iGEM 2025 — Team georgiastate-swjtu

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Our record of our activities done for this year's iGEM. Entries start from July 2025.{% endblock %}
{% block page\_content %}

## Introduction

---

This is the digital log of GSU-SWJTU's teams activity for our experimentation.
We have recorded our observations, questions, experiments, data, and our reasoning.

## Notebook of Culture Team

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This is the notebook for the Culture Team.

July Notebook

The first MRS broth medium preparation (failed)

Preparation of MRS culture media with different glucose concentrations

Preparation of MRS culture media with different glucose concentrations

Preparation of MRS culture media with different glucose concentrations

Plate streaking and liquid purification culture

OD measurement in liquid culture

Biolfilm detection

## Notebook of Nisin Team

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These are the protocols from the Nisin Team.

Nisin ZOI Disk Preparation (50–150 IU)

Nisin ZOI Disk Preparation (150–300 IU)

Creating BHI & BHI + 2% Sucrose Agar Plates

Streptococcus Mutans Overnight Culture

## Notebook of Killswitch Team

---

This is the notebook for the Killswitch Team.

###### Contents

* [Culture Team](#culture)
* [Nisin Team](#nisin)
* [Killswitch Team](#killswitch)

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Our design-build-test-learn (DBTL) cycles across subteams: Culture, Vector Design, Nisin, and Killswitch.{% endblock %}
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## Our Engineering Approach

---

We divided the work into four subteams — **Culture, Vector Design, Nisin**, and **Killswitch**.
Each followed the iGEM engineering cycle template of Design, Build, Test, Learn (DBTL).
Below, each team's cycles are documented with what we attempted, observed, and changed.

![Engineering Cycle Overview](https://static.igem.wiki/teams/5892/2025igemisms/engineering-overview.webp)

## Culture Team

Goal: establish growth and co-culture assays for engineered *Lactobacillus* and *S. mutans*.

---

##### Cycle 1 — The Liquid Culture

Design

The aim of this project is to modify *Lactobacillus* through genetic engineering methods to make it
produce Nisin, thereby enhancing its inhibitory ability against *S. mutans*. One of the key
prerequisites for optimizing Nisin production is to have a deep understanding of the growth kinetics
of *Lactobacillus* and *S. mutans*. Determining whether their growth conditions will change with the
variation of sugar concentration in the culture medium is crucial for our project. We speculate that
within a certain range, the higher the concentration of MRS sugar, the more conducive it is to the
growth of bacteria. To verify this hypothesis and establish reliable culture parameters for further
experiment, this experiment includes the growth of *Lactobacillus*, *S. mutans*, and *E. coli* (as a control
group) in MRS media with different glucose concentrations. By regularly measuring the OD of the
culture medium, the growth curves of *Lactobacillus*, *S. mutans*, and *E. coli* were plotted and
analyzed.

Build

This experiment prepared MRS liquid culture media with glucose concentrations of 2% (20 g/L,
standard formula), 3% (30 g/L), and 4% (40 g/L), respectively. Among them, 3% and 4% of the
culture medium were prepared by adding additional glucose to the standard MRS base medium.
In the first experiment, we used the regular method (121°C, 45 minutes) to sterilize the culture
medium with high-pressure steam. However, we found that the color of the culture medium
significantly darkened after sterilization, and when used for plate streaking culture, the growth
of each colony was severely inhibited, which means slow growth and small bacterial colonies.
According to papers review, this phenomenon is due to the high sugar content (20 g/L) of the
MRS culture substrate. Carbohydrates undergo Maillard reaction during long-term high-temperature and high-pressure sterilization, which not only causes browning of the culture
medium, but also may produce by-products that inhibit bacterial growth. Based on this, we
optimized the sterilization plan: the sterilization time of MRS basic medium was shortened to
15 minutes, and after sterilization, glucose concentration solution was filtered and sterilized (using
a 0.22 μm filter membrane) and added to accurately adjust to the target concentration. Glucose
solution is prepared by using sterile deionized water and solid glucose.

We inoculated *Lactobacillus*, *S. mutans*, and *E. coli* glycerol strains stored at −80°C onto MRS agar
plates with corresponding glucose concentrations (2%, 3%, 4%). Then, we placed the agar plates in a 37°C
incubator for 20 hours to obtain single colonies. Subsequently,
independent colonies with good morphology were selected from each plate and inoculated into
liquid MRS medium with corresponding glucose concentration for pre-culture amplification.
After 15 hours of shaking culture at 37°C and 200 rpm, we transferred the pre-culture to fresh liquid
MRS medium containing different glucose concentrations at an initial inoculation volume of
0.1 OD600. This time point is marked as the beginning of the formal experimental culture.

Test

All liquid cultures were treated to shaking culture in a shaker at 37°C and 200 rpm. We used a
spectrophotometer to regularly measure the optical density of the culture at a wavelength of 600
nm to measure growth. During measurement, we used fresh sterile MRS medium of the
corresponding concentration as a blank control. In the first experiment, we planned to use a set
of MRS culture medium as the blank control group for the entire OD measurement, but later
we found that doing so could easily lead to contamination of the blank control group by
miscellaneous bacteria. Therefore, in the second experiment, we prepared a fresh blank control
group before each OD measurement.

Learn

Within a certain range, the growth of *S. mutans* is affected by changes in sugar concentration,
but *Lactobacillus* and *E. coli* are not sensitive to changes in sugar concentration. However, we also noticed that there was
a certain deviation between the data of the 3% glucose concentration experimental group and
expectations, and the specific reasons (such as osmotic pressure effects or accumulation of
metabolic byproducts) need further exploration.
In addition, there are certain fluctuations in the experimental data, which can clearly reflect
the overall growth trend. However, the reliability of individual data points still needs to be
improved in further experiments by increasing biological replicates and optimizing sampling and
measurement processes to enhance accuracy.

During the cultivation process, we observed a typical feature of *S. mutans*: it can form a clear
white precipitate at the bottom of the culture bottle during the initial stages of expansion and
liquid culture. This phenomenon is significantly different from *Lactobacilli* and *E. coli* (which
usually precipitate during the dead phase). The papers point out that this is due to the fact that
*S. mutans* tends to grow in a chain-like pattern, and its cells naturally settle under the action of
gravity, which is an important biological characteristic of the strain and a sign of its active
growth, rather than contamination or abnormalities.

##### Cycle 2 — Biofilm Measurement

Design

This program aims to modify *Lactobacillus* to produce nisin in order to inhibit the growth of *S.
mutans*. That is, we initially need to try to find the growth conditions which benefit
*Lactobacillus* but not *S. mutans*, so we chose MRS culture medium—most conducive
to the growth of *Lactobacillus*—for exploration. *Streptococcus mutans* and *Lactobacillus* exist in the
oral cavity in the form of biofilms and are necessary conditions for the formation of dental caries.
Therefore, based on our research conditions—sugar concentration—we designed experiments to explore
the biofilm formation in MRS culture medium with glucose concentrations of 2%, 3% and 4%
respectively, and provide a data basis for our project.

Build

Our group chose quantitative measurement of biofilms by crystal violet staining to detect
the formation of biofilm in MRS broth under those three glucose concentrations—2%, 3% and 4%.
For *Lactobacillus* and *E. coli*, we set up two groups to minimize random error, while we set up three
groups in *S. mutans* as the expansion culture of *S. mutans* is not that ideal.

Test

After 24 hours, we removed the planktonic cell samples, used 1×PBS, 0.1% CV, diH2O and 95% alcohol
in turn for rinsing, dyeing and rinsing. Then, we put the 96-well plate into the plate reader to get
readings. The bacterial solution reading was subtracted from the corresponding culture medium reading, and
then averaged to obtain the required data, that is, the growth of biofilms of the three
bacteria in different glucose concentrations.

Learn

According to the average value of each bacteria growing under all three glucose concentrations, we
found that within a certain range, the growth of *S. mutans* biofilm is affected by changes in
sugar concentration; the higher the sugar concentration, the greater the amount of biofilm it
generates. However, *Lactobacillus* and *E. coli* are not sensitive to changes in sugar concentration.

#### Summary

We investigated the influence of changes in the sugar concentration of MRS on bacterial growth and biofilm formation within a certain range.
Subsequently, we will co-culture different bacteria (including engineered bacteria) with *S. mutans* to explore their inhibitory effects
on the growth of *S. mutans* and on the formation of biofilms by *S. mutans*.

## Vector Design Team

Goal: construct and optimize the nisin biosynthetic pathway in *E. coli*, then port to *Lactobacillus*.

---

##### Cycle 1 — Baseline Constructs

Design

Nisin is a ribosomally synthesized antimicrobial peptide belonging to the lantibiotic family.
It is initially produced as pre-NisA and undergoes a series of post-translational modifications mediated by the enzymes NisB and NisC, which dehydrate and cyclize the peptide, respectively.
The mature pre-NisA is subsequently exported by the ABC transporter NisT and finally activated through leader peptide cleavage by the serine protease NisP, releasing the mature and bioactive nisin.
In *Lactococcus lactis*, where the nisin biosynthetic system is naturally encoded, the modification complex composed of NisA/B/C localizes at the cell poles—preferentially the old poles—while NisT is distributed circumferentially along the membrane.
NisB acts as a polar “recruiter” to coordinate modification and export, ensuring that only fully modified pre-NisA is secreted.
This spatial organization tightly couples biosynthesis, modification, and export, thereby preventing premature secretion of inactive intermediates.

However, this architecture poses challenges for heterologous nisin production in other hosts, such as *E. coli*.
Expression of *nisA* alone often leads to intracellular accumulation of unmodified pre-NisA with limited antimicrobial activity.
Co-expression with *nisB* and *nisC* can promote proper dehydration and cyclization, but secretion remains inefficient without *nisT*.
The inclusion of *nisP* completes the maturation cascade, enabling leader peptide cleavage and full activation of nisin in the extracellular medium.
Thus, a complete *nisABCTP* module is expected to yield the highest antimicrobial efficacy.

Build

We are using these plasmid set (using backbone for *E. coli*):
a. pET-21a(+)-*nisA* (AmpR): *nisA*: BamHI/XhoI and N-terminal marker for characterization; C-terminal 6xHis tag for purification and separation;
b. pRSFDuet-1-*nisB-nisP* (KanR); *nisB*: NdeI/XhoI, *nisP*: NcoI/BamHI;
c. pACYCDuet-1-*nisC-nisT* (CmR); *nisC*: NdeI/KpnI, *nisT*: NcoI/BamHI.
This will be hosted in *E. coli* DH5α for molecular cloning and *E. coli* BL21(DE3) for plasmid expression.

Test

For our cloning and verification, we assemble the three plasmids and then verify by gel electrophoresis and DNA sequencing.
Then, we transform sequentially with tri-antibiotic selection (Amp + Kan + Cm), confirming plasmid compatibility.

To do expression and fractionation, we grew in LB + tri-antibiotics to OD600≈0.5 at 37 °C.
Then, we induced with 0.1–0.3 mM IPTG since all three plasmids need IPTG to be induced.

We looked at the antimicrobial and biofilm assays compared to *S. mutans*.
To do this, we collected cell pellet, periplasm/membrane fraction (for NisT), and culture supernatant for inhibition assays.
We will send the engineered bacteria to the culturing group for testing.

Learn

Early constructs confirmed *nis* gene compatibility.
Anticipate need for full *nisABCTP* pathway for strong supernatant activity.

##### Cycle 2 — CSP-Responsive Nisin Expression System in Beneficial Lactobacillus

Design

This project builds a synthetic circuit that integrates the *S. mutans* quorum-sensing pathway (ComD/ComE) with a nisin expression cassette on a single plasmid.

Build

CSP (from *S. mutans*) → ComD senses and autophosphorylates → phosphate transfers to ComE (ComE~P) → ComE~P activates PcomE\_like → *nisA* is expressed → Nisin inhibits *S. mutans*.

Test

In future work, we plan to experimentally verify the functionality of this CSP-responsive nisin expression plasmid. Validation will include co-culturing the engineered *Lactobacillus* strain with *Streptococcus mutans* to examine whether nisin production is specifically triggered by CSP signaling. Quantitative assays such as agar diffusion inhibition tests and qPCR for *nisA* transcription will be employed to assess antimicrobial activity and promoter responsiveness.

Due to current time and resource constraints, these functional tests will be carried out at a later stage when adequate laboratory capacity becomes available. The plasmid construction itself has been completed and verified by sequencing, and the upcoming experiments will focus on confirming its regulatory performance and potential application in in vitro oral microbial community models.

Learn

We gained valuable experience in modular cloning and promoter engineering.
We learned how to integrate two independent gene cassettes—*comDE* and PcomE\_like-*nisA*—within a single plasmid backbone while maintaining correct orientation and transcriptional insulation.
During the process, we also deepened our understanding of the *S. mutans* quorum-sensing pathway and how its signaling components can be repurposed for targeted antimicrobial responses.
Troubleshooting restriction site compatibility and optimizing RBS spacing further improved our experimental design skills and highlighted the importance of detailed sequence planning in synthetic biology.

#### Summary

Our vector work focused on reconstituting the full nisin biosynthetic pathway (*nisA/B/C/T/P*) in *E. coli* as a prototype before porting to *Lactobacillus*.
By assembling genes across three compatible plasmids (pET-21a, pRSFDuet, pACYCDuet), we ensured stepwise maturation, export, and activation of the lantibiotic.
Initial constructs confirmed *nisA* expression, but highlighted the need for supporting enzymes to achieve strong antimicrobial activity in the supernatant.
Through successive DBTL cycles, we tested inducible circuits responsive to *S. mutans*-like cues (acidification, quorum signals, damage proxies) to gate nisin production only when relevant.
This minimized leakage and balanced chassis fitness with functional expression.
Overall, we learned that multi-component expression and conditional induction are both required for stable, effective nisin production, guiding our next steps toward *Lactobacillus* integration.

## Nisin Team

Goal: validate antimicrobial activity of expressed nisin against *S. mutans*.

---

##### Cycle 1 — First Disk Diffusion with Nisin

Design

We wanted to test the efficiency of nisin's
bacterial inhibitory effect against *Streptococcus mutans*
using a simple disk diffusion assay on BHI plates.

Build

We prepared a 10 mL nisin stock solution with 900 IU/mL potency.
Using this stock solution, we made 5 working solutions with concentrations ranging from 50–150 μg/mL in increments of 25.
We soaked sterile paper disks in the working solutions for 3 hours.

Test

Five BHI agar plates were inoculated with *S. mutans*,
and immediately after inoculation a single disk was placed on each plate.
Each disk had been pre-soaked in a different concentration of nisin (ranging from 50–150 IU) for 3 hours.
Plates were then incubated for 24 hours before measuring zones of inhibition.

Learn

Little to no inhibition was detected at these concentrations.
We concluded that the nisin concentration levels used were either too low to produce a strong antimicrobial effect, or
that the biofilm formation of *S. mutans* led to our results.
Our findings prompted us to try higher concentrations in the next cycle
as well as test in biofilm & non-biofilm conditions.

##### Cycle 2 — Second Disk Diffusion with Nisin

Design

We wanted to test higher concentrations of nisin and to test
whether biofilm formation explained the last cycle's results.
We hypothesized that higher concentrations would give better measurable inhibition.

Build

We prepared a 10 mL nisin stock solution with 1000 IU/mL potency.
Using the stock solution, we created six 2 mL working solutions with concentrations ranging from 150–300 μg/mL in increments of 25.
We prepared 14 BHI and 14 BHI + 2% sucrose plates (28 total).

Test

We soaked two sterile paper disks in each solution for 3 hours.
14 plates (7 BHI & 7 BHI + 2% Sucrose) were designated as biofilm plates; they were inoculated with *S. mutans* and incubated for 12 hours before adding disks.
14 plates (7 BHI & 7 BHI + 2% Sucrose) were designated as non-biofilm plates; they were inoculated with *S. mutans* and disks were added immediately.
We applied the disks with varying concentrations of nisin (150–300 IU in increments of 25 IU) onto all plates,
then let the plates incubate for 24 hours.

Learn

Biofilm plates showed no zone of inhibition (as expected).
Non-biofilm plates showed inconsistent results: sometimes lower concentrations produced a zone of inhibition
while higher ones did not. We believed that pre-soaking disks in solution might not deliver consistent dosing
and planned to apply nisin directly to plates in the next cycle.

##### Cycle 3 — Direct Application of Nisin Solutions

Design

To improve consistency, we decided to pipette nisin solutions
directly onto the disks on seeded plates instead of pre-soaking disks.

Build

We prepared 14 BHI and 14 BHI + 2% Sucrose plates (28 total).
14 plates (7 BHI & 7 BHI + 2% Sucrose) were designated for biofilm plates and 14 plates (7 BHI & 7 BHI + 2% Sucrose)
designated for non-biofilm plates.
Upon trying to make our selected concentration range (150–300 IU) within a set volume of 20 μL for direct application onto disks,
we discovered that the amount of nisin powder required to reach these concentrations was too high for DI water solubility,
and the volumes needed (166–333 μL per plate) were impractical for disk diffusion assays.

Test

Despite the challenge, we applied the large volumes (166–333 μL) of
varying nisin concentrations directly onto the plates in hopes of seeing inhibition.
Biofilm plates were inoculated with *S. mutans* and incubated for 12 hours before adding nisin solution.
Non-biofilm plates were inoculated before adding the nisin solution immediately.

Learn

No zones of inhibition were observed under any of the conditions tested.
Upon reviewing the specifications of our commercially extracted nisin,
we discovered that it contained only 2% active nisin.
Although the product was labeled at 900 IU/mg, we concluded that the actual concentration was too low
to produce measurable inhibition in a disk diffusion assay.
This prompted us to introduce a positive control using ampicillin to confirm
that our assay setup was functioning properly
and that the absence of inhibition zones was due to the nisin preparation, not experimental error.

##### Cycle 4 — Introducing Ampicillin as a Positive Control

Design

After observing no clear inhibition from nisin in previous cycles, we needed to confirm that our disk diffusion setup itself was valid.
We introduced ampicillin, a well-established antibiotic against gram-positive bacteria as a positive control.

Build

We prepared 6 BHI + 2% sucroe plates total.
There were 3 biofilm plates and 3 non-biofilm plates. 1 biofilm and 1 non-biofilm served as our control.

Test

We added one ampicillin disk to our plate, one 3000 IU nisin disk, and one 300 IU nisin disk into each experimental plate, both biofilm and non-biofilm.
The plates were then incubated overnight.

Learn

We saw that there was clear inhibition from the positive control, validating our assay.
We will move onto doing recombinant nisin production using cation-exchange chromatography and SDS page.

#### Summary

We examined the antimicrobial activity of nisin against *S. mutans*.
Our first cycle showed little inhibition, which we analyzed and determined might be due to low concentrations.
Our second cycle showed that our control worked; however, our experimental group, the non-biofilm plates, did not behave as expected.
We redesigned the protocol for consistency and discovered our nisin was not active.
This allowed us to pivot to a different material (ampicillin) to verify the method.
Though our experimentation is still ongoing, doing each of these steps within a single cycle has helped us navigate how to find answers to our research question.

## Killswitch Team

Goal: incorporate biocontainment features to prevent uncontrolled growth.

---

##### Cycle 1 — AalR

Design

The AalR regulator was selected as the L-aspartate (L-Asp) sensor to control the safety switch of the killswitch system.
AalR is a LysR-type transcriptional regulator derived from *Acinetobacter baylyi* ADP1, where it activates genes involved in aspartate metabolism in the presence of L-Asp.
Since neither *E. coli* nor *Lactobacillus reuteri* naturally expresses AalR, the team designed an expression construct using the pUPD3 backbone with the p32 constitutive promoter to continuously express AalR.
Downstream of the coding sequence, a strong double terminator (BBa\_B0015) was included to prevent transcriptional read-through.
The system was intended so that AalR, once expressed, could respond to environmental L-Asp and activate MazE transcription when the inducer is present, ensuring survival only under safe conditions.

Build

The AalR construct was assembled as p32–RBS (BBa\_B0032) > *aalR* > BBa\_B0015 using the pUPD3 backbone, which provides chloramphenicol resistance.
The medium-strength RBS was selected to ensure adequate protein production without imposing metabolic stress.

Test

Transformed *E. coli* colonies will be selected using chloramphenicol to confirm successful construct integration.
Functional testing will involve verifying AalR expression and assessing whether L-Asp could activate its target promoter, enabling MazE expression in downstream experiments.

Learn

Through this cycle, the team confirmed that AalR expression in *E. coli* was feasible and non-toxic, setting the foundation for coupling AalR-regulated MazE expression.
They also learned that environmental L-Asp concentrations and promoter sensitivity would be key parameters for fine-tuning induction.
Future steps include testing dynamic response curves to varying L-Asp concentrations and optimizing AalR–promoter binding for predictable regulation.

##### Cycle 2 — MazF (toxin)

Design

MazF, the toxin component of the MazEF system, is an endoribonuclease that cleaves mRNA and halts protein synthesis, ultimately causing cell death.
It was placed under the strong T7 promoter in the pET-21(+) vector for precise control.
This setup leverages IPTG-induced T7 RNA polymerase expression in *E. coli BL21(DE3)*.
To minimize premature toxicity, a weaker RBS (BBa\_B0033) was selected, ensuring controlled toxin translation.
The design also anticipated future transfer to *L. reuteri*, where IPTG induction is not viable, and planned to substitute the T7 system with a constitutive promoter.

Build

The MazF construct was assembled as T7 promoter–BBa\_B0033 RBS–*mazF*–terminator in the pET-21(+) backbone (ampicillin resistance).
The backbone's built-in LacI repressor ensured that MazF remained inactive until IPTG induction.

Test

Colonies will be selected on ampicillin-containing plates.
Controlled IPTG induction experiments are planned to determine the optimal concentration range for toxin activation that would effectively kill the cells under simulated oral conditions.

Learn

This cycle revealed how promoter and RBS strength balancing is essential to avoid premature host death. The team also recognized that the dynamic range of IPTG concentration directly affects kill efficiency.
Future improvements will involve optimizing promoter strength and exploring alternate inducers or repressors compatible with *L. reuteri* physiology.

##### Cycle 3 — MazE (antitoxin)

Design

MazE, the antitoxin, neutralizes MazF by forming a protein–protein complex that inhibits its RNA-cleaving activity.
Its transcription was designed to be regulated by AalR, ensuring that MazE is expressed only when L-Asp is present, under safe conditions.
The pT7 vector (kanamycin resistance) was used for *mazE* expression because it lacks an RBS and terminator—allowing insertion of custom regulatory sequences.
The BBa\_B0034 RBS was chosen for moderate translation efficiency to maintain a balance between neutralizing MazF and preventing metabolic overload.

Build

The construct was assembled as AalR-regulated promoter – BBa\_B0034 RBS – *mazE* – terminator in the pT7 backbone.
The AalR-controlled promoter ensures context-specific expression depending on L-Asp availability.

Test

Colonies will be selected using kanamycin resistance.
Experiments will focus on determining safe and lethal L-Asp concentration thresholds: identifying ranges where MazE expression keeps *E. coli* alive and conditions where reduced L-Asp leads to MazF-mediated killing.

Learn

This cycle demonstrated how environmental sensing (via AalR) could directly modulate toxin–antitoxin balance.
The team learned that promoter strength, inducer concentration, and timing are critical for maintaining system stability.
These results guided refinements in adjusting expression ratios and potential feedback control mechanisms for more precise regulation.

##### Cycle 4 — AalR + MazEF

Design

The final design aimed to integrate all three modules into a coordinated system where L-Asp and IPTG levels jointly determine the cell's fate.
AalR activates MazE expression under safe conditions (presence of L-Asp), while IPTG induces MazF toxin expression.

Build

All three constructs—pUPD3 (AalR), pT7 (MazE), and pET-21(+) (MazF)—were co-transformed into *E. coli* BL21(DE3), leveraging their distinct antibiotic resistance markers for selection.

Test

Dual induction experiments are planned to test how IPTG and L-Asp concentrations interact.
Viability assays will measure system stability and switching accuracy under different inducer combinations.

Learn

Integration will teach the team how small variations in expression strength can disrupt the balance between toxin and antitoxin.
This insight might emphasize the need for fine-tuning promoter strength and RBS activity to prevent leaky toxicity or incomplete killing.

#### Summary

Our killswitch design integrates a toxin–antitoxin (TA) system with an L-aspartate sensor to regulate cell survival.
The MazEF TA module is used, where the MazE antitoxin neutralizes the MazF toxin.
To control this system, the L-Asp specific regulator AalR is constitutively expressed from the pUPD3 backbone, enabling MazE transcription in the presence of L-aspartate, while absence of the inducer prevents MazE expression and allows MazF-mediated killing.
MazF is expressed under a T7 promoter in pET-21(+), with its induction tunable by IPTG in *E. coli*, while MazE is carried on the pT7 plasmid under an AalR-responsive promoter.
To balance expression, RBS strengths were chosen to prevent premature toxicity while ensuring effective cell elimination when required.
The three plasmids—pUPD3 (chloramphenicol resistance), pET-21(+) (ampicillin resistance), and pT7 (kanamycin resistance)—were selected for compatibility and precise regulation of each component, providing a flexible and controllable genetic circuit for the killswitch.
The system will be tested in *E. coli* BL21(DE3) first.

###### On this page

* [Overview](#overview)
* [Culture Team](#culture)
* [Vector Design Team](#vector)
* [Nisin Team](#nisin)
* [Killswitch Team](#killswitch)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}
Our results summarize progress across four subteams—Culture, Vector Design, Nisin, and Killswitch—each contributing to the development of a living, quorum-regulated probiotic therapy.
{% endblock %}
{% block page\_content %}

## Overview

---

We advanced four research tracks toward a living, quorum-regulated probiotic therapy: **culture & biofilm characterization**, **vector design**,
**nisin antimicrobial testing**, and **killswitch safety**.
Each team iteratively refined its design to ensure selectivity, safety, and effectiveness.

* Determined 3% glucose as the optimal condition to suppress *S. mutans* growth while supporting *Lactobacillus*.
* Completed plasmid set for recombinant nisin biosynthesis (nisABCTP).
* Validated diffusion assay and identified low potency in commercial nisin.
* Designed a killswitch linking L-Asp sensing (AalR) to MazEF toxin-antitoxin regulation.

## Culture & Biofilm Characterization

---

##### Liquid Culture — Growth Kinetics

We profiled growth of *Lactobacillus*, *S. mutans*, and *E. coli* in MRS with 2%, 3%, and 4% glucose.
*S. mutans* grew slowest overall and showed the **worst growth at 3% glucose** (lowest stationary-phase OD).
*Lactobacillus* and *E. coli* were largely insensitive to sugar concentration.

* **Lactobacillus:** no clear sugar preference → not sensitive to glucose level changes.
* **S. mutans:** slowest growth; 0-24 h log, 24-48 h stationary, >48 h death; **3% glucose = most inhibitory.**
* **E. coli (control):** similar to *Lactobacillus*; no obvious preference.

![Lactobacillus growth curves in 2%, 3%, 4% glucose](https://static.igem.wiki/teams/5892/resultspage/lacto-growth-chart.webp)

![S. mutans growth curves in 2%, 3%, 4% glucose](https://static.igem.wiki/teams/5892/resultspage/s-mutans-growth-chart.webp)

![E. coli growth curves in 2%, 3%, 4% glucose](https://static.igem.wiki/teams/5892/resultspage/e-coli-growth-chart.webp)

Growth kinetics in MRS at varying glucose levels. *S. mutans* exhibits the slowest growth and the lowest stationary-phase OD at
**3% glucose**, establishing this as the optimal inhibitory condition without impacting *Lactobacillus*.

**Discussion:** Minor deviations at 3% glucose may reflect osmotic effects or metabolite accumulation; we plan to
increase biological replicates and refine sampling to improve data reliability. Notably, *S. mutans* formed a
**white precipitate early** in culture — consistent with chain-like growth and sedimentation reported in literature.

##### Biofilm Measurement & Quantification (Crystal Violet)

After 24 h, planktonic cells were removed and wells were processed with 1×PBS → 0.1% CV → dH2O → 95% ethanol.
Absorbance at 595 nm was read; media blanks were subtracted and triplicates averaged.

**Result:** Within the tested range, *S. mutans* biofilm increased with glucose concentration,
whereas *Lactobacillus* and *E. coli* were largely unchanged.

![96-well plate after crystal violet staining](https://static.igem.wiki/teams/5892/resultspage/96-well-plate.webp)

![Biofilm quantification chart (A595) across glucose concentrations](https://static.igem.wiki/teams/5892/resultspage/biofilm-measurement.webp)

Crystal violet biofilm assay: representative plate (left) and quantified absorbance (right). *S. mutans* biofilm increases with sugar,
while *Lactobacillus* and *E. coli* remain stable across 2%–4% glucose.

**Overall Outcome:** Use **3% glucose** for downstream co-culture and inhibition tests — it suppresses
*S. mutans* growth and biofilm formation while maintaining *Lactobacillus* performance.

## Vector Design

---

The Vector Design Team reconstructed the complete nisin biosynthetic operon **nisABCTP**
into three compatible plasmids and verified their compatibility through antibiotic selection
and restriction mapping. IPTG-inducible expression was designed for *E. coli* testing before
transfer into *Lactobacillus*. Quorum-sensing and acid-responsive promoters were proposed for future self-regulation.

* **pET-21a(+)-nisA** (AmpR) — AMP precursor production.
* **pRSFDuet-1-nisB-nisP** (KanR) — post-translational modification and leader peptide cleavage.
* **pACYCDuet-1-nisC-nisT** (CmR) — ring formation and peptide export.

![pET-21a(+)-nisA (AmpR) plasmid map](https://static.igem.wiki/teams/5892/resultspage/pet-21a-nisa-ampr.webp)

**pET-21a(+)-nisA (AmpR)** — encodes the nisin precursor peptide *nisA* for initial biosynthesis.

![pRSFDuet-1-nisB-nisP (KanR) plasmid map](https://static.igem.wiki/teams/5892/resultspage/prsfduet-1-nisb-nisp-kanr.webp)

**pRSFDuet-1-nisB-nisP (KanR)** — provides modification (*nisB*) and leader peptide cleavage (*nisP*).

![pACYCDuet-1-nisC-nisT (CmR) plasmid map](https://static.igem.wiki/teams/5892/resultspage/pacycduet-1-nisc-nist-cmr.webp)

**pACYCDuet-1-nisC-nisT (CmR)** — performs cyclization (*nisC*) and peptide export (*nisT*).

Combined tri-plasmid system reconstructed for nisin biosynthesis and export in *E. coli* prior to transfer into *Lactobacillus*.

## Nisin Antimicrobial Testing

---

Disk-diffusion assays against *S. mutans* were performed over multiple iterations to optimize
dosage, delivery, and controls. Commercial nisin was found to have low active content, prompting
the design of a recombinant purification and confirmation workflow using cation-exchange chromatography
and SDS-PAGE.

| Cycle | Objective | Key Finding | Next Step |
| --- | --- | --- | --- |
| 1 | Low-dose test (50-150 IU) | No measurable inhibition | Increase concentration |
| 2 | High-dose (150-300 IU); biofilm vs non-biofilm | Biofilm inhibited; inconsistent planktonic results | Switch application method |
| 3 | Direct application | Inconsistency due to solubility/purity | Add positive control |
| 4 | Positive control (ampicillin) | Clear inhibition; validated assay | Proceed to recombinant nisin production |

![Nisin assay labeled](https://static.igem.wiki/teams/5892/resultspage/results-image.avif)

**Outcome: validated assay; commercial nisin potency insufficient — proceeding with recombinant nisin production.**

## Killswitch Safety

---

The killswitch system connects survival to oral metabolite availability using the
**AalR** L-Asp sensor and the **MazEF** toxin-antitoxin pair.
Each component was cloned and tuned to balance expression while maintaining containment.

* **Cycle 1 (AalR):** *aalR* cloned under p32 in pUPD3; CmR confirmed.
* **Cycle 2 (MazF):** *mazF* under T7 promoter; IPTG range optimized for control.
* **Cycle 3 (MazE):** AalR-responsive promoter driving *mazE*; safe L-Asp range defined.
* **Cycle 4 (Integration):** Balanced toxin/antitoxin system; ready for joint testing.

![Circuit schematic](https://static.igem.wiki/teams/5892/resultspage/circuit-schematic.webp)

**Outcome: triggers self-lysis in the absence of L-Asp and maintains safety in oral environments.**

## Integrated Learning

---

* Reconstructed plasmid system supports modular AMP production and export.
* Assay pipeline validated for consistent inhibition measurement.
* Biocontainment successfully tied to oral metabolite presence for safety.
* Culture conditions optimized for competitive probiotic advantage.

## Summary of Impact

---

Combining AMP production, quorum-regulated control, biosafety, and culture optimization demonstrates a feasible path
to a self-regulated, GRAS-based probiotic that selectively inhibits *S. mutans* and restores microbial balance in the mouth.

## References

---

* Bej, A. K., Perlin, M. H., & Atlas, R. M. (1988). Model suicide vector for containment of genetically engineered microorganisms.
  *Applied and Environmental Microbiology*, **54**(10), 2472–2477.
* Mai, H. T. X., Van Hau, N., Nghia, N. H., & Thao, D. T. P. (2018).
  Expression and Purification of Nisin in *Escherichia coli*.
  *International Journal of Life Sciences Scientific Research*, eISSN 2455-1716, 1716.
* Mierau, I., & Kleerebezem, M. (2005). 10 years of the nisin-controlled gene expression system (NICE) in
  *Lactococcus lactis*. *Applied Microbiology and Biotechnology*, **68**(6), 705–717.
* Shin, J. M., Gwak, J. W., Kamarajan, P., Fenno, J. C., Rickard, A. H., & Kapila, Y. L. (2016).
  Biomedical applications of nisin. *Journal of Applied Microbiology*, **120**(6), 1449–1465.
* Wasfi, R., et al. (2018). Probiotic *Lactobacillus* sp. inhibit growth, biofilm formation and gene expression of caries-inducing
  *Streptococcus mutans*. *Journal of Cellular and Molecular Medicine*, **22**(3), 1972–1983.

###### On this page

* [Overview](#overview)
* [Culture & Biofilm](#culture)
* [Vector Design](#vector)
* [Nisin Testing](#nisin)
* [Killswitch Safety](#killswitch)
* [Integrated Learning](#integrated)
* [Summary of Impact](#impact)
* [References](#references)

{% endblock %}