/2020/1/14/T--NCKU_Tainan--results_nos_diffusion.png)

Fig. 5. The concentration of NO in aqueous humor of the porcine eyes with engineered bacteria and necessary substrates compared to the ones without engineered bacteria and necessary substrates.

We used the IPTG inducible system to control NOS expression and nitric oxide production. From all the experimental results above, we have proven that our system is functioning well. Starting from NOS construction, confirmed NOS expression, and NOS functional test, we have provided convincing results to support our ideas.

---

## Biofilm Production

**Goal:**

Enhance biofilm production and improve the binding affinity of our engineered bacteria.

**Achievements:**

1. Construction of *csgD* and *csgA*
2. Run SDS-PAGE to confirm expression
3. Functional test to measure the amount of biofilm production and binding affinity

We are very grateful that one of our PIs, Professor Masayuki Hashimoto, from the Institute of Molecular Medicine, with generosity, provided us a BW25113 wild type strain, BW25113 *△csgD*, and PCA24N plasmid to support our *csgD* and *csgA* experiment.

As the first step, we constructed plasmid that only contains *pLac-csgD* on BW25113 and an improved version that contained *NOS*, *csgD* and *csgA* using gBlock provided by IDT. After that, we transformed into *E. coli* BL21(DE3) strain. Then, we used PCR to confirm that our bacteria carried *csgD* and *csgA*. The results are shown in Fig. 1.

Furthermore, we ran SDS-PAGE to check the protein expression of CsgD and CsgA. We cultured the bacteria for 2 hours, and added IPTG. We induced the bacteria for 12 hours long, then adjusted the OD600 to 3. We expected to see CsgD protein size around 25 kDa[[2](#ref2)] and CsgA around 15 kDa[[3](#ref3)]. The results below have shown the size as expected for CsgD and CsgA protein expression.

[![](https://static.igem.org/mediawiki/2020/2/24/T--NCKU_Tainan--results_csgd_improvement.png)](https://static.igem.org/mediawiki/2020/2/24/T--NCKU_Tainan--results_csgd_improvement.png)

Fig. 6. SDS-PAGE of E.coli BL21(DE3). M: Marker; Lane 1: Wild type *csgD* in PCA24N; Lane 2: Wild type BW25113 (control); Lane 3: Knockout *csgD* in PCA24N; Lane 4: Knockout BW25113 (control); Lane 5: BL21(DE3)-*NOS-csgA-csgD*. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).

Next, for further confirmation, we used a congo red dye to see the curli expression. If there is a biofilm, the absorbance value at 500 nm will be higher[[4](#ref4)]. We used BW25113 as control, and compared it to BW25113 with *csgD*, and *NOS-csgA-csgD* on BL21(DE3). First, we cultured the bacteria for 2 hours and induced them with IPTG. After culturing it overnight, we added congo red to 1 ml of bacteria and left it for 2 hours. Next, we centrifuged the stained culture to separate the supernatant and pellet. By using a microplate reader, we measured the absorbance value at 500 nm and normalized by 600 nm wavelength. Through Fig. 7 below, we can see that the amount of biofilm increased after adding *csgA*.

[![](https://static.igem.org/mediawiki/2020/2/2c/T--NCKU_Tainan--Measurement-congored.png)](https://static.igem.org/mediawiki/2020/2/2c/T--NCKU_Tainan--Measurement-congored.png)

Fig. 7. Amount of biofilm being produced by bacteria with different genetic backgrounds at different times.

After literature research, we found a biofilm staining protocol using congo red to stain bacteria growing in 96-well plate. After several attemps repeating this protocol, we soon discovered that most of the experimental error came from the staining procedure and the steps of removing supernatant which decrease the accuracy. We tried to remove the supernatant by hitting the plate to the table. As shown in Fig. 8, the results show large standard deviation.

[![](https://static.igem.org/mediawiki/2020/6/68/T--NCKU_Tainan--results_96_array.png)](https://static.igem.org/mediawiki/2020/6/68/T--NCKU_Tainan--results_96_array.png)

Fig. 8. Overexpression of *csgA-csgD* increases biofilm production. Three asterisks denote 0.0001 value, and four asterisks represent ＜0.0001 value.

However, the standard protocols to test biofilm formation cannot directly support our claims to increasing binding affinity. Therefore, we come up with a simple and quantifiable measurement protocol for evaluating biofilm binding ability. To observe the adherence of bacteria to the contact lens surface, we cultured the bacteria for 2 hours then induced with IPTG. Next, we proceed into a 96-well plate and leave for 12 hours of incubation. Afterward, we used external forces to test the bacteria binding affinity by free falling an object - a book, from different heights and measuring the OD600 value to determine its concentration. As the concentration increases, the bacteria will have a greater binding affinity.

[![](https://static.igem.org/mediawiki/2020/e/e9/T--NCKU_Tainan--Results_biofilmformula.png)](https://static.igem.org/mediawiki/2020/e/e9/T--NCKU_Tainan--Results_biofilmformula.png)

Fig. 9. (A)The formula and graph of the experiment. (The weight of book is 1.6 kilogram, the collision is considered as completely inelastic collision, and the collision time is presumed as 0.1 second.) (B) The binding affinity of control, *csgD*, and *csgA-csgD* with given force from different heights.

The results show the bacteria overexpressing CsgA-CsgD can withstand greater force. Thereby, we can prove that overexpressing CsgA-CsgD not only enhances biofilm production, but our bacteria can also bind properly to the contact lens.

---

## Kill Switch

#### Tetracycline (TC), Chlortetracycline (CTC)

**Goal:**

To prevent our engineered bacteria surviving outside the contact lens.

**Achievements:**

1. TC and heat inactivated CTC characterize

Our first kill switch design used TetR fused with VP16, tet-off system[[5](#ref5)] to control the Colicin E7(BBa\_K117000) as our biosafety measure. We will put tetracycline analog into the contact lens, this will prevent the VP16 to activate the lysis gene. If the bacteria leave the contact lens, where the environment has low concentration of tetracycline analog, the lysis gene will be activated. (Fig. 10)

There are several options when it comes to tetracycline analog that can act on the tetR protein, changing its DNA binding affinity, however, according to Biomol GmbH-Life Science Shop, 50 mg of Anhydrotetracycline costs 54 US dollars[[6](#ref6)]. In search of a cheaper alternative, we turned to tetracycline(TC)[[7](#ref7)] and Chlortetracycline(CTC)[[8](#ref8)], which has been reported to bind to tetR protein and affect its affinity to tetO.

[![](https://static.igem.org/mediawiki/2020/c/c7/T--NCKU_Tainan--Contribution_kill_switch_first_design.png)](https://static.igem.org/mediawiki/2020/c/c7/T--NCKU_Tainan--Contribution_kill_switch_first_design.png)

Fig. 10. Design schematic of the kill switch using tet-off system.

After many attempts constructing this plasmid resulted in failure. So, we turned to gene knockout as our biosafety. However, we can still characterize the effect of TC and CTC on TetR using iGEM BioBrick BBa-K611059. (Fig. 11)

[![](https://static.igem.org/mediawiki/2020/0/0b/T--NCKU_Tainan--Contribution_GFPnew.png)](https://static.igem.org/mediawiki/2020/0/0b/T--NCKU_Tainan--Contribution_GFPnew.png)

Fig. 11. iGEM BioBrick BBa-K611059.

BBa-K611059 has two open reading frames, a *pTet* promoter controlled GFP, and pBAD promoter controlled TetR. As we used arabinose to produce TetR to repress pTet, this allowed us to try different tetracycline analogs and observe GFP signal recovery to assess their potency. Since most tetracycline analogs have antimicrobial activity, we tested two different ways to compensate for this effect, heat inactivation of tetracycline analogs and co-transform tetracycline resistance plasmid.

We first transformed the plasmid carrying BioBrick BBa-K611059 into BW25113, which cannot metabolize arabinose, and added different concentrations of heat inactivated CTC[[9](#ref9)] to see if the GFP signal is restored.

After 12hr of culture with different concentrations of heat-inactivated CTC and 0.0001% of arabinose, we can see as arabinose was added into the culture, the normalized GFP signal slightly decreased, suggesting BBa-K611059 functions correctly. As 20µg/ml heat-inactivated CTC can rescue the GFP signal, and 30µg/ml will result in higher GFP signal. Also, through the OD600 results we can also conclude the heat-inactivated CTC does not affect *E. coli* growth.(Fig. 12)

[![](https://static.igem.org/mediawiki/2020/b/b8/T--NCKU_Tainan--Contribution_ctc.png)](https://static.igem.org/mediawiki/2020/b/b8/T--NCKU_Tainan--Contribution_ctc.png)

Fig. 12. GFP value according to different concentrations of heat inactivated CTC with 0.0001% of arabinose and the OD600 value.

Despite showing that heat-inactivated CTC can activate pTet, we were not satisfied with the dynamic range, so we decided to challenge using TC itself, since this operon originated in response to antibiotic resistance. In concerns of TC will be deadly to E. coli itself, also there has not been reported heat-inactivation TC can activate pTet, we co-transformed a TC resistance plasmid, pACYC184, into BW25113 to conduct further experiment.(Fig. 13)

[![](https://static.igem.org/mediawiki/2020/6/6f/T--NCKU_Tainan--Contribution_tc.png)](https://static.igem.org/mediawiki/2020/6/6f/T--NCKU_Tainan--Contribution_tc.png)

Fig. 13. (A) GFP value according to different concentrations of TC with 0.002% of arabinose and the OD600 value; (B) GFP value according to different concentrations of TC with 0.0002% of arabinose and the OD600 value.

We first tried two different arabinose concentrations, 0.002% and 0.0002%, both concentrations show repress GFP signal. However, bacteria experienced growth retardation in 0.002% of arabinose, we speculate that the high arabinose concentration induces osmotic stress to the bacteria. Also we can see as TC concentration increases, although the GFP signal remains the same, the OD600 decreases.This indicates that TC can induce pTet to produce GFP but suppress bacteria growth. We speculate that pACYC184 can not withstand such high concentration of TC.

From these results, since both TC and heat-inactivated CTC can activate pTet, but TC seems to affect cell growth at higher concentrations although we supplied the bacteria with resistance plasmid, we can conclude that heat-inactivated CTC is a better inducer compared to TC.

#### Diaminopimelate (DAP)

**Goal:**

To prevent our engineered bacteria from surviving outside the contact lens.

**Achievements:**

1. Transform plasmid with *T7* polymerase gene into WM3064
2. Successfully prove our bacteria will not survive without diaminopimelate (DAP)

We use the knockout strain WM3064 which deleted the gene *dapA* encoding for 4-hydroxy-tetrahydrodipicolinate synthase that is essential for cell wall synthesis[[10](#ref10)] and growth. For the growth, the strain WM3064 must rely on the supplement of diaminopimelate (DAP) in medium; in other words, the bacteria can’t survive without DAP. With this concept, we designed our kill switch for safety.

We transformed a plasmid carrying *T7* RNA polymerase into WM3064 to control the expression of *NOS* by the *T7-lacI* promoter. After that, we ran SDS-PAGE to ensure the protein expression of NOS under the *T7-lacI* promoter. The expected protein size of NOS is 40 kDa. As seen in Fig. 14, our WM3064 with *T7* RNA polymerase can successfully produce NOS.

[![](https://static.igem.org/mediawiki/2020/2/20/T--NCKU_Tainan--results_sds.png)](https://static.igem.org/mediawiki/2020/2/20/T--NCKU_Tainan--results_sds.png)

Fig. 14. SDS-PAGE of *E.coli* WM3064 after IPTG induction for 2 hours. M: Marker; Lane 1: Wild type; Lane 2: *T7* RNAP and NOS. The arrow indicates NOS (~40kDa).

In order to prove that *E. coli* WM3064 can’t survive without DAP, we spread our *E. coli* WM3064 with *T7* RNA polymerase to different plates. As seen in this result (Fig. 15), the bacteria can survive on the plate with exogenous DAP. When there is no more DAP supplied, the bacteria cannot survive.

[![](https://static.igem.org/mediawiki/2020/9/96/T--NCKU_Tainan--Results_daptwo.png)](https://static.igem.org/mediawiki/2020/9/96/T--NCKU_Tainan--Results_daptwo.png)

Fig. 15. Conformation of DAP-deficient strain WM3064. WM3064 was streaked on two different agar plates containing (A) Kanamycin (Km) and 0.3 mM DAP; (B) Kanamycin (Km) only.

---

## References

1. BRENDA - Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH). Brenda-enzymes.org.<https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM.> Published 2020. Accessed September 9, 2020.
2. csgD - CsgBAC operon transcriptional regulatory protein - Escherichia coli (strain K12) - csgD gene & protein. Uniprot.org. Published 2020. Accessed October 25, 2020. <https://www.uniprot.org/uniprot/P52106>
3. csgA - Major curlin subunit precursor - Escherichia coli (strain K12) - csgA gene & protein. Uniprot.org. Published 2020. Accessed October 25, 2020. <https://www.uniprot.org/uniprot/P28307>
4. Team:TAS Taipei - 2017.igem.org. 2017.igem.org. [https://2017.igem.org/Team:TAS\_Taipei.](https://2017.igem.org/Team%3ATAS_Taipei.) Accessed September 27, 2020
5. Kim, H J, et al. “Tetracycline Repressor-Regulated Gene Repression in Recombinant Human Cytomegalovirus.” *Journal of Virology*, vol. 69, no. 4, 1995, pp. 2565–2573
6. Anhydrotetracycline (hydrochloride). Biomol GmbH - Life Science Shop. Published 2020. Accessed October 25, 2020. <https://www.biomol.com/products/chemicals/antibiotics/anhydrotetracycline-hydrochloride-cay10009542-50>
7. Aleksandrov A, Schuldt L, Hinrichs W, Simonson T. Tetracycline-Tet Repressor Binding Specificity: Insights from Experiments and Simulations. *Biophysical Journal*. 2009;97(10):2829-2838.
8. Bochner BR, Huang HC, Schieven GL, Ames BN. Positive selection for loss of tetracycline resistance. *Journal of bacteriology*. 1980;143(2):926-933.
9. Pósfai G, Koob MD, Kirkpatrick HA, Blattner FR. Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. *Journal of bacteriology*. 1997;179(13):4426-4428.
10. McLennan N, Masters M. GroE is vital for cell-wall synthesis. *Nature.* 1998;392(6672):139-139.

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