iGEM 2023 — Team wist

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{% block title %}Notebook{% endblock %}
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Notebook

PARTS

# Basic Parts

## BBa\_K5006000

## BBa\_K5006011

## BBa\_K5006012

## BBa\_K5006014

## BBa\_K5006015

## BBa\_K5006016

## aiiA

## luxR

## sspB

## aiiA gRNA

## ysnE gRNA

## sspB gRNA

# Composite Parts

## BBa\_K5006010

## BBa\_K5006013

## BBa\_K5006018

## aiiA composite part

## LuxR system composite part

## Killswitch

TIMELINE

###### February, 2023

Recruit Team Member (1)
Brainstorming topics

###### March, 2023

Brainstorming topics (continue)
Basic research
First time presenting ideas with NCHU
Basic experiment training

###### April, 2023

Brainstorming topics (continue)
Basic research
Regular meeting with NCHU
Basic experiment training
Recruit Team Member (2)
iGEM Team Registration

###### May, 2023

Confirmation of topic (orchid)
Drafting the first draft of the project
Background research (solidification of research)
Regular meeting with NCHU
Basic experiment training
Recruit Team Member (2)
First Human Practice -- WIST Assembly Seminar

###### June, 2023

WIST iGEM Instagram
Targeting Dickeya fangzhongdai
Investigation (research)
Completed the first proposal

###### July, 2023

Solidifying and organizing research
Plasmid Assembly
Degron Invention
Employing killswitch
Start drafting Wiki page
Conduct 3 education events in 3 different school
Inoculation experiment
Beach cleanup collaboration with Taiwan iGEM

###### August, 2023

Growth Curve Experiment
Participated in the Taiwan-wide iGEM Seminar hosted by KCIS Linkou
Postcard Collaboration with KCIS
Visited three orchid farms:
Interview and consultation
Safety form
Writing title and abstract
Planning future experiment

###### September, 2023

Antagonism experiment
Shooting and making promotion video
Sponsor invitation
Team T-Shirt
Wiki Construction
Safety form
Check In form
Registry
Judging form
Complete story-line

###### October, 2023

Wiki Construction
Plasmid Extraction/transformation experiment
Inoculation experiment
Fundraising in WIST PTA
(Parent Teacher Assembly)
AHL Quantification Experiment
PCR Experiment
Killswitch Measurement experiment
Presentation Video

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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

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/unnamed.png)

PLASMID
 CONSTRUCTION

# Bacillus subtilis 168

We are modifying the bacterium *Bacillus subtilis* 168 in order to achieve desired features in our bacteria. This will be achieved through the design and transfer of a specialized plasmid, containing the targeted genetic material, into *Bacillus subtilis* 168. Once this genetically modified bacterium is introduced to Orchids, *Bacillus subtilis* 168 will interact with the orchids in a manner that leads to enhanced resilience against soft rot. Scroll down to navigate the elements we engineered in our plasmid.

QUORUM QUENCHING

## aiiA gene

The aiiA gene is one of the most crucial elements in our project as it is known for its ability to produce enzymes that binds with AHL molecules and degrade them.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/aiia-gif.gif)

## p43 promotor

Constitutive Promoter 43 continuously drives the expression. Therefore pivotal for ensuring the continuous expression of the aiiA gene in B. subtilis.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/p43.png)

## AHL molecule (OHHL)

OHHL (3-oxo-hexanoyl homoserine lactone is one of the AHL (acyl-homoserine lactone), and it is the dominant signal molecule responsible for the communication between Dickeya fangzhongdai in the production of plant cell wall degrading enzymes. This communication is known as quorum sensing.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/ahl-molecule.png)

## AHL

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/aiia-and-ahl-interaction.png)

The diagram depicts the expected scenario: the enzymes produced by the aiiA gene will bind to AHL molecules and subsequently break them down. This will complete the quorum quenching process.

# constantly producing AiiA is not energy efficient and may exhaust Bacillus subtilis

we need to save energy

scroll down to see how we manage to save the energy consumption and simultaneously bring more benefits to our project!

ENERGY SAVING

# what if AiiA is only produced when AHL is presented?

To conserve energy until the appropriate moment for the aiiA gene to be expressed, we've implemented an advanced degron system. This system is constructed using dCas9, CI repressor, and ssrA tag. The degron system serves the purpose of not only suppressing the expression of aiiA but also that of sspB and YsnE.

# the regulator: dCas9\_CI\_ssrA degron

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/degron-gif.gif)

To match the optimal moment for the aiiA gene to be activated, we implemented a groundbreaking degron system composed of dCas9, a CI repressor, and ssrA tag. This degron system is designed with the specific goal of suppressing gene expression until the appropriate time arrives. This degron system is also applied to other fragments that we’ve implemented on our plasmid.

## guide RNA

the guide RNA (gRNA) will help locate the degron system to the correct location (target gene).

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/guide-rna.png)

## dCas9

is a modified form of Cas9 protein that is responsible for finding target genomic DNA sequences, and is also responsible for inhibiting the expression of certain genes until the degron system is degraded

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/dcas9.png)

## CI Repressor

suppresses gene expression. This component is linked with dCas9 by the GSlinker. In our design, CI repressor works in tandem with the gRNA to guide the dCas9 to its precise destination, ensuring that the target genes remain suppressed until the appropriate moment of activation

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/ci-repressor.png)

## ssrA-tag

plays a critical role in the degradation process. Once the ssrA-tagged dCas9 and CI repressor are in place, they become recognizable by ClpXP, a protease complex. This complex is aided by sspB, a protein that can direct ClpXP to the ssrA-tag’s location (as illustrated in the image). The SspB assists the overall system by speeding up the process of recognizing the complex

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/ssra-tag.png)

the illustration of ssrA tag leading the degradation of the degron system when the SspB is presence.

McGinness, Kathleen E., Tania A. Baker, and Robert T. Sauer. "Engineering controllable protein degradation." Molecular cell 22.5 (2006): 701-707.

## target gene

the degron system we designed will lay itself on the gene we want to temporarily repress. This includes the aiiA gene, sspB and ysnE.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/target-gene.png)

additional benefits!

# ysnE is a gene that produces IAA, a plant growth promoting hormone. With the degron system, we can have IAA at expected time.

# degron trigger: LuxR and sspB

LuxR and sspB fragments are used to detect quorum sensing activities - OHHL in particular. When LuxR sensed the presence of AHL (OHHL), they combine, to triggering aiiA expression. sspB plays an essential role as it can speed up the combination of AHL with LuxR and aid ClpXP in degradation.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/luxr-sspb.png)

## luxR

luxR acts as a receptor for AHL molecules. When they are attached, they will bind to the pLux promotor, which allows sspB to continue carrying out the process.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/luxr-attarch-ahl.png)

## p23106

p23106 is the promotor that allows the constitutional expression of luxR.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/p23106.png)

## Lux promotor

the promoter, responsible for the production of sspB, will only be activated when LuxR binds with AHL.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/plux-promotor.png)

## sspB ClpXP

sspB serves as a crucial facilitator in the interaction between ClpXP and the protein associated with the ssrA-tag. In this particular stage of the degron system, dCas9 undergoes degradation, paving the way for the activation of the remaining three components to commence their respective functions.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/sspb-clpxp.png)

our idea is backed by the research done by McGiness as they proved the presence of SspB can facilitate the degrading process of ClpXP

McGinness, Kathleen E., Tania A. Baker, and Robert T. Sauer. "Engineering controllable protein degradation." Molecular cell 22.5 (2006): 701-707.

BIOSAFETY

# toxin antitoxin kill switch.

to avoid any artificial pollution to the environment, we designed a killswitch that can be operated by us to kill *Bacillus subtilis* after it completes its job. This killswitch system is inspired by previous iGEM team, iGEM17\_CCU\_Taiwan. We made improvements and modifications so that it is more compatible with our project.

# toxin and antitoxin

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/mazf.png)
![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/maze.png)

MazF induces cell death by precisely cleaving mRNA at a specific location, whereas mazE serves as an antitoxin, countering the effects of the toxic protein MazF.

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/pgrac.png)

pgrac promotor is responsible for the expression of both mazF and MazE

# how do we manipulate the killswitch?

# the killswitch is only activated when lactose is applied.

To exert additional control over this system, we've integrated a lactose inducer based on the lac operon, controlled by the constitutive promotor: pveg.

This operon allows the translation of the coding sequences only when lactose is present. In situations where lactose is lacking, both genes' translation is halted, resulting in the faster degradation of mazE compared to mazF. As a result, mazF is left unrestricted, ultimately leading to cell death. This design not only ensures the effective functioning of the kill switch but also enables activation within controlled timeframes.
![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/pveg-promoter.png)

# lac operon

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/laci.png)

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/killswitch-condition-1.png)

this diagram illustrates the condition where the killswitch is not activated

# the protein produce by LacI will block the way of mazE, mazF, therefore repressing the expression of the killswitch

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/repression-by-laci-protein.png)

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/killswitch-condition-2.png)

this diagram illustrates the condition where lactose is presented. The lactose will bind with the protein LacI produced, then leave the
operator. The killswitch is then activated.

# Our plasmid, pHT\_WIST168\_Bacillus

![](https://static.igem.wiki/teams/5006/wiki/project-page/engineerging-page/plasmid-map.png)
We use golden gate assembly technique to reconstruct the vector of modified pHT10, named as pHT100. We insert two fragments that are multifunction in terms of expressing the genes and triggering their funcitons, and can be replicate into B. subtilis.

# RESULT

Brining our ideas to life.

[Read](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D)

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## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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Result

INOCULATION

## 1.1 Inoculation of *Dickeya fangzhongdai*

This experiment is designed to evaluate different concentrations (optical density 0, 0.1, 0.3, 0.5, 0.7) of *Dickeya fangzhongdai* strain Ph3 and Ph21 have greater influence of soft rot.The impact of each strain is evaluated based on the resulting optical density (OD) values, specifically at 0, 0.1, 0.3, 0.5, and 0.7.

![](https://static.igem.wiki/teams/5006/wiki/project-page/experiment-page/inoculation-ph21.jpg)
strain Ph21

![](https://static.igem.wiki/teams/5006/wiki/project-page/experiment-page/inoculation-ph3.jpg)
strain Ph21

##### Following the experiment, our conclusion is that the *Dickeya fangzhongdai* strain Ph21 outperforms others in inducing soft rot, both in terms of infection severity and the speed at which it occurs.

## 1.2 Antagonism Testing *(Bacillus subtilis)*

The experiment is designed to evaluate the functionality/effectiveness of *Bacillus subtilis* 168 against *Dickeya fangzhongdai* without any prior modifications to *Bacillus subtilis* 168.

*Dickeya fangzhongdai*

*Bacillus subtilis 168*

## Positive

![](https://static.igem.wiki/teams/5006/wiki/project-page/experiment-page/positive-1-2-experiment.jpg)

If the experiment is positive, meaning that Bacillus subtilis has natural function for killing Dickeya fangzhongdai, their will be an apparent gap between the cross section between Bacillus subtilis and Dickeya fangzhongdai.

## Negative

![](https://static.igem.wiki/teams/5006/wiki/project-page/experiment-page/1-1-experiment-negative.jpg)

The image shows the initial stage of the plate where we cultivate Dickeya fangzhongdai Ph21 and Bacillus subtilis 168 on LB Agar plates for a duration of 12 hours.

![](https://static.igem.wiki/teams/5006/wiki/project-page/experiment-page/1-2-experiment-before.png)

## Result: Negative

The outcome indicates that *Bacillus subtilis*, on its own, lacks the ability to eliminate *Dickeya fangzhongdai*.

## 1.3 *Bacillus subtilis* 168 vs pathogen

Purpose

The experiment takes another step to to assess the performance and efficacy of *Bacillus subtilis* 168 against *Dickeya fangzhongdai* Ph21 without any prior alterations. In contrast to the previous experiment, this one is conducted directly on orchid leaves.

![](https://static.igem.wiki/teams/5006/wiki/1-3-experiment.jpg)

Result

The experiment takes another step to to assess the performance and efficacy of Bacillus subtilis 168 against *Dickeya fangzhongdai Ph21* without any prior alterations. In contrast to the previous experiment, this one is conducted directly on orchid leaves.

## 1.4 Re-isolated

The purpose of this experiment is to assess Bacillus subtilis's capability for endosymbiosis with orchid leaves, which serves as a rationale for our selection of *Bacillus subtilis* as the host for the plasmid.

Method

In order to assess the survival of *Bacillus subtilis* 168, two different application methods were employed: immersion of leaf samples and surface spraying of the bacterial suspension onto the leaves. After a 24-hour incubation period, the study aimed to determine the persistence of *Bacillus subtilis* 168 under each application method.

On Day 2, bacterial enumeration was done by applying *Bacillus subtilis* 168 (B+) to three plates and distilled water (B-) to one plate. Leaves were treated accordingly with B+ or B-, and bacterial samples from leaf squares were evenly spread on three LB Agar plates per treatment. On Day 3, bacterial colony counting was carried out.

Result
![](https://static.igem.wiki/teams/5006/wiki/reisolation-1.jpg)
![](https://static.igem.wiki/teams/5006/wiki/reisolation-2.jpg)

## Control (CK)

![](https://static.igem.wiki/teams/5006/wiki/control.jpg)

##### Implication

The results are highly successful, as they clearly illustrate that *Bacillus subtilis* possesses the capacity to establish endosymbiosis with orchid leaves, rather than perishing.

PLASMID CONSTRUCTION

![](https://static.igem.wiki/teams/5006/wiki/screenshot-2023-10-12-at-8-43-39-am.png)

This figure shows that the vector we extract has the correct backbone size of about 7900bp

![](https://static.igem.wiki/teams/5006/wiki/screenshot-2023-10-12-at-8-43-33-am.png)

The first well is the vector that has not been digested.
The second well is the vector that has been digested by one-cutting side restriction enzyme.
The third well is the vector that has been digested by two-cutting side restriction enzymes.
The fourth well is the result of primer checking the ampicillin by using PCR.

![](https://static.igem.wiki/teams/5006/wiki/.png)

Backbone of the plasmid Firstly, we construct the plasmid with golden gate technique, then we transform the plasmid into Bacillus subtilis W168 to express the gene and stored in E. coli DH5α.
We inserted our desired gene fragments into our plasmid through the restriction sites XhoI and Aflii.

Insert aiiA + Kill Switch Next, we insert the aiiA gene and MazE and MazF toxin (Kill Switch). After the Kill Switch is well-functioned, we use different concentrations of lactone to check the less amount that is needed for Kill Switch to function.

![](https://static.igem.wiki/teams/5006/wiki/16531035.png)

Insert LuxR + sspB and Degron System Lastly, we insert the LuxR with sspB and Degron system ( include gRNAs, dCas9, CI repressor and ssrA DAS+4 tag ) into the pWS4 plasmid, and we named this plasmid as pWS4-1. It has been completely constructed and is looking forward to verification.

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