iGEM 2023 — Team gems-taiwan

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NOTEBOOK

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Notebook

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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ENGINEERING

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Overview
Cloning
Csg Contruct
MetJ Gene Knockout
MetJ Construct
Future Improvements
Biosafety Contruct
Future Improvements
References

Overview

Our project aims to engineer a coral-localized *E. coli* to produce and regulate biofilm, specifically through the *mazE/F* genes,
the *csg* (or curli) operon (for biofilm production), and the *metJ* regulon(for biofilm regulation) respectively. Throughout our
experimentation, we followed an iterative engineering cycle, the steps include: Design, Build, Test, and Learn. This cycle is
especially apparent in our cloning experiments; in which we designed and assembled our desired plasmids.

Cloning

Csg Contruct

The Csg construct was designed in benchling and the sequence was provided to IDT for synthesis. Due to its prolonged length,
the construct was synthesized in two fragments with the separation occurring at 1028 bp, making fragment 1 (csg 1) 1027 base
pairs and fragment 2 (csg 2) 2103 base pairs. We used the backbone pSB1C3 as its vector due to its high copy number and
assembled the fragments using biobrick assembly.

First, we performed amplification PCR to amplify the fragments synthesized through IDT. We struggled to amplify the product,
and even when we obtained the product, they were all of low concentration and high impurity.

To optimize our PCR condition, we tried different DNA polymerases, such as Phusion, Q5, and Primestar polymerases. We also
altered the PCR cycles. Since we hypothesized that our construct might have higher GC content that might result in secondary
structures formed during annealing, we added DMSO, which is commonly used to reduce secondary structures. DMSO can also help
relax supercoiled plasmids by increasing single-stranded regions, which allows enhanced topoisomerase I activity.

Despite such changes, our colony PCR results continued to be negative, showing band lengths much lower than the expected 5177bp (Figure 1).

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-1.png)

Figure 1. Gel electrophoresis results for Csg construct colony PCR.

Because of the repeated failures in running amplification PCR and biobrick assembly after altering our experimental conditions
(PCR conditions), we decided to redesign our primers. Our main issue with our primers is that our overlapping primers had too
short of a homologous region (20bp), which made our primers anneal poorly. We redesigned our primers by increasing the homologous
regions of our overlapping primers to 30 bp and decreasing the GC content in the primers to further prevent non-specific binding
and formation of secondary structures during amplification PCR.

We then successfully performed biobrick assembly to ligate the 2 fragments and the vector (pSB1C3), which we then transformed into *E. coli* DH5α. Colony PCR was performed to confirm the fragment size (Figure 3).

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-2.png)

Figure 2. 1: csg fragment 1 2: csg fragment 2

| Fragments | Concentration (ng/ul) |
| --- | --- |
| Csg 1 | 45.942 |
| Csg 2 | 32.642 |

Table 1. Table of all concentrations for csg

We confirmed that we successfully obtained pSB1C3-csg after receiving the results from sequencing.

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-3.png)

Figure 3. Gel Result for csg colony PCR

Csg Gene Knockout

We attempted to knock out the *csg* genes from the *E. coli* genome using homologous recombination to increase the accuracy
of our assays involving biofilm. Because **E. coli** naturally produces biofilm, when we express our Csg construct in *E. coli*,
we cannot decipher whether the biofilm is produced naturally by *E. coli* or our Csg construct, nor quantify the potency in
producing biofilm by our synthetic Csg construct.

To test our Csg construct that produces biofilm, before we transform our Csg construct into **E. coli**, we need to knock out
original biofilm genes from the *E. coli* genome to increase the accuracy of our testing.

We attempted to knock out the *csg* operon, responsible for producing and secreting biofilm, from the *E. coli* genome using
homologous recombination [[1]](https://www.pnas.org/doi/full/10.1073/pnas.120163297).

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-4.png)

Figure 4. Homologous recombination diagram

The homologous recombination process involves:

1. Template plasmids (pKD13) carrying Kanamycin-resistance genes flanked by FRT (FLP recognition target) sites.
2. We generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the *csg* operon.
3. By electroporating the PCR product into *E. coli* strain MG1655 containing the helper plasmid pKD46 encoding the Red recombinase, the KmR gene swaps with the *csg* operon on the *E. coli* genome.
4. KmR was then eliminated by using the helper plasmid pCP20 encoding the FLP recombinase.

We obtained the materials used for KO homologous recombination from Dr. Masayuki Hashimoto of the Institute of Molecular Medicine at the National Cheng Kung University:

1. *E. coli* strain MG1655 contains pKD46 plasmid that aids in replacing the *csg* operon with our amplified Kanamycin-resistant gene.
2. pKD20 plasmid encoding the FLP recombinase to eliminate the Kanamycin-resistant gene.

Due to time constraints from delays to our cloning process, we did not complete the designed gene knockout process.

MetJ Construct

The MetJ construct was designed in benchling and the sequence was provided to IDT for synthesis. Due to its prolonged length, the
construct was synthesized in 5 fragments with the separations occurring at 1703 bp, 2441 bp, 3540 bp, 4301 bp, and 5054 bp.

We used pET28a as our plasmid because of its low copy number, to prevent an over-expressing of MetJ proteins that may plummet
biofilm production, too quickly and also because we plan on co-transforming both the plasmids for the Csg construct and MetJ
construct, so we needed a plasmid with a different antibiotic resistance gene from pSB1C3 for screening.

First, we performed amplification PCR to amplify the fragments synthesized through IDT. We struggled to amplify the product,
and even when we obtained the product, they were all of low concentration and high impurity.

To optimize our PCR condition, we tried different DNA polymerases, such as Phusion, Q5, and Primestar polymerases. We also altered
the PCR cycles. Since we hypothesized that our construct might have higher GC content that might result in secondary structures
formed during annealing, we added DMSO, which is commonly used to reduce secondary structures. DMSO can also help relax supercoiled
plasmids by increasing single-stranded regions, which allows enhanced topoisomerase I activity.

Despite such changes, we continued to obtain PCR products of low concentration and purity. We attempted to perform Gibson assembly to
assemble the fragments into the vector pET28a, but when we ran Colony PCR to confirm the fragment size, we did not see anything on the
gel. We hypothesized that the inability to obtain product from Gibson assembly is due to the high amount of fragments that may decrease
the efficiency of Gibson assembly, so we ran overlapping PCR to connect our fragments in pairs (1, 2+3, 4+5) before performing Gibson
Assembly. We failed to obtain any product (Figure 5), even after changing our PCR conditions: different # of PCR cycles, different
polymerase, and different amounts of DNA product.

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-5.png)

Figure 5. Overlapping PCR results with no product

Because of the repeated failures in running amplification PCR and overlapping PCR, even after altering our experimental conditions (PCR
conditions), we decided to redesign our primers. Our main issue with our primers is that our overlapping primers had too short of a
homologous region (20bp), which made our primers anneal poorly. We redesigned our primers by increasing the homologous regions of our
overlapping primers to 30 bp and decreasing the GC content in the primers to further prevent non-specific binding and formation of
secondary structures during amplification PCR (Table 2).

After our new primers arrived in late August, we successfully re-amplified our fragments (Figure 6). Our amplification PCR products’
concentrations improved significantly (Table 2).

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-6.png)

Figure 6. 1, 2, 3, 4, and 5 are the 5 different fragments after amplification.

| Fragments | Concentration (ng/ul) |
| --- | --- |
| MetJ 1 | 54.492 |
| MetJ 2 | 153 |
| MetJ 3 | 66.272 |
| MetJ 4 | 36.632 |
| MetJ 5 | 62.9 |

Table 2. Table of all concentrations for MetJ

We then performed Gibson assembly to ligate the 5 fragments and the vector (pET21A), which we then transformed into **E. coli** DH5α.
We failed to obtain the correct size during colony PCR to confirm the fragment size.

Due to time concerns, we decided to clone the fragment that contains the *metJ* gene (fragment 3) synthesized by IDT and into the pJET vector,
and transform it into *E. coli* strain DH5α. This allows us to at least test if the MetJ protein can successfully repress biofilm production.
We successfully cloned the MetJ fragment 3 into the pJET vector, and performed colony PCR to confirm the fragment size (Figure 7). The
sequencing results showed that our construct was successfully cloned.

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-7.png)

Figure 7. Gel pic of failed MetJ colony PCR

Future Improvements

To increase the efficiency of amplification PCR and Gibson assembly, we will review our fragments to possibly lower the GC content at the ends.

Biosafety Construct

The Biosafety construct was designed in benchling and the sequence was provided to IDT for synthesis. Due to its prolonged length, the
construct contains 4 fragments with the separations occurring at 775 bp, 2130 bp, and 2802 bp. We used the backbone pET21a as a vector.
We attempted to assemble the fragments using Gibson assembly. We were able to successfully amplify all but the third fragment (Figure 8),
even after redesigning our primers. Due to time concerns, we will further complete this construct in the future.

![](https://static.igem.wiki/teams/4852/wiki/engineering/figure-8.png)

Figure 8. Amplification for Biosafety. 1, 2, 3, and 4 are the 4 different fragments after amplification PCR.

Future Improvements

To improve cloning efficiency for future cloning success, we will redesign our fragments to lower the GC content at the ends, increasing the efficiency of amplification PCR and Gibson assembly.

References

[1] Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. *Proceedings of the National Academy of Sciences of the United States of America*, 97(12), 6640–6645.
<https://doi.org/10.1073/pnas.120163297>

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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EXPERIMENTS

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Overview
Cloning
Csg Construct
MetJ Contruct
Biosafety Construct
Functional Assays
Biofilm Production
Assay
MetJ Protein Assay
Biosafety Assay
MetJ Assay
Implementation Testing
References

Experimental Overview

To engineer bacteria with the capacity to produce and regulate biofilm, we utilized
the functions of the curli operon and the metJ regulon, respectively, to create two
constructs. Additionally, to address the risk of unwanted contamination by our
engineered bacteria, we have also designed an toxin-antitoxin system (inspired by the
[Johns Hopkins 2021](https://2021.igem.org/Team%3AHopkins) team).

To assess the functionality of these engineered bacteria and the impact of our
biofilm-related constructs, we have devised three key assays to evaluate the functionality
of our engineered bacteria: a biofilm production assay, a MetJ assay, and a biosafety assay.

Cloning

We performed multiple cycles to assemble our three desired plasmids, we will be
using the pSB1C3 for our biofilm-producing construct (Csg construct), pET28a for
the biofilm-regulating construct (MetJ construct), and the plasmid pET21a for Biosafety
construct.

Csg Construct[[1]](https://doi.org/10.1146/annurev.micro.60.080805.142106)

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-1-a.png)

Figure 1.a A diagram of our Csg construct

Our Csg construct is responsible for producing and regulating biofilm (together with
the MetJ construct). We designed our construct on benchling, added biobrick prefix and
suffix, and provided its sequence to IDT for synthesis; the synthesized product was
amplified via PCR and ligated with pSB1C3 via biobrick assembly. The assembled plasmid
was transformed into *E. coli* DH5Alpha, miniprepped, and sequenced for confirmation.

For cloning results, please visit the [engineering page](https://2023.igem.wiki/gems-taiwan/engineering)

During the design process, we conducted several literature reviews and found that *csg* genes
are responsible for biofilm production in *E. coli*, which are commonly used and studied, and
known for its highly resilient and adaptive qualities — which are crucial to survival in ocean
environments. Its **non-pathogenic qualities** also are key to our selection. These genes encode
for the major component of curli, which is a proteinaceous fiber that forms the structural
backbone of the biofilm. By engineering the *csg* genes into SAR11 bacteria, it allows SAR11
to produce biofilm and facilitate its **integration** into the **coral microbiome**. Research was
done to challenge certain strains of viruses containing *csgF* and *csgG*, cell lysis mechanisms
that would destroy the cell membrane, in SAR11. However, it was proved that the virus didn’t
infect any of the SAR11, confirming that Csg doesn’t affect SAR11
[[2]](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9004378//). Additionally,
it is also proven that SAR11 can allow the production of biofilm. Research showed that SAR11
was found in the RO biofilm communities, suggesting SAR11 cells are present within the biofilm
structure, confirming SAR11's capacity to facilitate the production of biofilm, along with the
production of probiotics [[3]](https://www.sciencedirect.com/science/article/pii/S2772421221000015).

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure2.gif)

Figure 2. Full animation of the Csg construct

The *csg* operon (curli operon)[[1]](https://www.annualreviews.org/doi/10.1146/annurev.micro.60.080805.142106)

In order to achieve this, we synthesized the *csg* genes found in ***E. coli* MG1655** and inserted it
into our Biofilm construct. We have only selected parts of the *csg* genes, which include
*csgB, csgA, csgC, csgE, csgF* and *csgG*. This selection of *csg* genes from *E. coli* MG1655 was based
on their known role in curli fiber expression, secretion, and biofilm [[4]](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1428138/).

The *csgB* gene initiates the process of curli fiber assembly by acting as a nucleator for the
polymerization of CsgA, the major curli subunit. Meanwhile, CsgC protein acts as a chaperone
that prevents the premature polymerization for CsgA. The *csgE* and *csgF* genes interact with the
*csgG* gene to increase the **efficiency** of curli assembly.

T7 promoter

By using **T7 promoter**, we can achieve high levels of expression of *csg*, which is responsible
for curli fiber production and biofilm formation in our SAR11 bacteria.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-3-animation-of-t7-promoter-in-csg-biofilm-construct-1.gif)

Figure 3. Animation of T7 promoter in Csg construct

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-4-animation-of-metbox-operator-in-csg-biofilm-construct-2.gif)

Figure 4. Animation of met operator in Csg construct

MetJ Construct

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-1-b.png)

Figure 1.b A diagram of our MetJ construct

Our MetJ construct is responsible for regulating biofilm. The construct sequence was designed
using benchling and then sent to IDT for synthesis. After receiving our synthesized construct,
we amplified and added complementary basepairs to pET28a and performed Gibson assembly. This
assembled plasmid was subsequently transformed into *E. coli* DH5Alpha cells, followed by miniprep
purification and sequencing analysis to confirm the accuracy of the construct's sequence.

For cloning results, please visit the [engineering page](https://2023.igem.wiki/gems-taiwan/engineering)

Met Operator

We also employed the regulatory protein MetJ to suppress biofilm production to mitigate the
suffocation of corals from the overgrowth of biofilm, and to aid in that process we have
integrated a met operator into our construct. By binding to the met operator, MetJ
effectively reduces the synthesis of biofilm.

The Met Regulon

Due to initially rapid production of biofilm required for the attachment of our engineered
bacteria, the intended probiotic, and the coral mucosal layer, we decided to implement T7
promoter, which acts as a strong constitutive promoter for the production of biofilm.
However, this opens the door to a variety of problems.

The most glaring of these problems is the possibility of disease. Biofilm acts as an ideal
environment for not only the bacteria that we intend to use as part of our solution, but also
to various pathogens that can develop on the biofilm. The accumulation of the pathogenic bacteria
on the biofilm increases rates of coral disease [[5]](https://www.sciencedirect.com/science/article/abs/pii/S004565352030758X).
The most outstanding example of this would be the possibility of it serving as a reservoir for
pathogens that causes the Stony Coral Tissue Loss Disease [[6]](https://www.frontiersin.org/articles/10.3389/fmars.2022.1009407/full).

Another problem that we face with biofilm is the blocking of sunlight to the coral. Corals are
dependent on a species of algae called zooxanthellae for the production of their food through
photosynthesis. In some species, up to 90% of their consumption could be dependent on zooxanthellae
[[7]](https://oceanservice.noaa.gov/education/tutorial_corals/coral02_zooxanthellae.html).
Consequently, the blocking on sunlight toward coral from biofilm may affect the coral’s health.

To address these issues, we utilized the MetJ regulatory protein in a quorum sensing mechanism,
a regulatory protein that represses the production of biofilm by attaching to the met operator.
As the bacterial population increases over time, so does the concentration of the autoinducer (AHL).
Autoinducers are signaling molecules that are produced in response to changes in cell-population density.
As the density of quorum sensing bacterial cells increases so does the concentration of the autoinducer
[[8]](https://www.science.org/doi/abs/10.1126/science.280.5361.295).
Under conditions of low population density, there will be a low concentration of AHL, resulting
in the low production of MetJ proteins. Due to the low binding affinity of MetJ, the production of
biofilm under these conditions will be high. Under high population density, the high AHL concentration
will induce the production of MetJ, which will then decrease biofilm production, allowing the effective
control of the gene expression responsible for biofilm formation.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure5.gif)

Figure 5. Full animation of MetJ construct

CinR-CinI-CinS & PraR [[9]](https://onlinelibrary.wiley.com/doi/10.1111/mmi.12670)

The CinR-CinI-CinS system is regulated through quorum sensing, which leads to the production of the MetJ
regulatory protein. The main function of CinR is to control **quorum sensing** by initiating the expression
of the cinIS operon. Meanwhile, the primary role of CinI is to synthesize **AHL**, while CinS works by inactivating
PraR, a repressor molecule, through the formation of a **1:1 dead-end complex**.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-6.gif)

Figure 6. Animation of PraR and CinS forming a 1:1 dead-end complex

AHL Synthesis [[10 ,](https://journals.asm.org/doi/10.1128/jb.01650-08)
[11]](https://www.academia.edu/25167010/Co_ordination_of_quorum_sensing_regulation_in_Rhizobium_leguminosarum_by_induction_of_an_anti_repressor)

The PraR repressor inhibits the activity of the praiI promoter, resulting in decreased production of MetJ
as a consequence of reduced RaiI and RaiR levels. **AHL synthesis** is primarily carried out by RaiI, while RaiR
functions as an **AHL receptor** that allows bacteria to respond to changes in population density. This response
includes increased expression of CinI induced by AHL, resulting in **higher levels of CinS expression**, ultimately
leading to **increased MetJ expression**. Furthermore, AHL can also be synthesized through the activity of the
CinR-CinI-CinS system.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-7-compressed.gif)

Figure 7. Animation of how AHL affects the relationship of CinR and CinI

CinS Expression Enhancement [[12]](https://www.mdpi.com/2072-6651/15/6/380)

Even though CinI and CinS proteins can be produced independently, the production of CinS is still relatively
low. Our team's objective is to avoid excess biofilm production, so we utilized the capability of the CinI
protein to increase the production of CinS. This enhances the binding of CinS to PraR, thereby upregulating
the *raiR* and *raiR* genes.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-8-compressed.gif)

Figure 8. Animation of relationship between CinI and CinS

Biosafety Construct

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-1-c.png)

Figure 1.c A diagram of our Biosafety construct

Our Biosafety construct is designed to localize in coral. The sequence of the construct was created by
[Johns Hopkins 2021 iGEM Team](https://2021.igem.org/Team%3AHopkins) and then sent to IDT for synthesis. After receiving the synthesized construct,
amplified and added complementary basepairs to pET28a and performed gibson assembly. This resulting plasmid
was then introduced into *E. coli* DH5Alpha cells, followed by purification using miniprep technique and sequencing
analysis to validate the correctness of the construct's sequence.

For cloning results, please visit the [engineering page](https://2023.igem.wiki/gems-taiwan/engineering)

The potential risks of GMOs in the natural environment and their impact on marine ecosystems have
prompted our team to draw inspiration from the biosafety mechanism developed by iGEM John Hopkins
2021 Team [[13]](https://2021.igem.org/Team%3AHopkins), which
utilizes **quorum sensing**. This mechanism enables us to regulate our bacteria based on **population
density. Antitoxins** are constantly produced in the bacteria in small amounts. When the bacterial
population reaches a high density, there is an increase in the concentration of autoinducers, specifically
**AHL molecules**. This triggers the repression of toxins by our bacteria to allow the bacteria to survive;
while the **anti-toxins(MazE)** will neutralize any remaining **toxins(MazF)**. MazF functions as an mRNA interferase,
and its activation under conditions of stress triggers a sequence-specific cleavage of mRNA molecules,
resulting in a cessation of cellular growth. In the absence of stress, MazF is deactivated by forming
a complex with its corresponding antitoxin, MazE.

Conversely, when the population density decreases due to factors like ocean currents causing bacteria
dispersion from corals, minimal or no AHL molecules are received, **toxins** are being produced inside
the bacteria at high amounts, overwhelming the amount of **anti-toxins** produced, leading to slow
self-destruction overtime. This mechanism acts as a biosafety killswitch, effectively preventing
unintended proliferation in other environments. Our project has undergone thorough consultation with
renowned experts in the field, including Dr. Tang Sen-Lin and Professor David Bourne who have given
their approval for its non-disruptive impact on the ecosystem. For more information, visit our
[Integrated Human Practice](https://2023.igem.wiki/gems-taiwan/human-practices) page.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-9-full-animation-of-biosafety-construct.gif)

Figure 9. Full animation of Biosafety construct

EsaI-EsaR-EsaBox [[14]](https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2958.2002.02987.x)

The EsaI-EsaR-EsaBox system functions as a regulatory mechanism for the production of **anti-toxins**
and **toxins** in bacteria. Previous studies
have indicated that EsaR acts as a repressor by binding
to the EsaBox region. Previous studies [[15]](https://www.pnas.org/doi/full/10.1073/pnas.95.13.7687) have demonstrated that upon binding to EsaR in a 1:1 ratio,
inducible molecules exhibit the ability to downregulate the production of our toxin, MazF. This is
achieved through an upregulation in the synthesis of TetR protein. The key component involved in this
process is the **cognate transcriptional regulator** encoded by EsaR, which **responds to AHL signals**.
Additionally, EsaI encodes an **AHL (N-acyl homoserine lactone) signal synthase** responsible for
**synthesizing AHL molecules (EsaI)**.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-10-animation-of-esai-esar-esabox-system.gif)

Figure 10. Animation of EsaI-EsaR-EsaBox system

TetR & MazF Interaction

As mentioned previously, it has been observed that the TetR protein serves as a repressor to the
tetR promoter (R0040). This interaction results in a decrease in MazF toxin production. The interaction
between the TetR repressor and the MazF toxin is crucial for regulating toxin levels.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-11-animation-of-tetr-mazf-interaction.gif)

Figure 11. Animation of TetR & MazF interaction

MazE & MazF Interaction

As specified, MazF functions as the toxin that prevents any potential environmental harm caused by our
engineered bacteria. **MazE** plays a crucial role as an **antitoxin**, maintaining a balanced and stable **toxin-antitoxin
system** in **high bacterial population density**. The interaction between MazE and MazF is vital for maintaining
a balanced toxin-antitoxin system and preventing potential environmental harm caused by the engineered
bacteria. The MazE/MazF toxin-antitoxin system has been widely used in many different organisms [[12]](https://www.mdpi.com/2072-6651/15/6/380).
Our biosafety system depends on regulation of the bacterial population.

![](https://static.igem.wiki/teams/4852/wiki/biobricks/figure-12-animation-of-maze-mazf-interaction-1.gif)

Figure 12. Animation of MazE & MazF interaction

J23113 & TetR Promoter

We opted for the **J23113 promoter** as a suitable choice to control the production of MazE and EsaI proteins,
primarily due to its characteristic as a **"moderate-to-weak promoter"**. This selection ensures that the
antitoxin is produced in limited quantities, effectively counteracting some of the MazF toxins. As a result,
it prevents immediate bacterial death under any circumstances. In a similar fashion, we selected the **tetR promoter(R0040)**
to regulate MazF and EsaR production based on its **repressible nature** and **moderate to strong strength**.
This specific promoter's function can be repressed by TetR protein, providing an effective mechanism for
regulating downstream gene expression.

For cloning results, please visit the [engineering page](https://2023.igem.wiki/gems-taiwan/engineering)

Functional Assays

Goal

The goal of our functional assays is to:

1. Prove that our Biofilm construct produces biofilm.
2. Prove that our MetJ construct can regulate biofilm growth rate.
3. Prove that our Biosafety construct kills the bacteria beyond a certain concentration.

For functional assay results, please visit the [proof of concept](https://2023.igem.wiki/gems-taiwan/proofofconcept) page

Biofilm Production Assay

We will culture *E. coli* DH5α containing the Csg construct and *E. coli* DH5α and compare their biofilm
growth using an ELISA reader and crystal violet staining to ensure that the Csg construct does produce biofilm.

MetJ Protein Assay

Because we were not able to finish cloning our MetJ construct, we decided to transform the fragment that contains
the *metJ gene* (MetJ fragment 3) into *E. coli* DH5α along with our Csg construct. This allows us to test whether
expression MetJ proteins can successfully repress biofilm production. In this assay, we plan on growing multiple
cultures of engineered *E. coli* containing MetJ fragment 3 and Csg construct, and a control group (only Csg construct),
and measuring their bacteria population and amount of biofilm through ELISA reader with different staining methods.
This allows us to see whether the MetJ regulatory protein can successfully repress biofilm gene expression. We expect
to see a significant difference in biofilm growth in the cultures containing only the Csg construct and the cultures
containing the *metJ gene*.

For functional assay results, please visit the [proof of concept](https://2023.igem.wiki/gems-taiwan/proofofconcept) page

Biosafety Assay [[14]](https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2958.2002.02987.x)

We want to demonstrate that our engineered *E. coli* bacteria die off at low culture concentrations and survive at
high concentrations. To do this, we will measure the growth of *E. coli* containing our biosafety plasmid across various
liquid culture concentrations. This not only allows us to perceive the effectiveness of our mechanism but also find whether
the concentration in which the bacteria population starts to die out is too high or too low, which will be used to assist
future optimizations of our project.

MetJ Assay

We aim to demonstrate that individual bacteria biofilm growth rates decrease at higher cell densities. To achieve this,
we will cultivate engineered *E. coli* cultures (with MetJ and Biofilm plasmids) for varying durations. We will measure
bacterial population and biofilm levels using an ELISA reader with different staining methods. This will enable us to
track changes in bacterial population and biofilm production over time, helping us to calculate the individual bacteria
biofilm production rate. We anticipate observing a decline in individual bacteria biofilm production rates as the bacterial
population increases.

For functional assay results, please visit the [proof of concept](https://2023.igem.wiki/gems-taiwan/proofofconcept) page

Implementation Testing

After we prove that our constructs work through our functional assays, we still need further testing before implementing
our mechanism in real life. To ensure that our mechanism functions as intended in the ocean, we will re-run our functional
assays in ocean water conditions. To ensure that our mixture of coral feed spray attaches to the mucus layer of the coral
and that our probiotics are properly absorbed by the corals, we will test our mechanism on corals under a controlled setting.

References

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[9] Frederix, M., Edwards, A., Swiderska, A., Stanger, A., Karunakaran, R., Williams, A., Abbruscato, P., Sanchez-Contreras, M., Poole, P. S., &
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[10] Edwards, A., Frederix, M., Wisniewski-Dyé, F., Jones, J., Zorreguieta, A., & Downie, J. A. (2009, March 6). *The cin and rai Quorum-Sensing Regulatory Systems
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