iGEM 2024 — Team ccu-taiwan

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Oct.

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## Week 1. (May 1st-5th)

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#### Wet Lab:

* Determining *E. coli* Nissle 1917 as a compatible probiotic for our project.
* Determining experimental methods for stomach acid carriers.
* Consulting with professors regarding probiotic sources.
* Integrating project information.

#### Dry Lab:

* Excluding the stress relief lamp as hardware application.
* Deciding to use beverages as the starting point for hardware development.
* Dividing the design of the film into two directions; a layer of film attached under the sealing film of the daily beverage and a film hanging on the wall of the beverage cup.
* Confirming the zinc finger binding domain sequences and preparing for protein modeling.

#### Human Practices:

* Investigating the evidence that serotonin supplementation can rapidly enhance mood.
* Gathering and analyzing global data on the prevalence of depression.

#### Wiki:

* Conducting color research for the Wiki.
* Selecting a Wiki style that aligns with our topic.

## Week 2. (May 6th-12th)

---

#### Wet Lab:

* Exploring the regulatory methods of 5-HTP production.
* Exploring the oxygen-induced suicide mechanism for the biosafety strategy.
* Planning the open lab summer camp.

#### Dry Lab:

* Discussing how to present hardware and software in oral presentation.
* Exploring materials that are more suitable for edible and water-soluble film.

#### Human Practices:

* Mining the causes of negative moods and the effects of prolonged negative emotions.
* Planning and brainstorming activities to enhance engagement and outreach to a broader audience.

## Week 3. (May 13th-19th)

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#### Wet Lab:

* Integrating the information of two plasmids for 5-HTP production and enhancement.
* Designing open lab summer camp content.
* Determining the role of protocatechuic acid as a regulator of our plasmid.
* Writing the safety form.

#### Dry Lab:

* Choosing duck feet gelatin, fish skin, maltodextrin and starch as candidates of edible film.
* The plasticizers that can be used for starch films are glycerin, sorbitol, and polyethylene glycol.
* Deciding to simulate the concentration and calculate the production of 5-HTP to obtain the amount of bacterial powder required for the purpose of mathematical modeling.
* Completing the calculation of 5-HTP production as intracellular tryptophan concentration increases.
* Completing the language conversion, basic user interface, Fourier transformation, and data export functions for APP.

#### Human Practices:

* Analyzing the specific objectives of each planned activity.
* Determining themes for upcoming social media posts.

## Week 4. (May 20th-26th)

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#### Wet Lab:

* Integrating information for the open lab summer camp.
* Determining the regulation of 5-HTP production.
* Writing the safety form.

#### Dry Lab:

* Confirming the film will be attached beneath the sealing film of daily beverage.
* Searching for the material and protocol of film.
* Mathematical modeling of 5-HTP production after increasing to three enzymes.
* Simulating mutTPH1 and finding out its similar sequence protein: 3HF6.
* Showing the stress level on software.
* Finding professors for suggestions for software improvement.
* Supporting Open Lab on the introduction of 3D printers.

#### Human Practices:

* Organizing workshops to enhance the public's understanding of emotions.
* Posting registration information for Open Lab sessions on Instagram.

#### Wiki:

* Deciding and planning Wiki homepage design.

## Week 5. (May 27th-2nd)

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#### Wet Lab:

* Analyzing the pros and cons of 5-HTP production from probiotics.
* Analyzing and formulating the biosafety strategy.

#### Dry Lab:

* Confirming the protocol of maltodextrin and starch film.
* Searching for the information of the 3D bioprinter to find out whether it can be used in hardware.
* Optimizing the simulation of tryptophan absorption and in vivo reactant concentrations.
* Confirming the necessary constant in mathematical modeling.
* Using BH4 data to simulate the relationship between MH4 concentration and reaction rate (TPH\_Vmax).
* Simulating the fusion protein of TPH1 + zif268.
* Simulating DHPR and finding out its similar sequence protein: 1DHRA.
* Simulating PCD and finding out its similar sequence proteins: 1F93.
* Adding the results of the pressure test and the notification that will pop up when the pressure is too high for software.

#### Human Practices:

* Exploring the frequency of hand-shaken drink consumption among Taiwanese individuals and identifying the demographics involved.
* Brainstorming ways to make foreigners understand about hand-shaken drinks.

## Week 1. (Jun. 3rd-9th)

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#### Wet Lab:

* Analyzing the differences between 5-HTP production from probiotics and supplements.
* Determining some proteins for biosafety.
* Designing a vector map in SnapGene.

#### Dry Lab:

* Writing a project description of our film.
* Organizing questions for a software interview.

#### Human Practices:

* Focusing on relationships, stress, and emotions.
* Deciding to organize separate activities for younger and older people.
* Planning to organize activities for the local community, focusing on emotional care for the elderly.

#### Wiki:

* Designing a detailed wiki page, especially for the “Project Description” page.
* Assigning color codes to each element on the wiki.

## Week 2. (Jun. 10th-16th)

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#### Wet Lab:

* Analyzing experimental method to replace the ampicillin resistance gene with a kanamycin resistance gene in pST39.
* Designing the open lab summer camp experimental course.

#### Dry Lab:

* Writing a project description for edible film.
* Optimizing questions for the software interview.

#### Human Practices:

* Arranging the schedule for activities and assigning responsibilities.
* Reconsidering the Free Hug campaign and simultaneously initiating a Hug Marathon event to promote our concept.

#### Wiki:

* Designing the loading page for the wiki.
* Determining the font styles for each section.
* Coding the menu and footer of wiki.

## Week 3. (Jun. 17th-23rd)

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#### Wet Lab:

* Replacing the ampicillin resistance gene in the pST39 vector with a kanamycin resistance gene.
* Designing the presentation and textbook for the open lab summer camp.
* Demonstrating the open lab summer camp experimental course.

#### Dry Lab:

* Listing out future progress schedules.
* Confirming the protocols of different kinds of film.
* Adding soy protein and sodium caseinate film as edible film candidates.
* Adding peptide linker for fusion protein modeling.
* Finishing simulating the fusion proteins with linkers: TPH1 + zif268, PCD + PSBll.

#### Human Practices:

* Deciding to conduct a survey due to the uncertainty of the workshop.
* Establishing the details of activity of Free Hug campaign.
* Scheduling the workshops.
* Inquiring about the timing, preparing the proposal, and approaching the manager of the community center regarding the Elderly Seniors Program.
* Proposing street surveys and interviewing local residents.
* Reviewing the objectives that the events need to achieve and providing an overview of the event.

#### Wiki:

* Typesetting the page of the project description.

## Week 4. (Jun. 24th-30th)

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#### Wet Lab:

* Replacing the ampicillin resistance gene in the pST39 vector with a kanamycin resistance gene.
* Completing the presentation and textbook for the open lab summer camp.
* Printing out the textbook for the open lab summer camp.

#### Dry Lab

* Searching the guideline of the film.
* Determining the dissolving rate, liquid viscosity and transparency as the selection criteria of edible film.
* Calculating the production of 5-HTP.
* Simulating the fusion protein of DHPR + ZFa.
* Finishing the powerpoint of the introduction of 3D printers for the Open Lab.

#### Human Practices:

* Canceling the workshop and transitioning to an "Emotional Awareness" lecture.
* Confirming the speaker for the lecture, as well as the time, location, and anticipated number of attendees.
* Determining the format of the questionnaire for the street survey.
* Planning to organize an online lecture.
* Preparing the survey for local chain beverage stores.

#### Wiki:

* Putting the content onto the page of the project description.

## Week 1. (Jul. 1st-7th)

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#### Wet Lab:

* Previewing the open lab course.
* Replacing Ampicillin resistance gene in plasmids with Kanamycin resistance gene.

#### Dry Lab:

* Performing the preliminary experiment of film.
* Searching for the protocol to examine viscosity of film.
* Optimizing experiments of dissolving rate, viscosity and transparency of the film.
* Modeling the production rate of 5-HTP.
* Preparing the interview with professors to improve protein modeling.
* Concluding results of modeling contents on wiki.
* Adding the function to alert users in the app when the users measured pressure exceeds the limit for the software.
* Adding the phone notification permission for the software.

#### Human Practices:

* Confirming team-building benefits and discounts, discussing the cost and quantity of cup sleeves, and finalizing team uniforms by July 15.
* Finalizing the survey content, focusing on recent events.
* Designing a poster for the international student free hug campaign, with an emphasis on cultural exchange.
* Planning an emotional care event for August 30, considering the involvement of potential psychological experts. Preparing content for store managers and designing an iGEM banner.
* Preparing to visit nearby beverage stores to promote our project and conduct surveys from the store;s perspective.
* Planning an emotional care event on August 30, considering potential psychological experts to involve. Preparing content for store managers and designing an iGEM banner.
* Preparing to visit beverage stores nearby to promote our project and conducting surveys from the store's perspective.
* Scheduling online lectures and finding speakers..
* Designing team logo.

## Week 2. (Jul. 8th-14th)

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#### Wet Lab:

* Attending the experimental course.
* Replacing Ampicillin resistance gene in plasmids with Kanamycin resistance gene.

#### Dry Lab:

* Finalizing the protocol of the dissolution rate experiment.
* Revising the experimental design for viscosity.
* Estimating the costs of different film types.
* Interviewing professors to improve software.
* Debugging based on the interview content for software.
* Finishing the design of the software icon.
* Adding the schedule planning function for the software.
* Redesigning the user interface for the software.

#### Human Practices:

* Finalizing the team logo and holding a vote.
* Replacing online lectures with an interview featuring a psychology professor.
* Visiting beverage stores nearby to promote our project and conducting surveys from the store's perspective.
* Revising the survey content and order, then posting it on social media after receiving approval from a supervisor.
* Coordinating the event schedule with store managers and discussing plans with a physical therapist, including banner creation and logistics.

#### Wiki:

* Discussing Wiki design details and positioning.
* Gathering information on team members for the 'Members' section.
* Revising minor details of the Wiki.

## Week 3. (Jul. 15th-21st)

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#### Wet Lab:

* Ligating TPH1 and QDPR inserts into the plasmid pST39 by Gibson assembly.
* Transforming pST39-TPH1 and pST39-QDPR into DH5a for selection.
* Completing the first batch of the open lab experimental course.

#### Dry Lab:

* Confirming the experimental methods and materials for the film and conducting the experiment.
* Finishing the first batch of dissolution rate experiments for film by using resistance measurements.
* Designing and developing a hardware device to measure the dissolution test.
* Using Spectrophotometry to test the dissolution rate for films of different materials.
* Testing the new software: VMD for protein modeling.
* Adding the function to remind users to practice diaphragmatic breathing for relaxation for software.
* Taking the frequency method of HRV out of the measurement.

#### Human Practices:

* Preparing to film a promotion video and discussing the storyline to ensure a compelling promotional video.
* Scheduling an appointment for the expert interview, finalizing details with the psychology professor.

#### Wiki:

* Designing the 'Team Members' Wiki page.
* Adding the loader for Wiki.
* Trying the flip cards of 'Team Members'.
* Fixing the position of the sidebar.

## Week 4. (Jul. 22nd-28th)

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#### Wet Lab:

* Sequencing plasmids pST39-TPH1 and pST39-QDPR.
* Completing the second batch of the open lab experimental course.

#### Dry Lab:

* Organizing the data for the first batch of dissolution rate experiments for film.
* Completing the second run of dissolution rate experiments by spectrophotometer.
* Deciding to exclude the experiment for the viscosity of film.
* Testing the alphaphold2 for protein modeling with the help of an adviser.
* Changing the measurement time to 3 minutes for the software.
* Keeping the last 10 seconds and deleting the first 10 seconds in data pre-processing for the software.
* Detailed planning of the progress for each item.

#### Human Practices:

* Compiling and reviewing the results from the beverage store visits to analyze the feedback and insights gathered.
* Planning to visit the Culture night market with posters, QR codes, and a lucky draw activity to engage with participants.

#### Wiki:

* Designing the notebook and sponsorships page.
* Setting deadlines for notebook content.
* Adding a progress bar for every page.

## Week 5. (Jul. 29th-Aug. 4th)

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#### Wet Lab:

* Culturing bacteria for microencapsulation experiments.
* Inserting the protocatechuic acid (PCA) regulatory cassette into the vector.

#### Dry Lab:

* Preparing interviews for film.
* Designing humidity experiment for film.
* Referring to the result from iTASSER, use AlphaFold2 to run again the protein modeling.
* Composing the menu and work and rest planning in the app.

#### Human Practices:

* Deciding on the next visit to the night market and adjusting the activities accordingly.
* Heading to the Culture Night Market for a free hug event, distributing questionnaires, and conducting a quick emotional state survey.
* Arranging team photoshoot session.

#### Wiki:

* Coding 'Team Member' page.

## Week 1. (Aug. 5th-11th)

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#### Wet Lab:

* Checking the expression of the following proteins: pST39 - TPH1, pST39 - PCBD1, pST39 - QDPR.
* Sequencing the plasmid pST39-PCA.
* Preparing materials for the microencapsulation experiment.

#### Dry Lab:

* Organizing the result of soy protein, sodium caseinate and starch film.
* Contacting the film sealing machine manufacturer for an interview.
* Designing the liquid viscosity test.
* Repairing the bar chart of the app.
* Designing the posters.

#### Human Practices:

* Organizing a 'Free Hug' event for international students at the university.
* Seeking fundraising support from several temples in Chiayi.
* Posting updates on social media to introduce our iGEM team and project.
* Developing questions for interviewing the experts.

#### Wiki:

* Coding 'Notebook' page.
* Adding May and June notebook into 'Notebook' page.

## Week 2. (Aug. 12th-18th)

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#### Wet Lab:

* Checking the expression of the following proteins: pST39 - TPH1 + PCBD1, pST39 - PCBD1 + QDPR, pST39 - QDPR + TPH1.
* Inserting the KanR cassette into the vector.
* Conducting a microencapsulation experiment.

#### Dry Lab:

* Organizing the result of sodium caseinate film liquid viscosity test.
* Formulating the preservation conditions for the film.
* Coding two mathematical models with scaffold and no scaffold.
* Designing the mathematical models of how PCA concentration affects gene expression.

#### Human Practices:

* Collaborating online with the CSMU x NCHU iGEM team.
* Collaborating online with the NYCU Formosa iGEM team.
* Contacting and reaching out to international iGEM teams for collaborations.
* Recording the second batch of the promotion video.
* Posting the results of the 'Open Lab' event, including pictures and videos, on social media.

#### Wiki:

* Designing Wiki homepage.
* Retype setting 'Members' page.

## Week 3. (Aug. 19th-25th)

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#### Wet Lab:

* Sequencing the plasmid pST39 - TPH1 + PCBD1 + QDPR.
* Inserting the KanR cassette into the vector.
* Analyzing the microencapsulation experiment using a Zetasizer.

#### Dry Lab:

* Designing the humidity test for film.
* Brainstorming the outline of 'Application'.
* Modifying the outline of protein models.
* Including the app background.
* Including the feature where the water wave moves according to the waveform during measurement.

#### Human Practices:

* Editing the second batch of promotional video.
* Participating in the Taiwan iGEM Seminar for two days.
* Preparing the questionnaire for the Emotion Awareness Seminar.
* Designing and ordering the team banner.
* Scheduling and brainstorming social media contents through to completion.

#### Wiki:

* Rescheduling wiki content deadline.

## Week 4. (Aug. 26th-Sep. 1st)

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#### Wet Lab:

* Checking the expression of the following proteins: pST39 - TPH1 + PCBD1 + QDPR.
* Inserting the RCR cassette into the vector.
* Preparing additional samples from the microencapsulation experiment for pH testing.

#### Dry Lab:

* Designing an experiment to observe microbial growth at different humidity levels over a period of time.
* Discussing future applications for film.
* Managing the app beta version.

#### Human Practices:

* Seeking fundraising support from Management College.
* Organizing a physical exercise activity for the elderly at a social center.
* Collaborating online with the HKUST iGEM team.
* Finalizing the promotion video.
* Finalizing 'Human Practices' Wiki outline.

#### Wiki:

* Finalizing the Wiki homepage design.
* Gathering information for attribution form.
* Confirming Wiki all pages outline.

## Week 1. (Sep. 2nd-8th)

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#### Wet Lab:

* Finalizing the analysis of experimental results for the induction of three fusion proteins.
* Conducting gel extraction, colony PCR, and restriction digestion to verify plasmid size.
* Testing encapsulated *E. coli* at different pH levels.
* Writing Wiki content outline.

#### Dry Lab:

* Deciding to exclude the experiment on bacterial growth in different humidity conditions.
* Composing a reminder for the user to check their HRV in the app at 3 p.m. daily.
* Finishing the meditation page and integrating the music page into the app.
* Releasing a software demo and download tutorial.

#### Human Practices:

* Completing and submitting the promotion video.
* Promoting the open registration for the Emotion Awareness Seminar.
* Introducing the iGEM competition and our current project through a campus campaign.
* Starting to write Wiki content.

#### Wiki:

* Gathering information for each page.
* Typesetting 'Member' pages.

## Week 2. (Sep. 9th-15th)

---

#### Wet Lab:

* Testing the strength of the PCA promoter region with green fluorescent protein.
* Recording the growth behavior of uncoated and coated *E. coli*.
* Starting to write the Wiki content.

#### Dry Lab:

* Conducting the Wiki content of the software.
* Changing the suggestion for releasing stress will only appear after closing the window, which indicates high or severe stress levels in the software.
* Changing the daily reminder time to 2 p.m. and adding the sound effect.
* Addressing the issue of large file size for the software.

#### Human Practices:

* Organizing a recruitment drive to enlist new members for the next iGEM team.
* Holding the Emotion Awareness Seminar to help more people identify their own emotions.

#### Wiki:

* Coding 'Home' page.

## Week 3. (Sep. 16th-22nd)

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#### Wet Lab:

* Testing the strength of the PCA promoter region with green fluorescent protein.
* Recording the growth behavior of coated *E. coli*.
* Finishing the first version of Wiki content.

#### Dry Lab:

* Using the precision instrument OMRON Intellisense HEM-7071 to verify the accuracy of the software measurement data.
* Finishing the wiki content of protein modeling, application, and software.

#### Human Practices:

* Finishing the first version of Wiki content.
* Finishing the presentation video script.

#### Wiki:

* Collecting pictures and information from each group.
* Adding animations for wiki.

## Week 4. (Sep. 23rd-29th)

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#### Wet Lab:

* Finalizing Wiki content.

#### Dry Lab:

* Finalizing Wiki content.
* Adding a collecting function on the App.
* Typesetting the measurement result page.

#### Human Practices:

* Recording the presentation video.
* Improving and finalizing Wiki content.

#### Wiki:

* Finalizing and editing all Wiki page designs.
* Gathering information for each page.

## Week 5. (Sep. 30th-Oct. 6th)

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#### Wet Lab:

* Reviewing all content on the Wet Lab Wiki.

#### Dry Lab:

* Finalizing software.

#### Human Practices:

* Editing the presentation video.

#### Wiki:

* Finalizing and editing all Wiki page designs.

## Week 1. (Oct. 7th-11th)

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Coming soon...

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{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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* [Overview](#en1)
* [Zif268-m-hTPH1 protein & BH4 regeneration](#en2)
* [DNA scaffold](#en3)
* [PCA-Induced Promoter](#en4)
* [Reference](#en5)

## Overview

Our primary objective is to engineer Nissle 1917 to produce 5-HTP, a precursor to serotonin that humans can utilize. We designed an improved Zif268-m-hTPH1 protein sequence within its plasmid to achieve this. Additionally, we aim for Nissle 1917 to regenerate the Zif268-m-hTPH1 protein autonomously, so we incorporated the sequences for ZFa-hQDPR and PBSII-hPCBD1 proteins. To enhance the efficiency of this process, we also designed a system to bring these proteins into close spatial proximity using Zinc finger proteins and a DNA scaffold.

## Zif268-m-hTPH1 protein & BH4 regeneration

**Design:**

To ensure continuous production of 5-HTP in Nissle 1917, we introduced the gene sequence for Zif268-m-hTPH1, which is essential for 5-HTP synthesis. Additionally, we inserted the gene sequences for PBSII-hPCBD1 and ZFa-hQDPR proteins, enabling Nissle 1917 to synthesize the coenzyme BH4 necessary for 5-HTP production.

**Result:**

![](https://static.igem.wiki/teams/5040/engineering/1figure.jpg)

▲ After confirming the insertion of the expression cassette into the pST39 vector, we induced protein expression by treating with 0.2 mM IPTG at 37°C. To determine whether the expression level increases over time, we conducted inductions for 0, 4, and 8 hours, using pST39 as the control group

## DNA scaffold

**Design:**

We designed a DNA sequence containing a zinc finger protein binding motif to optimize the spatial arrangement of Zif268-m-hTPH1, PBSII-hPCBD1, and ZFa-hQDPR proteins. This sequence is then replicated into circular ssDNA using the rolling circle replication (RCR) mechanism.

**Build:**

Initially, we utilized modeling to predict the feasibility of the fusion proteins and designed the corresponding sequences. We constructed a plasmid to express the three fusion proteins and designed the DNA scaffold sequence on a separate plasmid.

**Learn:**

Through this design, Nissle 1917 may produce an excess of 5-HTP, potentially leading to symptoms like irritable bowel syndrome. Therefore, we decided to incorporate a switch within the DNA scaffold system to regulate 5-HTP production more effectively.

![gene](https://static.igem.wiki/teams/5040/engineering/2gene.png)

▲ We used the T7 promoter to drive the sequences of Zif268-m-hTPH1, PBSII-hPCBD1 and ZFa-hQDPR proteins.

## Switch for Enhancing 5-HTP Production—PCA-Induced Promoter

**Design:**

To align with our stress reduction theme, we identified a sensor responsive to protocatechuic acid (PCA) and its associated promoter. We integrated two promoters within one plasmid: one for continuous expression of the sensor, PcaUAM protein, and the other for the PCA promoter. A terminator was also included to separate the two promoters.

**Build:**

We positioned the PcaUAM gene downstream of the LacIQ promoter to ensure its constant expression. Following the PcaUAM gene, the T7Hyb1 terminator was inserted to demarcate the two promoters. When PCA enters Nissle 1917, it binds to the PcaUAM sensor, activating the PCA promoter and triggering the downstream expression of RepA genes. Ultimately, the RepA protein recognizes the RCORI 105 sequence and initiates the RCR reaction, amplifying the DNA scaffold to enhance 5-HTP production.

![dna](https://static.igem.wiki/teams/5040/engineering/3dna.png)

▲ The three fusion proteins bound to their respective motifs on the DNA scaffold, bringing them closer together.

![gene](https://static.igem.wiki/teams/5040/engineering/4gene.png)

#### Reference

---

1. Metabolic pathway engineering for high-level production of 5- hydroxytryptophan in Escherichia coli (Wang et al, 2018)
2. Escherichia coli “Marionette ” strains with 12 highly optimized small-molecule sensors. (Nat Chem Biol. 2018)
3. Rolling-circle replication of bacterial plasmids. (SA Khan, 1997)

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* [Main Enzymes](#rs1)
* [5-HTP production](#rs2)
* [RCR and PCA - regulatory system](#rs3)
* [Encapsulation](#rs4)

## Expression of Main Enzymes and Coenzymes in *E. coli*.

We employed rolling circle replication (RCR) to produce DNA scaffolds and fused three enzymes—m-hTPH1, hPCBD1, and hQDPR—with zinc finger binding domains. We simulated and designed fusion proteins through protein structure modeling: Zif268-rigid linker-hTPH1, PBSII-rigid linker-hPCBD1, and Zfa-hQDPR, which recognize DNA scaffolds. Subsequently, we verified the expression of these fusion proteins in *E. coli* strain BL21, both individually and in combination, confirming successful protein production. Finally, we analyzed 5-HTP production using an ELISA kit.

### Protein structure simulations

---

![](https://static.igem.wiki/teams/5040/result/1tables.png)

▲ Figure 1: Protein structure simulations using AlphaFold2 and iTASSER.

We incorporated a rigid linker (rLinker) into the fusion proteins, revealing that the N-terminal Zif268 and PSBll (pink) fused with rLinker (yellow) and the C-terminal m-hTPH1 and h-PCBD1 (blue) achieved the best structural predictions. Despite using different linkers, they did not enhance ZFa protein expression, with the best structural predictions observed when ZFa (pink) was fused at the N-terminus of hQDPR (blue).

For more details, please see our [Protein Modeling](https://2024.igem.wiki/ccu-taiwan/model) page.

### Optimal enzyme expression

---

After confirming successful cloning, we assessed the optimal expression of our fusion proteins—Zif268-rigid linker-hTPH1, PBSII-rigid linker-hPCBD1, and Zfa-hQDPR. We tested different induction times and IPTG concentrations at 37°C to induce protein expression.

![](https://static.igem.wiki/teams/5040/result/2figure.jpg)
![](https://static.igem.wiki/teams/5040/result/3figure.jpg)

![](https://static.igem.wiki/teams/5040/result/4figure.jpg)

▲ Figure 2: Coomassie Blue-stained SDS-PAGE analysis of the three fusion proteins at 0, 4, and 8 hours of IPTG induction. (Sup: supernatant; P: pellet)

We observed that the individual expression of PBSII-rigid linker-hPCBD1 and Zfa-hQDPR fusion proteins was successful. However, the Zif268-rigid linker-m-hTPH1 fusion protein did not express well, leading us to extend the induction time and increase IPTG concentration.

We conducted inductions for 0, 12, and 24 hours to investigate whether increased induction time would improve expression. Additionally, we assessed the impact of IPTG concentration by testing 0.2 mM, 0.4 mM, and 0.8 mM IPTG.

![](https://static.igem.wiki/teams/5040/result/5figure.jpg)
![](https://static.igem.wiki/teams/5040/result/6figure.jpg)

![](https://static.igem.wiki/teams/5040/result/7figure.jpg)

▲ Figure 3: Coomassie Blue-stained SDS-PAGE analysis of the fusion proteins at 0, 12, and 24 hours of IPTG induction.

![](https://static.igem.wiki/teams/5040/result/8figure.jpg)
![](https://static.igem.wiki/teams/5040/result/9figure.jpg)

![](https://static.igem.wiki/teams/5040/result/10figure.jpg)

▲ Figure 4: Coomassie Blue-stained SDS-PAGE analysis of the three fusion proteins with IPTG concentrations of 0.2 mM, 0.4 mM, and 0.8 mM.

Increased induction time and IPTG concentrations did not improve the expression level of the Zif268-rigid linker-m-hTPH1 fusion protein. Thus, we maintained the initial induction conditions for subsequent experiments. After confirming the successful insertion of three expression cassettes (Zif268-rigid linker-m-hTPH1, PBSII-rigid linker-hPCBD1, and Zfa-hQDPR) into the pST39 vector, we induced protein co-expression using 0.2 mM IPTG at 37°C.

![](https://static.igem.wiki/teams/5040/result/11figure.jpg)

▲ Figure 5: The Coomassie Blue staining of SDS-PAGE analysis of three fusion proteins' co-expression with IPTG induced.

We successfully co-expressed the fusion proteins, establishing optimal expression conditions for our constructs.

## Analyzing 5-HTP production using ELIZA kit.

After preparing the probiotics, we will analyze 5-HTP production using an ELISA kit. Results will be provided once the kit arrives.

## Plasmid construct of RCR and PCA-regulatory system in E.coli.

To regulate DNA scaffold formation, we employed the PcaUAM-pPCA3B5b Sensor to control the expression of RepA, the replication initiation protein in RCR. The RCR cassette includes RCORI 65, RCORI 105, and ZNF motifs. The PCA-regulated DNA scaffold formation aims to promote a 5-HTP biosynthesis pathway in *E. coli*.

### The RCR cassette: RCORI 65-ZNF motifs-RCORI 105.

---

![rcr](https://static.igem.wiki/teams/5040/result/12rcr.png)

▲ Figure 6: DNA digestion of the RCR cassette inserted into the pST39 vector.

After confirming the RCR cassette insertion into pST39, we proceeded with DNA sequencing and the next steps of the experiment.

### PCA-regulatory system

---

We identified point mutations in the PcaUAM sequence after confirming the insertion of the PCA-regulatory cassette into pST39.

![pca](https://static.igem.wiki/teams/5040/result/13pca.png)
![pca](https://static.igem.wiki/teams/5040/result/14pca.png)

▲ Figure 7: DNA sequencing results of the PCA-regulatory cassette inserted into the pST39 vector.

Repeated experiments showed persistent point mutations, prompting us to use a different strain for plasmid transformation.

![pca](https://static.igem.wiki/teams/5040/result/15pca.png)
![pca](https://static.igem.wiki/teams/5040/result/16pca.png)

▲ Figure 8: DNA sequencing results of the PCA-regulatory cassette, inserted into pST39, transformed into *E. coli* strains DH5α and StbIII.

Mutations persisted even after changing the bacterial strain for transformation. We will proceed with further experiments using alternative strains and varying culture temperatures to reduce mutation rates.

## Efficiency of microencapsulation

We applied chitosan and sodium alginate in a layer-by-layer (LbL) assembly on EcN cells to protect probiotics from harsh gastrointestinal conditions. We enhanced the stability of these layers by adding calcium chloride, forming a gel layer via ionic cross-linking. We assessed the coating efficiency via zeta potential analysis and evaluated bacterial growth behavior after incubation at pH 2.5 and 7.0, simulating gastric and intestinal environments.

### Zeta potential

---

Chitosan provides positive charges, and sodium alginate provides negative charges. By detecting the zeta potential after coating each layer on *E. coli*, we confirmed the successful coating of the bacteria.

![](https://static.igem.wiki/teams/5040/result/17graphic.png)

▲ Figure 9: Zeta potential of the chitosan-alginate layers (measured by Dynamic Light Scattering).

### Efficiency Test: Gastrointestinal Simulation

---

To simulate the gastrointestinal environment, we incubated encapsulated strains at pH 2.5 for 1, 2, and 4 hours and then at pH 7.4 for 3 hours to test bacterial survival.

![uncoated](https://static.igem.wiki/teams/5040/result/18uncoated.jpg)

(pH2.5 for 1 hour)

![uncoated](https://static.igem.wiki/teams/5040/result/19uncoated.jpg)

(pH2.5 for 2 hour)

![uncoated](https://static.igem.wiki/teams/5040/result/20uncoated.jpg)

(pH2.5 for 4 hour)

▲ Figure 10: [uncoated *E. coli*] pH test after incubating in pH 2.5 for 1, 2, and 4 hours.

![coated](https://static.igem.wiki/teams/5040/result/21coated.jpg)

(pH2.5 for 1 hour)

![coated](https://static.igem.wiki/teams/5040/result/22coated.jpg)

(pH2.5 for 2 hour)

![coated](https://static.igem.wiki/teams/5040/result/23coated.jpg)

(pH2.5 for 4 hour)

▲ Figure 11: [coated *E. coli*] pH test after incubating in pH 2.5 for 1, 2, and 4 hours.

While no uncoated *E. coli* survived in the acidic environment, we observed survival rates of 1.302 x 10⁷ cfu/mL, 3.74 x 10⁶ cfu/mL, and 3.7 x 10⁶ cfu/mL for coated strains at 1, 2, and 4 hours of incubation, respectively. The encapsulated strains successfully passed through the acidic environment and were released into an alkaline environment, indicating that our carriers protect probiotics effectively.

### Efficiency comparison and summary

---

We compared coated and uncoated strains by incubating them at pH 2.5 and pH 7.0 for 1 hour, followed by observation of their growth behavior in TB broth with glass beads to simulate peristaltic movement.

![](https://static.igem.wiki/teams/5040/result/24graph.png)
![](https://static.igem.wiki/teams/5040/result/25graph.png)

▲ Figure 12 (left): Bacterial growth behavior after water bath at pH 2.5 for 1 hour.
▲ Figure 13 (right): Bacterial growth behavior after water bath at pH 7.0 for 1 hour.

The coated strain entered the log phase earlier than the uncoated strain, demonstrating higher survival rates in both acidic and neutral environments.

![](https://static.igem.wiki/teams/5040/result/26graph.png)

▲ Figure 14: Growth behavior comparison between uncoated and coated strains after water bath incubation.

The coated strain showed consistent growth behavior, while the uncoated strain experienced delayed log-phase entry in acidic conditions, highlighting the protective effect of the encapsulation process.

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