iGEM 2023 — Team zju-china

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Mar. 2023

March 1st

March 4th

March 8th

March 18th

March 29th

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

Apr. 2023

April 17th

April 20th

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

May 2023

May 3rd

May 17th

May 24th

May 27th

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

June 2023

June 3rd

June 7th

June 14th

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

July 2023

July 7th

July 12th

July 15th

July 23rd

July 26th

July 29th

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

Aug. 2023

August 9th

August 12th

August 16th

August 23rd

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

Sept. 2023

September 6th

September 16th

September 20th

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

Oct. & later 2023

# March 2023

## March 1st

### Agenda

(1) Team members' safety training

(2) Introduction of supervising teachers

### Notes

**from Professor Ming Chen**

- **Respect:** mutual respect among team members.

- **Responsibility:** Emphasize the importance of taking responsibility for our actions.

- **Risk:** Foster a culture of innovation and encourage taking calculated risks.

- **Resilience:** Stress the need for strong resilience and the ability to bounce back from setbacks.

**from Professor Fan Yang**

Encourage us not to be burdened by past achievements but to boldly pursue innovation.

## March 4th

### Agenda

(1) Explanation of iGEM scoring criteria

(2) Sharing outstanding iGEM projects from previous years

(3) Sharing of previous projects by ZJU-China team

## March 8th

### Agenda

Conduct a brainstorming session based on the specific track

### Notes

**Conservation - Shiyi Lin**

**Track ideas:**

- Plant disease monitoring

- Plant virus defense

- Plant-based recombinant vaccines

- Plant communication

- Plant microplastics

- Introduction of ethnobotany

- Endangered species detection

- Livestock disease treatment

- Antibiotic use in livestock farming

- Livestock vaccines

- Livestock mosquito control

**Fundational Advance - Zhenning Yang**

**Track ideas:**

- Modular Receptor Platform Development

- Orthogonal optogenetic systems

- Protein targeting for synthetic biology research

- Artificial organelles

- Optimization of chassis organisms or development of new chassis organisms

**Other Inspiration:**

- Bioproduction of melanin

- Weight loss interventions

**Biomanufacture - Wenqing Ding**

**Track ideas:**

- Fire-resistant wall coating

- Toilet cleaner

- Bacterial pets

- Non-damaging hair dye/permanent wave solution

- Safety airbags

**Other Inspiration:**

- Microbial production of meat substitutes

- Emergency biological drinking water

- E. coli modification and engineering

- Control of miRNA entry into exosomes through adapter modification RNA live cell in situ staining

- Drug addiction treatment (neurology)

**Diagnostic - Linghao Xia**

**Track ideas:**

- Vaginal infection test

- Trinucleotide repeat biosensor

- Visual bacterial biosensor

- RNA m6A sequencing

- Contrast agent

- Complementary peptides

- RNA sensor

**Environment - Junjie Li**

**Track ideas:**

- Pine wood nematode treatment

- Red fire ant pheromone

- Activated carbon and biological removal of methyl perchlorate

- Coral reef restoration

- Degradation of polycyclic aromatic hydrocarbons in asphalt roads

**Therapeutics - Mingxuan Zhang**

**Track ideas:**

- Brief overview of the RASER (Abnormal Signal Rewiring Effector Release) system and improvement directions

- Bacterial tryptophan reduction for immune suppression mitigation

- Orthogonal IL-2-controlled signal response

- Tooth decay treatment

**Climate Crisis - Hanzhang Li**

**Track ideas:**

- Main focus on Microbial Fuel Cells (MFC) for energy-saving and emission reduction

- Artificial cement

- Plastic recycling - transformation of a specific item

## March 18th

### Agenda

Conduct a brainstorming session based on the specific track

### Notes

**Shiyi Lin**

- Brucellosis prevention and control

- Microplastic enrichment

- Bacterial imaging

- Bacterial neural networks

- E.coli with sound production function

- E.coli with hearing ability

- Bacterial long-distance signal transmission (quantum entanglement)

- Biopolymer films & bacteria color imaging & scroll repair

- Bacterial high-intensity thin films

**Junjie Li**

- Bacterial warfare/gene transfer

- Hangover cure

- Mosquito control

- Physical contact signaling

- Magnetic field-controlled engineering bacteriopharmaceutical delivery

- Antiviral bio-coatings

- Air purification wall coatings

- pH-regulating bacteria

**Linghao Xia**

- Yeast artificial organelles

- Modularization of photosensitive receptors

- Mirror information storage, conversion of chiral D to L

- Phase separation mechanism

**Mingxuan Zhang**

- RASER for the treatment of EGFR mutations

- Tumor hyperthermi

- Adenovirus treatment for hair loss

- Treatment of land desertification & salinization

- Tumor vaccines

**Wenqing Ding**

- Adenovirus treatment for hair loss

- Plant-derived animal antibodies

- Treatment of psoriasis

- Plant injection of animal antibodies

- Reprogramming of cancer cells

- Meningitis treatment

- Novel intestinal chip

- Drug delivery through the blood-brain barrier

- Optogenetics cage

- Endoplasmic reticulum stress

**Qingyao Yan**

- Tumor bomb

- Alcohol detoxification + alcoholic hepatitis

- Gut microbiota and thrombosis

- Artificial photoperiod in plants

- DNA barcodes to track food poisoning

- Electroactive microorganisms

- Smell robo

- DNA origami

- Bacterial sound collector

- Probiotic fermentation and transformation of traditional Chinese medicine

- Genetic code rewriting

- Bioadhesive for rapid hemostasis

**Zhengning Yang**

- Melanin synthesis

- T6SS

- DNA molecular encryption

- Microbial fertilizers for soil improvement

- Space synthetic biology

**Hanzhang Li**

- Hangover cure

- Hair loss

- New reporting methods

- Anti-aging

- Livestock disease monitoring

**Yueran Shen**

- Adenovirus treatment for hair loss

- In-body microplastics

- Biochips x synthetic biology

## March 29th

### Agenda

Summary of project design progress

### Notes

- Brucella - phage therapy

- Introduction of animal antibodies-NLR protein in plants, vaccine delivery methods

- Blood-brain barrier delivery - improve RNA stability

- DNA encryption technology - signal-induced DNA inversion

- Space water cycle

- Biomineralization and dental caries treatment

- Bacterial timed bomb - time-controlled suicidal plasmid

# April 2023

## April 17th

### Agenda

(1) ZJU-China 2023 project topic voting

(2) Discuss project feasibility with mentors

### Notes

The RNA vaccine technology for plants has received unanimous approval from the members of the
Wet team.
Space water cycle and biomineralization project design have become alternative projects.

## April 20th

### Agenda

(1) Report project topic to our PI.

(2) Discuss project feasibility with our PI.

### Notes

**[First Project - saRNA]**

(1) Adding a signaling mechanism - Is it too burdensome for systemic delivery? For example, targeting rice
blast disease in leaves No need for systemic delivery in space No need for continuous delivery over time

(2) Is NLR a mechanism in plants? What is the innovation point? Using antibodies produced by animals for
enhanced specificity and resistance The antibodies we use are also derived from animals and their sequences
can be traced

(3) How is it different from reference literature? The article still relies on transgenic methods (the
principle of disease resistance remains unchanged, mainly focusing on implementation methods)

**Issues:** Complexity, need further clarification, feasibility needs to be thoroughly explored
Improvement direction: Precision control rather than passive control, expressing at the right place and time
(right place, right time to express and suicide - also applicable to the third project)

**[Second Project - Mars]**

(1) Only the initial story is related to Mars, the subsequent experiments are not strongly linked to Mars...
Consider conditions of anoxic environment and extreme temperatures (suggestion: enhance the local environment,
no need to react in an exposed environment)

(2) Are there no other aspects on Mars that need improvement besides modifying perchlorates?

(3) Based on this, further exploration can be done - determining water production

(4) (This implies creating artificial organisms that are entirely adapted to the Martian environment, which
seems extremely challenging)

**Improvement direction:** Early investigation - Identify the remaining issues with soil composition
Biosafety considerations for humans; Biosecurity mainly targeting the environment

**[Third Project - Cartilage]**

(1) Heart stent (rhythm heartbeat)

(2) Shaped cartilage, e.g., nose reconstruction, ear reconstruction

(3) Need to add new highlights (otherwise, it's a bit ordinary)

(4) Cartilage can be integrated using in-body 3D printing

(5) Cell 3D printing, I have heard reports where integrins are used to guide structure growth of cells

**Improvement direction:** Add shape control, benchmark current cartilage repair and treatment mechanisms

**[Future Work]**

**Feasibility exploration:** Technological roadmap, involved species, antigen-antibody expression system,
biosafety concerns, environmental pollution (First project has higher risks but is more innovative; second and
third projects have minimal risks)

# May 2023

## May 3rd

### Agenda

Preliminary experiment design

### Notes

(1) Self-replication-related sequences of plant positive-sense RNA viruses, such as Sindbis virus, TMV, BMV
**[by Wenqing and Junjie]**

(2) Rice NLR/ Tobacco NLR/ Arabidopsis thaliana NLR: Further research and selection + specific sequence identification
**[by Qinyao and Mingxuan]**

(3) VHH sequences: Fluorescent proteins, VHHs targeting pathogens **[by Linghao and Hanzhang]**

(4) TLS sequences **[by Shiyi and Zhenni]**

(5) Chassis research **[by Yueran and Lu Qian]**

## May 17th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- PCR of pikm gene

- Primer design (3 types of TLS, Arabidopsis thaliana, Arabidopsis thaliana full, tobacco full)

- Gene PCR of TLS-GFP

- Homologous recombination

- Transformation into E.coli

- Internal innovation and entrepreneurship fund application

## May 24th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- Agrobacterium-mediated transformation of TLS

- Alignment of pikm2 gene

- Preparation of Agrobacterium competent cells

- Primer design for cuticle protein

- Completion of multiplex PCR with multiple cloning sites

## May 27th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- Went to Professor Liang Yan's place to collect the extracted cDNA

- Purchased tobacco seeds and growth medium

- Successfully conducted PCR amplification of the pikm2 gene

- Received primers for viral coat protein and fluorescent protein, ready for ligation

# June 2023

## June 3rd

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- Construction of pikm2 plasmid

- Construction of 3 TLS plasmids

- Learning tobacco cultivation techniques

- Learning transient expression techniques

## June 7th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- **Software:** This discussion mainly revolves around the functional prediction model based on amino acid
sequence and structure, involving parameter learning, structure influence, chemical reactions, and discussing
issues such as computational capabilities and structure file formats.

- Discussion on the issues of Agrobacterium, handling methods for variables, and testing results.

- Discussion on rice cultivation plan, progress of experiments, and next steps' arrangements.

## June 14th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

File handling, format confirmation, error debugging, and performance optimization in the
process of model deployment and testing.

# July 2023

## July 7th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- Summer work arrangement

- Preparation for the Conference of China iGEMer Community

- Presentation of the project design

- Discussing project feasibility with iGEM teams from other universities in China

## July 12th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

(3) Literature sharing

### Notes

- Design and order the remaining 19 TLS DNA sequences for Arabidopsis thaliana

- Attempt to produce multiple copies of TLS, around a dozen

- The previous TMV clone recovery concentration was a bit low.

- The pgreen-pikm1 sample has been received

- Primers for ID sequence are ready; infect plants with TMV and use immunosuppressants

- Literature sharing by Zhenning

## July 15th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

(3) Literature sharing

### Notes

- Agrobacterium activation completed

- Injected 4 sets of TLS into 16 tobacco plants

- Performed tRNA experiments on all 20 groups of Arabidopsis thaliana, connected them to GUS

- Completed ordering of repetitive tandem sequences

- Removed TMV coat protein+movement+pgreen for testing, only backbone without target gene, waiting for
sequencing results for the remaining points

- Completed replacement of ID sequence for pikm1

- Designed a hardware cursor caliper puncher

- Changed the strategy of the model

- **Software:** Predicting fragments based on the chemical properties, hydrophobicity, and electric charge
of exposed bases, real-time fragment simulation of compound/amino acid natural growth process, selecting
method - computational efficiency, pure random process

- Literature sharing by Linghao and Yueran: Linghao - RDRP truncation; Yueran - downstream pathway under ETI

## July 23rd

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- Injected TLS into tobacco plants, preparing to send 16 plants to the College of Agriculture to observe
fluorescence.

- **Human practice:** Contacting the Food and Agriculture Organization of the United Nations and the
Department of Agriculture and Rural Affairs of Zhejiang Province.

- Contacting the mentor.

- Failed to construct the rdrp plasmid.

- Modified the pikm1 sequence into nanobody; completed Agrobacterium-mediated transformation of pikm1, pikm2,
and gfp.

- TMV plasmid construction.

- Brainstorming on the design of the biosafety module.

## July 26th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- TLS fluorescence observation, qPCR preparation.

- Tobacco transient expression injection (with GFP stimulant), observe immune phenotype after 4-5 days.

- **Software:** Diffusion model used for protein structure prediction.

- **Hardware:** Puncher design completed.

- Idea for fluorescence tracking, capable of visualizing fluorescent movement.

## July 29th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

(3) Literature sharing

### Notes

- Plant RNA extraction, negative control missing.

- Construction of TLS plasmid, already performed homologous recombination.

- Reconstructing the rdrp plasmid.

- Spottings phenotype, if not present, uses reactive oxygen species.

- **Model:** Infection model supplemented with invasion and invasion kinetics.

- **Model:** Considering not only the flow of RNA in the straight tube system, but also constructing a
more realistic system.

- **Literature sharing:** Model of spore transmission and infection in the air.

# August 2023

## August 9th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

(3) Literature sharing

### Notes

- The replicating tobacco plants for the four TLS groups have been prepared.

- Several new TLS constructs are not successful.

- **Biosafety:** Promoters, elements, terminators; validation of promoters and terminators.

- **Software:** Parameter tuning phase, running the model requires a server; software developed as a web
tool.

- **Hardware:** Found a good approach, referring to a master's thesis from Zhejiang University.

- **Literature sharing:** Zhenning, Yueran.

## August 12th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

(3) Literature sharing

### Notes

- New construction of TLS GUS

- Review of problems in rdrp plasmid construction

- Linearization of pgreen-tmv

- Activation and injection of pikm1-2

- New detection method for TLS

- Validation of promoter and terminator

- Enterprise contact for Human Practice; Discussion of project design with Professor Chai Jijie

- Consultation on legal regulations and related issues

- Contact with transformation author on WeChat public account

- **Literature sharing:** Ling Hao's paper on NLR modification; Qin Yao - the impact of aerosols on
farmers (allergens)

## August 16th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- TMV-GFP plasmid to be constructed, successful bacterial transformation, tobacco injection, immune
phenotype; the previous weeks were preliminary experiments, phenotype expression will start this weekend.

- Verify TMV load by measuring GFP expression level, assess immune clearance; can observe photosynthetic
changes and macroscopic changes.

- **rdrp:** new plasmid construction.

- **TLS:** 8 plasmids, transformed and plated for testing; different reporters, GFP, GUS, RUBY - plasmid
construction and injection.

- **Advanced modification:** deletion of certain structures to investigate their impact on mobility.

- **Professor Chen's suggestion:** after obtaining results, combine them with dry experiments to predict
which key structure affects RNA mobility.

- Construction of plasmids combining TLS and rdrp.

## August 23rd

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- RUBY plasmid construction completed.

- GUS plasmid construction.

- GFP protein extraction finished.

- Tobacco injection for NLR, exploring concentration conditions.

- **Software:** data processing.

# September 2023

## September 6th

### Agenda

(1) Summary of recent work

(2) Discussion of project issues

### Notes

- **Hardware:** Purchase plan and intentions to make a microscope and punching device

- The TLS experiment photos are still being processed.

- TLS mobility verification.

- Summary of wiki documentation and preparation for project closure.

## September 16th

### Agenda

Summary of recent work

### Notes

Verification of the biosafety module and exploration of research using more toxic genes.

Conducting sunlight exposure experiments on the biosafety module, observing different time
gradients, and placing 16 plates for observation.

The addition of local features with only single-point mutations has resulted in a decline in
model performance, indicating that our data processing is still insufficient. I am currently contemplating
implementing new advanced image processing methods through literature and other models, as well as exploring
new approaches to handle this data.

## September 20th

### Agenda

Summary of recent work

### Notes

It has been discovered that the design issue near the DMP protein has led to the deletion of
the replication protein region while retaining this segment may potentially trigger an ATG initiation problem.
Efforts are currently underway to resolve this issue.

By experimenting to expose the protein samples under sunlight, the lethal curve was observed.
Currently, we are purchasing new toxic proteins and simultaneously attempting new promoters, but due to the
inability to obtain inducers, the experiment has not yet begun.

# October and later 2023

Organize project experimental results, write final documents, and create web pages.

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Overview

Project Design

Cycle 1 - Based on Bacillus subtilis

Cycle 2 - Based on plant immunity

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

tRNA-like sequences(TLS)

Cycle 1 - TLS from Arabidopsis thaliana

Cycle 2 - TLS from different species

Cycle 3 - Characterizing TLS Mobility

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

The module of biosafety

Cycle1 - KillerRed

Cycle2 - miniSOG

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

# Overview

“Failure is central to engineering. Every single calculation that an engineer makes is a failure calculation.
Successful engineering is all about understanding how things break or fail.”
-- Henry Petroski

"Failure is central to engineering." This sentiment perfectly encapsulates the essence of engineering biology, a
discipline that constantly learns from both successes and setbacks.

Biological engineering, through the rigorous application of knowledge, aims to solve problems and develop new
technologies and products. It offers an unbiased lens that enables systematic thought and analysis of
assumptions and approximations in design. This mindset not only defines what is known, but also embraces the
unknown, fostering innovation and creativity.

Just as mature engineering fields like aircraft engineering have advanced through the years, we too are on the
path to achieving enhanced predictability and control in biology. We are excited to contribute to this frontier
as an iGEM participant and be part of the development of biological engineering's tools and practices. With
perseverance and dedication, we aim to demonstrate engineering excellence while pushing the boundaries of what
is possible in synthetic biology.

![](https://static.igem.wiki/teams/4628/wiki/engi/1.png)

Figure 1: The iteration cycle of biological engineering

Have they used models to meaningfully predict the behavior of their system or guide their experimental or design
choices, or alternatively, have they subsequently built models that characterize and explain how their system
works?

We adhere to the bioengineering iterative cycle of brainstorming, conducting experiments, and discussing
results, learning from failures and refining our project design and experimental design through the
“design-build-test-learn” cycle.

What experiments did the team do, and were the data replicated or built upon?

We conducted various tests throughout their cycles. These include modifying plant immune receptors with
nanobodies, performing RNA secondary structure predictions, conducting phenotypic observations, using qPCR,
protein expression assays, GUS staining, and utilizing fluorescence expression.

How rigorous are their experimental designs and measurements?

Our experiments are conducted with extreme rigor. You can see the safety guidelines we follow on the [safety page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D%20%20%20%20%20%20%20%20), and you can browse the standardized procedures we follow for our experiments on the [protocol page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D).
At the same time, we use a variety of ways to measure the results of our experiments to explore the best
measurement criteria. You can learn more about this in the [RESULTS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

Teams may have built software tools to help either with the simulation of their system, to design functionality
or to predict behavior.

We have optimized and improved nanobodies through bioinformatics analysis and computer technology. We have also
developed user-friendly software to assist their biopesticide products. We propose that targeted nano-antibodies
can be designed for any pathogenic bacteria in the future, which could offer a potential solution to the issue
of pathogen resistance caused by conventional pesticides.

You can learn more about this in the [software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D).

How much attention have the teams given to making the progress they have made reusable? For parts, or part
collections, how well characterized are they? Is this clearly documented in the Registry? Would you be happy to
use these parts?

We have standardized all the components involved in our project to meet the requirements of the igem community
and have completed the part upload. You are more than welcome to use our part and contact us about its usage.
Any suggestions will be appreciated.

To learn more, click [part](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27part%27%29%20%7D%7D).

# Project Design

## Cycle 1 - Based on Bacillus subtilis

### Design

Compared with traditional chemical pesticides, biopesticides have lower risk of environmental pollution and
less toxic side effects, which have less impact on human body and environment. Therefore, we focused on the
development of new biopesticides in the early stage of brainstorming, hoping to provide new inspiration for
this.

After our research, we found that Bacillus subtilis is a more widely used engineering bacteria in
biopesticides. We obtained a specification for a biopesticide using Bacillus subtilis as a biological chassis
and designed a Bacillus subtilis-based biopesticide based on it.

### Build

**Targeting - Enrichment**

(1) Strong motility using the natural chemotaxis ability of engineering bacteria targeting,
do not have to design.

(2) Enrichment at infestation

**Advantage:**

(1) Compared with the spore display technique, steps such as spore purification are
eliminated and the cost is lower.

(2) It can take full advantage of the strong motility of the bacteria itself to enhance the
targeting ability.

About BslA

The N-terminus is located on the cytoplasmic membrane of Bacillus subtilis. It then passes through the cell
wall, and the C-terminus is located in the extracellular region. the N-terminus is a signal peptide of the Sec
transporter system for transporting BslA from the cytoplasm to the cell wall.

![](https://static.igem.wiki/teams/4628/wiki/engi/2.png)

Figure 2: The module of engineering bacteria enrichment

### Mass inductive priming

The Agr system of Staphylococcus aureus (S. aureus) was chosen as the module for signal emission and
reception, consisting of an auto-inducing peptide (AIP), a receptor (AgrC), and a transcription factor (AgrA).
AIP is generated and secreted by AgrD, AgrB ([BBa\_K3447010](https://parts.igem.org/Part%3ABBa_K3447010)) When the number of transgenic Bacillus subtilis
increases to a certain level, AIP signaling accumulates, and AgrC senses the signaling molecule and
phosphorylates AgrA, activating the expression of promoters P2 ([BBa\_I746104](https://parts.igem.org/Part%3ABBa_I746104)), P3 ([BBa\_K212003](https://parts.igem.org/Part%3ABBa_K212003)).

![](https://static.igem.wiki/teams/4628/wiki/engi/3n.png)

Figure 3: The module of mass inductive priming

**Functional module**

Expression of asparagus antimicrobial peptide GAFP-1, synthesized antimicrobial peptide D4E1 ([BBa\_K1364013](https://parts.igem.org/Part%3ABBa_K1364013)),
secreted through SamQ signal peptide ([BBa\_K1074014](https://parts.igem.org/Part%3ABBa_K1074014)), and connected by protein junction ([BBa\_K2934005](https://parts.igem.org/Part%3ABBa_K2934005)).

![](https://static.igem.wiki/teams/4628/wiki/engi/4.png)

Figure 4: The functional module I

Via isobranchial acid pyruvate lyase and isobranchial acid synthase (pchBA, [BBa\_J45017](https://parts.igem.org/Part%3ABBa_J45017)); converts branchial
acids to salicylic acid (SA) and pyruvic acid, the former of which induces systemic acquired resistance (SAR)
in plants.

![](https://static.igem.wiki/teams/4628/wiki/engi/5.png)

Figure 5: The functional module II

**Biosafety module**

(1) The chassis strain of Bacillus subtilis is a common soil microorganism and plant
symbiotic bacteria, which itself will not cause greater damage to the ecological environment.

(2) The designed functional genes need to be activated by group induction after the
enrichment of two strains at the same time in the pathogenic bacteria infestation, which will limit the
proliferation of engineering bacteria and the impact on the ecology to a certain extent.

(3) The selected antimicrobial peptides should be biological components existing in nature,
or modified and designed for plant pathogenic fungi, with the action site in the cell wall of the fungus,
which is harmless to human beings and plants.

(4) Enhance the stability of the system through the use of integrating plasmids to limit
horizontal gene transfer.

(5) Add MazEF toxin antitoxin system ([BBa\_K302032](https://parts.igem.org/Part%3ABBa_K302032)), add the toxin gene MazF gene within the
gene of the modified module, and place the antitoxin gene MazF in a more distant position to reduce the
possibility of horizontal gene transfer.

![](https://static.igem.wiki/teams/4628/wiki/engi/6.png)

Figure 6: The biosafety module

### Test

As a result of our analysis, we have summarized the feasibility and shortcomings of our project as follows:

Feasibility

- Nanobodies have enabled expression in Bacillus subtilis

- The BslA protein is associated with biofilm formation and has been shown to enrich heavy
metals on surfaces by fusion tagging

- The selected group sensing system is derived from Staphylococcus aureus and is not
affected by the group sensing system of Bacillus subtilis itself

- The selected antimicrobial peptides have little effect on B. subtilis itself

- Except for the targeting module, the rest of the components are already present in PARTS
and can be optimized for Bacillus subtilis

Shortcomings

- Service targets are lower yielding crops and may require cost control

- Essentially a biopesticide, it may be difficult to demonstrate core competitiveness in
terms of effectiveness compared to existing chemical pesticides and biopesticides.

- Autonomous chemotaxis and target anchoring ability to be verified, need to prove the
effectiveness of the program design through experiments.

- Nanoantibodies need to rely on existing research to select surface antigens of pathogenic
fungi, expandability depends on the progress of existing research.

- Population sensing thresholds and effects need to be experimentally verified, and the
ability of engineered bacteria to survive and compete with field strains needs to be verified.

### Learn

We endorsed the idea of biopesticide development, but with a deeper consideration of the chassis. We began to
read more literature in an effort to keep our thinking from being limited by the biopesticide products
currently available.

## Cycle 2 - Based on plant immunity

### Design

We started reading more literature and came across one that provided the original inspiration for our flora
sentinel. Researchers from the University of East Anglia in the UK have devised a novel approach to enhance
plant disease resistance using animal antibodies. However, the current ID sequence lacks tight binding and
specificity. Therefore, our proposed solution involves substituting the ID sequence with a specially
engineered nanobody capable of precise effector binding, while still retaining the ability to recruit
signaling NLR and trigger downstream immune responses.

### Build

![](https://static.igem.wiki/teams/4628/wiki/engi/7.png)

Figure 7: The biosafety module

Our design is to replace the ID sequence with a nanobody that specifically binds to effects by directly
replacing the ID sequence fragment (base 509-730) of pickm-1 with enhancer, and trying to retain its ability
to repeat signaling NLR and activate downstream immediate responses after modification.

### Test

We successfully modified a natural plant immune receptor (NLR), and the method is to replace its original ID
sequence (the original pathogen-recognizing sequence in plant) with a nanobody that specifically recognizes
the effector. This modified NLR would improve plant’s ability to recognize pathogenic bacteria while retaining
original NLR’s ability to stimulate downstream immune responses, thereby enhancing the resistance of plants to
pathogenic bacteria. Please click on our Result for more information.

### Learn

Our future work will focus on improving the binding specificity of nanobodies and expanding their recognition
of different effectors. By optimizing and improving the nanobodies, we can ensure that it binds tightly and
specifically to the desired effector molecules, thereby enhancing plant defense against pathogens. In
addition, expanding the range of effectors that can be recognized by engineered nanobodies will further
enhance the immune response of the plant and improve the overall disease resistance.

We have already optimized and improved the nanobodies through computer technology by means of bioinformatics
analysis and developed user-friendly software to assist our biopesticide products. In the future, there is
hope that targeted nano-antibodies can be designed for any pathogenic bacteria through computer technology,
which could be a good solution to the problem of pathogen resistance caused by conventional pesticides.

# tRNA-like sequences(TLS)

## Cycle 1 - TLS from Arabidopsis thaliana

### Design

Our initial inspiration for this module came from the article "tRNA-Related Sequences Trigger Systemic mRNA
Transport in Plants". In this article, the researchers found that mRNAs containing tRNA-like structures (TLSs)
were enriched in the phloem stream and could move across graft junctions.

Therefore, we would like to introduce TLS module into the project, equipped our RNA with the ability to
transport in plant vascular tissues.

### Build

For early screening, we obtained 20 tRNA sequences of Arabidopsis thaliana from the ncbi database, and
performed RNA secondary structure prediction, using tRNA containing the above structure for subsequent
experiments.

### Test

We utilize phenotypic observation to assess the functionality and mobility of the transported mRNAs.

### Learn

To further validate the functionality of the TLS module, we can obtain additional tRNA sequences from
different species and perform RNA secondary structure prediction on them. By testing these additional TLS
elements, we can select the elements that perform best in mRNA transport across plant vascular tissues.

## Cycle 2 - TLS from different species

### Design

Based on our initial screening of 20 tRNA sequences from Arabidopsis thaliana, we have selected a set of TLS
elements for further experimentation. Additionally, we expanded our analysis to include tRNA sequences from
different plant species to maximize the diversity of TLS elements.

The next phase of our project will involve experimental validation of the selected TLS elements.

### Build

To verify the TLS-triggered mobility of fused transcript in vascular bundle of Nicotiana tabacum，we amplified
EGFP gene from plasmid pcDNA3.1-T2A-EGFP. 20 tRNA sequences of Arabidopsis thaliana and Nicotiana tabacum are
synthesized. Then 4 plasmids pGREENII-GFP(-TLS) is constructed, which contains 35S promoter and T-DNA repeat
to efficiently express our mRNA in plant cell.

### Test

We judged the intensity of protein expression by observing the amount of leaf fluorescence expression, but we
did not get a better observation because of the low intensity of fluorescence expression and the high
interference of experimental observation. Therefore, we began to explore new characterization modes.

### Learn

We will explore new characterization methods to improve the observation and quantification of protein
expression levels. This will allow us to better understand the effects of TLS elements on gene expression and
manipulation in plants.

## Cycle 3 - Characterizing TLS Mobility

### Design

We wanted to characterize the mobility of TLS by phenotypic observations, qPCR and protein expression assays.
In terms of phenotypic observation, we have constructed a total of three expression systems, GFP, GUS, and
RUBY, by reviewing literature to explore the best phenotypic expression effect.

### Build

![](https://static.igem.wiki/teams/4628/wiki/engi/8.png)

Figure 8: Plasmid profile of pGREENII 0800-MCS-GFP-TLS(A), pGREENII 0800-MCS-GUS-TLS(B) and
pGREENII 0800-kozak-RUBY-TLS(C)

### Test

We conducted qPCR experiments to confirm the movement of TLS-triggered GFP mRNA in plants. The results
indicated that mRNA containing TLS had moved further away from the injection site.

Additionally, the researchers performed GUS staining and used RUBY to visualize the movement and expression of
mRNA in plant cells. The results revealed that the mRNA sequence containing the Ala TLS was able to move
within the leaves, as evidenced by the presence of blue GUS catalytic products in the vascular bundles.

See more details in [Result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

### Learn

Further analysis and experimentation are needed to gain a deeper understanding of TLS mobility and its
implications in plant biology. This study provides a foundation for future research in this area, and
continued investigation may lead to valuable insights into the mechanisms and potential applications of TLS
elements in plants.

# The module of biosafety

## Cycle1 - KillerRed

### Design

Our engineered strains are mainly used in plants, and plants need sunlight to grow, so we chose sunlight as
the way to initiate suicide. We began screening and researching toxic proteins to select the most appropriate
ones for the biosafety module.

### Build

We initially considered KillerRed ([BBa\_K1184000](https://parts.igem.org/Part%3ABBa_K1184000)). According to extant research, KillerRed is a red fluorescent
protein that generates reactive oxygen species (ROS) under yellow-green light (540-585 nm). Given that plants
typically absorb blue-violet and red light, reflecting light near 540nm (green), KillerRed is ideally suited
as a biosafety 'kill switch'.

### Test

Other proteins in the KillerRed family, such as Supernova, were also validated. It has been shown to reduce
dimerization tendency and maintain the ability to generate ROS. We obtained its fragment by point mutation,
but light exposure showed that its suicide inducing effect was less stable than that of KillerRed.

![](https://static.igem.wiki/teams/4628/wiki/engi/9.png)

Figure 9: Results of light experiments of Agrobacterium with KillerRed V.S. empty plasmid
(on a sunny day)

### Learn

In order to ensure the success and reliability of our biosafety module, we deemed it necessary to explore
additional protein options. By switching to a different protein, we hope to find one that not only
demonstrates stability in inducing suicide but also aligns with the specific needs and requirements of our
engineered strains in plants.

This decision to replace Supernova serves as an important learning experience for future research and
development projects. The stability and consistency of the chosen protein should be thoroughly tested and
validated before implementation, ensuring that it can effectively execute the desired actions in the given
context.

## Cycle2 - miniSOG

### Design

By reading more literature and consulting with more senior researchers, we identified another phototoxic
protein.

### Build

By reading more literature and consulting with more senior researchers, we identified another phototoxic
protein.

### Test

our plate light experiment demonstrated a noticeable reduction of engineered bacteria under natural light
irradiation, regardless of whether the conditions were sunny or cloudy.

![](https://static.igem.wiki/teams/4628/wiki/engi/10.png)

Figure 10: Results of light experiments of Agrobacterium with empty plasmid V.S. minisog (On
a cloudy day)

### Learn

We have gained knowledge about the significance of considering the specific needs of the target organism and
the importance of rigorous testing and validation. Moving forward, we can use this experience to improve our
designs and expand our understanding of biosafety measures in genetically engineered organisms.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

1 tRNA-like sequences(TLS)

Secondary structure prediction of TLS

Plasmid construction

GFP qPCR result showing TLS-triggered transcript mobility

RUBY to visualize the movement and expression of mRNA

GUS staining showing mobility of other TLS

Future plan: multiple TLS

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

2 RNA dependent RNA polymerase(RdRp)

RNA dependent RNA polymerase(RdRp)

Plasimid Construction & Modification

Fluorescence microscopy of Transformed Leaves

Detection of Protein expression

Detection of RNA expression

Prediction of RNA secondary structure

Future Plan

Time-Expression Curve

Integration of RdRp and TLS

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

3 The module of plant immunity

PART1 Construction of pGreen-Pikm1 (enhancer)

PART2 Experiments for results

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

4 The module of biosafety

Testing of the promoter

Directed evolution of promoters

Selection of phototoxic proteins

Construction of plasmid vectors

Illumination experiments

Further experiments

![](https://static.igem.wiki/teams/4628/wiki/footer/divide.svg)

Reference

# 1 tRNA-like sequences(TLS)

## Secondary structure prediction of TLS

Research indicates that the folding structure of the TLS motifs triggers the movement of transcript in plant
vascular bundle. A predicted long hairpin motif with a stem (8 to 12 nucleotides)–variable bulge(s)–stem (4 to 7
nucleotides)–variable loop is found crucial to the mobility of transcript, And the D loop deletion enhances the
transport activity.[1]For early screening, we obtained 20 tRNA sequences of Arabidopsis thaliana from the ncbi
database, and performed RNA secondary structure prediction (done on [RNAfold web server](http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi%29)),
using tRNA containing above structure for
subsequent experiments.

![](https://static.igem.wiki/teams/4628/wiki/res/1.png)

Figure 1: Secondary structure prediction of natural tRNA sequence and artificially modified
tRNA sequence. tRNA of Met(A) and Met ΔΔT(B) are proven to be mobile, both of which share stem–variable
bulge(s)–stem–variable loop structure. Ala tRNA(C) share the same structure while His tRNA(D) doesn't.

## Plasmid construction

To verify the TLS-triggered mobility of fused transcript in vascular bundle of Nicotiana benthamiana，we amplified
EGFP gene from plasmid **pcDNA3.1-T2A-EGFP**. 20 tRNA sequences of Arabidopsis thaliana and
Nicotiana benthamiana are
synthesized. Then 4 plasmids **pGREENII-GFP(-TLS)** is constructed, which contains 35S promoter and
T-DNA repeat to
efficiently express our mRNA in plant cell.

To visualize the evaluation of mobility, we constructed a series of plasmid **pGREENII-GUS-TLS**
and
**pGREENII-RUBY-TLS**, where RUBY( [BBa\_K3900028](https://parts.igem.org/Part%3ABBa_K3900028) ) is a effective reporter for
noninvasively monitoring plant gene
expression and plant transformation[2]. Kozak sequence are added to the 5' terminal of CDS to enhance
translation efficiency.

![](https://static.igem.wiki/teams/4628/wiki/res/2.png)

Figure 2: To visualize the evaluation of mobility, we constructed a series of plasmid
pGREENII-GUS-TLS and pGREENII-RUBY-TLS, where RUBY( [BBa\_K3900028](https://parts.igem.org/Part%3ABBa_K3900028) ) is a effective reporter for noninvasively
monitoring plant gene expression and plant transformation[2]. Kozak sequence are added to the 5' terminal of
CDS to enhance translation efficiency.

## GFP qPCR result showing TLS-triggered transcript mobility

First, in order to verify that our mRNA can move in plants driven by TLS, Agrobacterium containing our
pGREEN-GFP-TLS shuttle vector was injected into the marked area of plant leaves for transient expression.
Unfortunately, no significant fluorescent signal was detected outside the injection region under excitation
light. However, relative GFP mRNA level of leaf samples outside the injection area showed that the mRNA
containing TLS moved further.

![](https://static.igem.wiki/teams/4628/wiki/res/3.png)

Figure 3: First, in order to verify that our mRNA can move in plants driven by TLS,
Agrobacterium containing our pGREEN-GFP-TLS shuttle vector was injected into the marked area of plant leaves
for transient expression. Unfortunately, no significant fluorescent signal was detected outside the injection
region under excitation light. However, relative GFP mRNA level of leaf samples outside the injection area
showed that the mRNA containing TLS moved further.

## RUBY to visualize the movement and expression of mRNA

Since GFP mRNA can be detected by qPCR, we speculate that the fluorescence signal of GFP is too weak to be
observed. Therefore, we seek more sensitive reporter. We constructed plasmid PGREENII-RUBY-TLS, transiently
expressed RUBY-TLS at the tip of tobacco leaves, and observed after 4-5 days of injection. The results showed
that TLS could trigger the transport of RUBY mRNA, and mRNA was successfully expressed in plant cell.

For quantitative measurement, qPCR experiments were also used to detect the movement of RUBY mRNA. The mobility
of RUBY was weaker than that of GFP, probably because RUBY mRNA (~4kbp) was much longer than that of GFP
(~800bp).

![](https://static.igem.wiki/teams/4628/wiki/res/4.png)

Figure 4: RUBY to visualize and expression of mRNA.
A) Movement of RUBY mRNA triggered by TLS. Colored spots were observed at positions outside the injection area
and close to the vascular bundle.
B) qPCR result. All three TLS showed mobility in 1cm and 2cm away from the injection aera.

## GUS staining showing mobility of other TLS

Considering the large difference in the length of the transcripts of GFP and RUBY, we used another commonly used
reporter : GUS(~2kbp), and we also wanted to explore the contribution of other TLS to mRNA mobility. A series of
PGREENII-GUS-TLS plasmids were constructed and transiently expressed in tobacco. The results showed that in
addition to the above TLS, Ala tRNA from Arabidopsis thaliana can also drive mRNA movement.

![](https://static.igem.wiki/teams/4628/wiki/res/5.png)

Figure 5: GUS staining result. After ethanol decoloring, obvious blue GUS enzymatic reaction
products were observed around the vascular bundle, indicating the successful transportation and expression of
mRNA.

Since we synthesized tRNA sequences of 20 amino acids, we will further explore the mobility of different TLS.
However, due to time constraints, we will improve this part in future work.

## Future plan: multiple TLS

In RNA imaging systems for MS2/MCP [3], sequence-specific RNAs that bind to MS2 viral capsid proteins are often
used to enhance binding to proteins by increasing the copy number, and such RNAs are characterized by the
ability to maintain a stable secondary structure after multiple repeats of the stem-loop. pCR4-24xMS2SL-stable
is a commonly used plasmid for the expression of MS2-specific binding RNA, which produces an RNA(SL24)
containing 24 stem-loop repeat structures[4]. Expecting to enhance mRNA binding to RNA motility-associated
proteins by increasing the copy number of the TLS sequence, we replaced some of the repeats of SL24 with the RNA
sequence of TLS (Ara.Mer.ΔΔt).

Due to the quality of our ligase, we failed to complete this part of the verification. We will explore whether
the superimposed TLS can further enhance the mobility of mRNA in subsequent experiments.

![](https://static.igem.wiki/teams/4628/wiki/res/6.png)

Figure 6: Secondary structure prediction of TLS(Ara.Met.multi). The sequence can form a
stable multiple stern-loop structure.

# 2 RNA dependent RNA polymerase(RdRp)

## RNA dependent RNA polymerase(RdRp)

This part attempts to use TMV replicon to extend the action time of RNA products. While expressing TMV RNA
dependent RNA polymerase, we detect transcription and translation levels of target proteins expressed using two
subgenomic promoters of TMV. Further, we try to predict the possibility of combining TLS and RdRp.

## Plasimid Construction & Modification

Considering that TMV is a mature vector, we try to modify it directly based on TMV genome. First, we
successfully cloned the complete genome of TMV from pUC18-TMV and the annotation showed that TMV had two
subgenomic promoters (subPromoter MP & subPromoter CP) respectively expressing movment protein and capsid
protein. On the one hand, we chose an existing mature transformation solution: we inserted GFP ORF after
subPromoter CP to replace CP ORF for expression (Fig.1 A). Because part of the coding sequence of capsid protein
constitutes the sequence of subPromoter CP, we mutant the start codon in the subpromoter(5,712-5,714,from ATG to
AGA) in case of expressing a nonsense peptide(Fig.1 C) [5].

On the other hand, to reduce the pressure on TLS of transporting RNA, we want to minimize the size of the RNA
product. Therefore, we try to delete the sequences of MP ORF and subPromoter CP and express the target protein
after subPromoter MP (Fig.1 B). Although part of the movement protein coding sequence is also included in the
TMV RNA-dependent RNA polymerase coding sequence, To ensure the functional and structural integrity of the
RNA-dependent RNA polymerase, we chose to preserve a sequence at the beginning of the movement protein ORF (17
bp) and verify its effect later (Fig.1 D).

Figure 1 Construction of plasmid for RdRp verification. A. the plasimid of pGreenⅡ -TMV RNA dependent RNA
polymerase-movement protein-subPromoter CP-EGFP; B. the plasimid of pGreenⅡ -TMV RNA dependent RNA
polymerase-subPromoter MP-EGFP; C. the sequence of CP start codon; D. the sequence of MP start codon.

![](https://static.igem.wiki/teams/4628/wiki/home/n7.png)

Figure 7: Construction of plasmid for RdRp verification.
A. the plasimid of pGreenⅡ -TMV RNA dependent RNA polymerase-movement protein-subPromoter CP-EGFP; B. the
plasimid of pGreenⅡ -TMV RNA dependent
RNA polymerase-subPromoter MP-EGFP; C. the sequence of CP start codon; D. the sequence of MP start codon.

## Fluorescence microscopy of Transformed Leaves

After successfully constructing the plasmid, we infected tobacco with Agrobacterium that transformed the
plasmid. At day 4, 9, 14, Fluorescence microscopy was used to observe GFP fluorescence at leaf infection sites
to determine the successful transfection of the plasmid and the amount of target protein expression. At the same
exposure intensity, it was found that the two groups expressing the RNA dependent RNA polymerase could directly
observe stronger fluorescence than the group expressing the protein directly with the CaMV 35s promoter (Fig.2
A).

Further, 5 fields of view at low magnification were selected for each group, and the mean fluorescence intensity
of each field was calculated and analyzed by Image processing software (Image J). The results showed that mean
fluorescence of the two groups expressing RNA-dependent RNA polymerase was significantly higher than that of the
control group (Fig.2 B). In addition, the fluorescence intensity of the subPromoter MP group (average 15.23) is
about 2.5 times that of the control group (average 35.04), and the subPromoter CP group is about 3.5 times
(average 50.412). Based on these results, phenotypically we demonstrate the effectiveness of two subgenomic
promoters, as well as the role of RNA-dependent RNA polymerase.

![](https://static.igem.wiki/teams/4628/wiki/res/8.png)

Figure 8: Microfluorescence of transient transformed leaves at day 4.
A. microfluorescence
image of transformed leaves, 4x means a quadruple objective, 10x means a quadruple objective;
B. mean fluorescent of multiple views at 4x, t test was used (GFP group as control, \*\*\* p<0.001, \*\*\*\*
p<0.0001).

## Detection of Protein expression

Next, to determine that the RNA dependent RNA polymerase enhances the expression of the target RNA at the
protein level, we took tissue from the transfection of leaves site and extracted the protein. Leaf tissue (50
mg) expressing GFP was harvested and GFP accumulation in extracts of total soluble protein was determined by
spectrofluorometry as described in Man and Epel [6]. GFP accumulation in tissues expressing GFP derived from
control group is significant inferior to that obtained from the subPromoter MP group and subPromoter CP group
(Fig.3 A). Tissue expressing GFP from subPromoter MP produced a mean value of 19.13 relative fluorescence
units., 4-fold more GFP in comparison to control group that produced a mean of 4.87 units. While that from
subPromoter CP produced a mean value of 30.07 relative fluorescence units., 6-fold more GFP in comparison to
control group. In addition, Western Blot was used to further verify changes in protein expression and the
results were consistent with the fluorescence intensity of the protein extract(Fig.3 B).

![](https://static.igem.wiki/teams/4628/wiki/res/aaaaaa.jpeg)

Figure 9: Function detection at protein level. fluorescent analysis of protein extract, t test
was used (GFP group as control, \*\*\*\* p<0.0001); B. protein immunoblotting, actin as the loading control.

## Detection of RNA expression

At the same time, the function of RNA-dependent RNA polymerase at the RNA level was also verified. 9 leaves
from
each group were taken for RT-qPCR to detect the transcribed RNA. The results showed that the two groups
expressing RNA-dependent RNA polymerase had significantly higher RNA expression levels (Fig.4). After
normalization with the control group, the average relative expression of the subPromoter MP group was about 4
and that of subPromoter CP was about 10. The results showed that RNA-dependent RNA polymerase can play a good
role in replication and increase the expression of target RNA, suggesting the possibility of prolonging the
lifespan of RNA.

![](https://static.igem.wiki/teams/4628/wiki/res/9.png)

Figure 10: RT-qPCR detects function of RdRp at the RNA level. t test was used (GFP group as
control, \*\*\*\* p<0.0001), n=9

## Prediction of RNA secondar1y structure

It has been confirmed that the self-replication of RNA dependent RNA polymerase requires the secondary
structure of TMV 3’ UTR, especially the stem-ring structure plays an important role in TMV replication [7].
The mobility of the TLS sequence also depends on the stem ring in the secondary structure and is inserted near
the TMV 3’utr in our design. To determine whether the combination of the two affects the correct formation of
the secondary structure, we utilized the RNA secondary structure prediction tool ([RNAFold Web Server](http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)
) to forecast the transformation sequence. The
results show that the secondary structure of TLS and TMV 3’UTR is basically not affected, and the stem-ring
structure is still preserved regardless of whether TLS is inserted directly after or before 3’ UTR(Fig.5).
This indicates that our transformation will not affect the normal realization of the structure and function of
the two.

![](https://static.igem.wiki/teams/4628/wiki/res/10.png)

Figure 11: Prediction of secondary structure after insertion of TLS

## Future Plan

At present, we have verified the replication function of TMV RNA-dependent RNA polymerase and the feasibility
of such modification. Due to time constraints, although Agrobacterium transfection of tobacco has been carried
out, we have not had time to sample and detect RNA and protein expression levels. We hope to draw the
time-expression curve of target RNA and protein under RdRP in the future to characterize the half-life and
action time of target RNA and protein.

At present, we have verified the replication function of TMV RNA-dependent RNA polymerase and the feasibility
of such modification. Due to time constraints, although Agrobacterium transfection of tobacco has been carried
out, we have not had time to sample and detect RNA and protein expression levels. We hope to draw the
time-expression curve of target RNA and protein under RdRP in the future to characterize the half-life and
action time of target RNA and protein.

## Time-Expression Curve

Finally, in order to verify that TLS and RdRp can work together, we chose the TLS (Ara.Met.Full) with the best
comprehensive performance, and tried to install two sets of RdRp systems of different sizes respectively. The
results show that although TLS mobility is reduced after RdRp is installed, it is still retained.

![](https://static.igem.wiki/teams/4628/wiki/res/n12.png)

Figure 12: Half-life of the product after loading RdRp
A.Curve of RNA expression over time. RNA relative expression in leaf tissues was measured at 4,9,14 days post
infection.we take 4 d.p.i as the peak expression of protein and RNA.
B.Curve of protein expression over time. Fluorescence intensity in leaf tissues was measured at 4,9,14 days
post infection.we take 4 d.p.i as the peak expression of protein and RNA

## Integration of RdRp and TLS

Finally, in order to verify that TLS and RdRp can work together, we chose the TLS (Ara.Met.Full) with the best
comprehensive performance, and tried to install two sets of RdRp systems of different sizes respectively. The
results show that although TLS mobility is reduced after RdRp is installed, it is still retained.

![](https://static.igem.wiki/teams/4628/wiki/res/n13.png)

Figure 13: Microfluorescence of infection after RdRp and TLS integration
A.Statistical table of positive field of view under microfluorescence. We selected tobacco leaves 4 days after
infection, counted 100 visual fields at different distances from the injection site under 40 microscope times,
and recorded the visual fields with fluorescent cells as positive.
.Microfluorescence image. Representative fluorescence images of different groups1cm and 2cm away from the
injection site were selected.

# 3 The module of plant immunity

## PART1 Construction of pGreen-Pikm1 (enhancer)

### Acquisition of sequences

**Acquisition of Pikm1 and Pikm2 sequences**

At first, we did not notice that the Pikm gene is not found in all rice blast-resistant rice varieties, so our
initial attempt to obtain the gene from general rice varieties was unsuccessful. Fortunately, we later
obtained rice seeds with Pikm resistance genes from Prof. Pan Qinghua, South China Agricultural University,
and obtained Pikm1 and Pikm2 genes by PCR.

**Cultivation of rice seeds**

We select full and complete seeds, then soak them in 75% ethanol for 1min, and disinfect them in 15% KC1O for
30min. After that, we rinse them repeatedly with distilled water for 5-6 times, then place them in 90mm petri
dish covered with 2 layers of filter paper, wet the filter paper in each culture, and place the petri dish in
a 28°C light incubator for germination experiments, with a light cycle of 14h/10h (day/night)

After the rice germinates, it is transferred to MS medium, and the culture is continued under the above
conditions. After the rice grows a certain length of roots, they are cut as the experiment materials rather
than leaves, because the PIKM gene is expressed more in the roots, which may be explained by that the rice
blast will infect roots first in the soil.

![](https://static.igem.wiki/teams/4628/wiki/res/11.png)

Figure 13: Rice (Oryza Sativa) that we grew in MS

**Plant RNA extraction**

Consider that the expression of the Pikm gene in roots is much higher than in leaves, we only take rice root
tissue. At the beginning, when we extracted it using the commonly used Trizol method, the extract was in a
viscous state. We speculated that was because there were a large number of polysaccharides in it, and the
Trizol method could not completely remove it. Therefore, later we used the RNAprep Pure kit (DP432, from
Tiangen) and got better results.

**Acquisition of Pikm1 and Pikm2 genes**

Using the RNA obtained above as a template, We obtained rice cDNA by rt-pcr method. The Pikm1 and Pikm2 genes
were then obtained by conventional PCR methods.

![](https://static.igem.wiki/teams/4628/wiki/res/12.png)

Figure 14: Nucleic acid electrophoresis results. Pikm is about 3200bp long, so we used a
15000bp ladder as a reference, and found that all the 4 lanes at the corresponding location had results.
After sequencing, we can be sure that we have successfully obtained the Pikm sequence

**Acquisition of pGreen plasmid skeleton**

Thanks to igem2022 member Yan Zhijian for providing the pGreen plasmid, which is a Ti plasmid of
Agrobacterium, which is compatible with and can be well expressed in Agrobacterium.

**Acquisition of nanobody (named enhancer) sequence**

We purchased the sequence of enhancer from Genscript Biotech Corporation. It was originally a protein that
enhance the fluorescence intensity of GFP, but later it was found to be a nanobody that specifically binds to
GFP, which fits our design

**Acquisition of GFP: experimental materials from this laboratory**

**Acquisition of pUC18-TMV plasmid**

We purchased this plasmid from the Hangzhou TOP Biotechnology Itd company.

### Plasmid construction

**Construction of pGreen-pikm2/GFP plasmid**

We use the pcr method for pGreen linearization, and then the seamless cloning method connects the fragments
with the skeleton.

![](https://static.igem.wiki/teams/4628/wiki/res/13.png)

Figure 15: PCR results of pGreen-pikm2, pGreen-GFP colonies

a) the results of colony PCR were from the Agrobacterium transforming pGreen-pikm2 plasmid,
and the PCR detection area was about 7000 bp at both ends of pikm-2, and 15000 bp Marker was used as a
reference, and all 8 lanes had bright bands.

b) The figure shows the results of colony PCR of the Agrobacterium transformed pGreen-GFP
plasmid, the PCR detection area is at both ends of the GFP sequence, a total of about 700 bp, using 2000 bp
Marker as a reference, and all 8 lanes have bright bands.

**Modification of pGreen-pikm1 (enhancer)**

The pGreen-pikm1 plasmid is constructed by the above method, the plasmid is linearized oppositely from the
two
sides of the ID sequence, and then the fragment and the skeleton are connected by seamless cloning.

![](https://static.igem.wiki/teams/4628/wiki/res/14.png)

Figure 16: PCR results of pGreen-pikm1 (Enhancer) colonies

The figure shows the results of colony PCR of Agrobacterium that transformed the pGreen-pikm1 (Enhancer)
plasmid. The PCR detection area was the Enhancer region, a total length of 351 bp, and we use the 2000 bp
Marker as a reference. Obvious bands are seen in the region of corresponding size.

**Construction of Pgreen-TMV-GFP**

Since we have not been able to obtain the corresponding nanobodies of some pathogens (such as viruses, etc.),
we can only display GFP protein outside the coat protein of TMV, which is equivalent to making GFP a
pathogenic effector, allowing the modified immune receptor (which targets at GFP) can specifically recognize
GFP and complete immunity

Initially, our strategy was to insert GFP into the coat protein of TMV in the pUC18-TMV plasmid (before the
stop codon [8]) firstly, but when we transformed the constructed plasmid to E. coli DH5α and did sequencing,
we found that the 3' UTR at the end was lost, and we speculated that recombination of LTR (Long terminal
repeat) may have occurred. Therefore, we had to change the strategy. We firstly construct pGreen-TMV
(**When
transformed into and amplified in Agrobacterium, this is a plasmid skeleton that have less ability to
conduct
LTR recombination with Agrobacterium**). In terms of transformation and amplification, we adopt a
more stable E.
coli stbl3 to avoid the possibility of LTR recombination. After all of that, we then insert GFP into the
constructed pGreen-TMV and do the sequencing to make sure we got the right sequence of TMV-GFP.

### Agrobacterium transient transient expression on tobacco

**Transfer plasmids into Agrobacterium by electroporation**

- Mix the GV3101 Chemically Competent Agrobacterium and plasmid in a certain proportion

- Pre-cool the Gene Pulser with ice for 10 min

- Transfer the mixture to the Gene Pulser, cover the cup, dry the wall of the cup, and put
it in the electroporator

- Start the electroporator (parameters - Arg: C=25uF, PC=2000hm, V: 2400V

- Quickly return to ice, add 500 uL LB medium, and transfer to a 1.5 ml centrifuge tube, 28
°C, 250 rpm, incubate for 2-3h

**Agrobacterium transient transient expression on tobacco**

- Prepare the transient buffer

Table 1: The component of the transient buffer

| 100mM MES(pH=5.6) | 1ml |
| 1M MgCl2 | 100μL |
| 150 mM acetyl syringone in DMSO | 10 μL |
| ddH2O | to 10ml |

- Value the OD of Agrobacterium, and calculate the volume we need for tobacco transfection
(V=1mLx0.3/0D600)

- Centrifuge at 4000rpm for 10min and aspirate the supernatant

- Transient buffer 1 mL to resuspend Agrobacterium at room temperature and keep in the dark
for 3 h

- Select tobacco for injection at 5-6 weeks’ age, and the bacterial solution is injected
from the back of the leaf with a sterile syringe

## PART2 Experiments for results

### GFP alone as effector to stimulate immunity

**Material**

- Agrobacterium containing pGreen-pikm1 (Enhancer) plasmid→ later referred to as pikm-1
(Enhancer)

- Agrobacterium containing pGreen-pikm2 plasmids → referred to as pikm-2

- Agrobacterium containing pGreen-GFP plasmids → referred to as GFP

**Design**

- Experimental group→ pikm-1 (enhancer) + pikm-2 + GFP

- Control group→ pikm-1 + pikm-2

- Positive control → GFP

- Negative control → Agrobacterium empty

Mark the injection area with a circle, inject according to the method mentioned above, and then placed in a
greenhouse under suitable conditions for culture, and the phenotype was recorded one week later

**Result detection**

**Phenotype observation**

Tobacco was irradiated with 488 nm excitation light to observe the removal of GFP fluorescence

It can be seen that compared with the positive control, the negative control has a clear fluorescence
clearance.

![](https://static.igem.wiki/teams/4628/wiki/res/rp1.png)

Figure 17: Phenotypic observations after transient tobacco

**Negative control:** Injection of Agrobacterium with
empty plasmid;

**Positive control:** GFP injection;**Experimental group:** NLR (enhancer) + GFP

**Immune clearance**

Next, we will quantify the clearance of GFP (Total protein fluorescence intensity & WB) at the molecular level
and confirm that it is cleared by plant’s immune system (ROS detection)

**Protein Fluorescent Analysis**

After one week of injection, we took leaf tissue from the injection area, extracted a fixed amount of total
protein, and measured its fluorescence intensity.

Result shows that compared with NLR\*+GFP group, GFP only and NLR+GFP had significantly higher fluorescence
intensities, which indicates that our modified NLR\* did produce effective clearance of GFP.

However, NLR+GFP also has a tiny reduction in fluorescence intensity compared to GFP only. We believe that this
does not prove that unmodified NLR also has the ability to clear GFP, but can be explained by the increase in
spontaneous dimerization after NLR overexpression [9].

Table 2 Measuring Fluorescent Intensity in in Plant Leaves

| Group | n | Fluorescent Intensity / (10000RFU) |
| Empty vector | 3 | ≈0 |
| GFP only | 3 | 13.39±0.2689 |
| NLR+GFP | 3 | 12.40±0.4821\* |
| NLR\*+GFP | 3 | 8.993±0.2650\*\*\*\* |

![](https://static.igem.wiki/teams/4628/wiki/res/16.png)

Figure 18: Measurement of Fluorescence intensity in the leaves

Note

\*P<0.05, \*\*\*\*P<0.0001, Positive control: GFP only

NLR represents the original unmodified immune molecules.

NLR\* represents an artificially modified molecule whose ID sequence has been replaced with a nanoantibody
Enhancer targeting GFP

**WB**

Since the Enhancer nanobody has the property of increasing fluorescence intensity upon binding GFP, we were not
sure of the immune activation function of the artificially modified NLR only by fluorescence intensity.

Thus, we further designed WB experiment for GFP to directly measure the change of GFP content and thus we can
measure the immune response more rigorously.

![](https://static.igem.wiki/teams/4628/wiki/res/wb1.png)

Figure 19: WB results for GFP

According to the information shown in the figure above, there was a more significant decrease in GFP content in
the NLR\*+GFP group compared to the GFP only group (positive group) and the empty vector group (negative group).

Note:

NLR\* represents an artificially modified molecule whose ID sequence has been replaced with a nanoantibody
Enhancer targeting GFP.

**ROS Measurement**

Reactive oxygen species (ROS) signaling plays an important role in the innate immune response of plants, can
directly inhibit the growth of pathogens, and can also participate in the disease resistance process as a
signaling molecule, and its rapid production is an important marker of plant defense system activation. ROS
production is mainly mediated by NADPH oxidase, also known as Respiratory Burst Oxidase homologous Proteins
(RBOHs) in plants, so plant ROS can be detected by elisa to determine the status and strength of the plant's
immune response. We used the ROS kit (MM-43700M1) to experiment on plant tissues after 1 week of injection

Table 3 Measuring Plant Immune Level through ROS Content in Plant Leaves

| Group | n | content / (pg·ml(-1)) |
| Normal tissue | 6 | 1.716±0.004 |
| Empty vector | 6 | 1.807±0.067\* |
| GFP only | 6 | 1.803±0.045\* |
| NLR+GFP | 6 | 1.835±0.057\*\*\* |
| NLR\*+GFP | 6 | 1.835±0.057\*\*\* |

![](https://static.igem.wiki/teams/4628/wiki/res/17.png)

Figure 20: Measurement of ROS content in the leaves

Note

\*P<0.05, \*\*\*P<0.001,\*\*\*\*P<0.0001

NLR represents the original unmodified immune molecules.

NLR\* represents an artificially modified molecule whose ID sequence has been replaced with a nanoantibody
Enhancer targeting GFP.

As shown in the table and figure above, it can be seen that compared with normal tissue, 3 groups respectively
injected with empty plasmids (1.807±0.067 vs. 1.1.716±0.004, P<0.05), GFP
(1.803±0.045vs.1.716±0.004,P<0.05),and
unmodified NLR+GFP(1.835±0.057 vs. 1.716±0.004, P<0.001) all had significant ROS elevations and remained
similar levels of immunity. This can be explained by the plant's own immune response against Agrobacterium

The experimental group injected with the modified immune receptor + GFP showed a higher immune response
intensity than the uninjected leaves (2.014±0.043 vs. 1.716±0.004, P< 0.0001). Even compared with other
experimental groups (empty plasmid, GFP, unmodified NLR), there was a significant increase in ROS, indicating
that our modified NLR by Agrobacterium transiently exactly enhanced the immune response of plant

### TMV-GFP as pathogen to stimulate immunity

**Material**

- Agrobacterium containing pGreen-pikm1 (Enhancer) plasmid→ later referred to as pikm-1
(Enhancer)

- Agrobacterium containing pGreen-pikm2 plasmids → referred to as pikm-2

- Agrobacterium containing pGreen-TMV-GFP plasmids → referred to as TMV-GFP

**Design**

- Experimental group→ pikm-1 (enhancer) + pikm-2 + GFP

- Control group→ pikm-1 + pikm-2

- Positive control → TMV-GFP

- Negative control → Agrobacterium empty

**Result detection: the method is the same as above**

At first, we did not observe a phenotype in naked eye. We thought that the viral plasmid construction was
unsuccessful, resulting in GFP not being displayed on the surface of the virus. Later, we found that
fluorescence can actually be seen under a fluorescence microscope. Considering that most of the various
references also observe the fluorescence of TMV-GFP virus under a microscope, we speculate that TMV-GFP
constructed by this method may be inherently weak fluorescence and invisible to the naked eye.

However, due to the time limit, we did not have time to complete the phenotypic detection under fluorescence
microscopy and subsequent ROS and WB.

# 4 The module of biosafety

## Testing of the promoter

### Selection and acquisition of promoters

In our understanding, there is a paucity of studies focusing on the promoters of Agrobacterium. To bridge
this gap, we experimented with four promoters.

The first one is a constitutive promoter, 50Spro( [BBa\_K4628022](https://parts.igem.org/Part%3ABBa_K4628022) ), which is responsible for encoding the
50S
ribosomal protein L28 in the Agrobacterium C58 genome [10].

In addition to this constitutive promoter, we also explored three inducible promoters. The first, Pvbp2, is
induced by acetosyringone [11]; the second, PsiA( [BBa\_K2448025](https://parts.igem.org/Part%3ABBa_K2448025) ), is triggered by D-Psicose; and the
third,
Ptac/cuo( [BBa\_K4628024](https://parts.igem.org/Part%3ABBa_K4628024) ), is activated by
4-Isopropylbenzoic acid [12].

It is noteworthy that the first two promoters were derived from Agrobacterium strains and were procured
through colony PCR. The third promoter was a synthetic construct obtained via overlap extension PCR.

### Characterization of the promoter

In our experiments, we characterized promoters using Green Fluorescent Protein(GFPuv, [BBa\_M45116](https://parts.igem.org/Part%3ABBa_M45116) ) as a
reporter gene. The expression of this gene was visualized using a fluorescence microscope and a
multifunctional imager.

Our observations revealed that the constitutive promoter 50Spro exhibited pronounced green fluorescence. The
inducible promoters only Pvbp2 displayed weak fluorescence.

For the characterization of 50Spro, we employed a dual antibiotic LB solid medium containing rifampicin and
kanamycin resistance. After a cultivation period of 48 hours, colonies exhibiting green fluorescence were
observed under UV or blue light.

For inducible promoters, we used a dual antibiotic LB solid medium supplemented with corresponding inducers.
Specifically, PsiA was induced by 150mM D-Psicose, Pvbp2 by 150μM acetosyringone, and Ptaccuo by 150μM
4-Isopropylbenzoic acid. After a period of 48 hours, colony PCR was performed for identification purposes.
Despite successful transformation in all cases, only Pvbp2 showed weak fluorescence. The other two promoters
did not exhibit any observable fluorescence even after centrifugation in liquid medium.

### Result

We observed colonies and bacterial fluid under a fluorescence microscope using blue light as the excitation
source.

![](https://static.igem.wiki/teams/4628/wiki/res/18-1.png)

![](https://static.igem.wiki/teams/4628/wiki/res/18-2.png)

Figure 21: Photographs of colonies under a 4x objective (control on the left and
Agrobacterium with GFP protein on the right).

![](https://static.igem.wiki/teams/4628/wiki/res/19.png)

Figure 22: A photograph of the bacterial solution on a slide under a 10x objective.
(control on the left and Agrobacterium with GFP protein on the right)

Gray values representing relative fluorescence intensity were obtained using a multifunction imager at
consistent exposure times. Examination revealed significant differences. The basal fluorescence of the culture
was subtracted in this analysis. These disparities are discernible between different promoters or distinct
proteins.

![](https://static.igem.wiki/teams/4628/wiki/res/a1-1.png)

![](https://static.igem.wiki/teams/4628/wiki/res/a1-2.png)

![](https://static.igem.wiki/teams/4628/wiki/res/a1-3.png)

Figure 23: FIG.1 Agrobacterium tumefaciens without fluorescent protein; FIG.2 50Spro-GFPuv;
FIG.3
Pvbp2-GFPuv;

![](https://static.igem.wiki/teams/4628/wiki/res/a-2.png)

Figure 24: Comparison of relative fluorescence intensity between the different promoters,
50Spro-GFPuv was very significantly different from the control group (p<0.001), and Pvbp2-GFPuv was
significantly different from the control group (p<0.05).

## Selection of phototoxic proteins

Our engineered strains are mainly used in plants, and plants need sunlight to grow, so we chose sunlight as the
way to initiate suicide.

### KillerRed

We initially considered KillerRed ( [BBa\_K1184000](https://parts.igem.org/Part%3ABBa_K1184000) ).
According to extant research, KillerRed is a red fluorescent
protein that generates reactive oxygen species (ROS) under yellow-green light (540-585 nm). Given that plants
typically absorb blue-violet and red light, reflecting light near 540nm (green), KillerRed is ideally suited
as a biosafety ‘kill switch’.

Other proteins in the KillerRed family, such as Supernova, were also validated. It has been shown to reduce
dimerization tendency and maintain the ability to generate ROS. [13] We obtained its fragment by point
mutation, but light exposure showed that its suicide inducing effect was less stable than that of KillerRed.

### miniSOG

We incorporated the more observable protein miniSOG as a complementary measure. This phototoxic protein,
derived
from LOV protein, produces toxic substances under blue light excitation to kill bacteria [14]. It also emits
green fluorescence for easy observation. To facilitate the use of this protein in Agrobacterium, its codon has
been optimized.

![](https://static.igem.wiki/teams/4628/wiki/res/19-1.png)

![](https://static.igem.wiki/teams/4628/wiki/res/19-2.png)

![](https://static.igem.wiki/teams/4628/wiki/res/19-3.png)

Figure 25: The relative fluorescence intensity of miniSOG and the control group was very
significant (p<0.001)

## Directed evolution of promoters

### Promoter analysis and mutation

We extracted the 40bp sequence before each Agrobacterium tumefaciens gene in NCBI as the key sequence of
promoters and analyzed whether each base was conserved. Then we selected two of the sequence with a total of
12 bases to be mutated using degenerate primers to construct a mutation library. GFPuv was used as a reporter
gene for the screening. In total, we obtained thousands of colonies with different fluorescence intensities
and extracted four of them for amplification. Surprisingly, sequencing results showed that three of the
brightest mutations in our screened strains were the same mutation sequence. We called it 50Spro-e1. This
proved that we had successfully evolved stronger Agrobacterium promoter and stably expressed it.

![](https://static.igem.wiki/teams/4628/wiki/res/30.png)

Figure 26: a. Nonconserved sites in Agrobacterium promoter near the -10 and -35 regions; b.
The sequencing results of Agrobacterium with higher brightness.

### fluorescence detection

we observed the colonies under the UV light, extracted the protein, and detected their fluorescence
intensities using a fluorescent microplate reader. This achievement will not only enhance the control of kill
switches, but also contribute to the iGEM community by providing stronger constitutive promoters for future
teams using Agrobacterium.

![](https://static.igem.wiki/teams/4628/wiki/res/31.png)

Figure 27: The protein fluorescence observation under ultraviolet light

![](https://static.igem.wiki/teams/4628/wiki/res/32.png)

Figure 28: The results of protein fluorescence quantitative detection which were
standardized by OD600 of bacterial solution, and empty plasmid was used in the control group. All
experimental groups showed extremely significant differences from the control group(p<0.0001).

## Construction of plasmid vectors

The plasmid we chose was pGREENll. all of our vectors were accomplished using Gibson Assembly [15]. Given the
unique ability of Agrobacterium species to deliver T-DNA to plants, we strategically designed the biosafety
module to be located outside the LB and RB sequences. This was done to prevent the inadvertent transfer of genes
to plants.

After experimenting with various sites, we ultimately selected to insert the fragment between the origin of
replication and the kanamycin resistance gene. This decision was informed by a range of factors, including
efficiency and stability.

To ensure the success of our transformations, we employed Sanger sequencing for confirmation. This allowed us to
verify that the strains had been positively transformed, thereby validating our experimental approach.

![](https://static.igem.wiki/teams/4628/wiki/res/20.png)

Figure 29: The plasmid we constructed (take 50Spro-GFPuv as an example)

## Illumination experiments

Due to the weak expression of inducible promoter Pvbp2 in promoter characterization experiments, and a high
quantity of acetosyringone has toxic effects [16]. We finally selected 50Spro as the promoter to verify the
suicide effect of phototoxic proteins. and sunlight was selected as the light source for simulating the plant
planting environment.

In the experiment, we used ice packs and foam to control the plate temperature below 30 degrees. After different
times of light experiments, the bacterial culture plates were incubated at 28 °C in the dark for 48 to 72h. Then
count the number of colonies, and the control group treated with empty plasmid in the dark light was used as a
benchmark.

**Results:**

Our experiments with the bacterial solution under light exposure revealed that the bacteria did not exhibit
significant reduction. We hypothesize that the accumulation of toxins within the bacteria may have a buffering
effect on the liquid environment, which could be beneficial for the preservation and transportation of our
engineered bacteria.

In contrast, our plate light experiment demonstrated a noticeable reduction of engineered bacteria under natural
light irradiation, regardless of whether the conditions were sunny or cloudy. However, the rate of bacterial
reduction varies under these two conditions. Given the variability of sunlight in cloudy weather, we opted to
use the cumulative light intensity (lux) over time as a variable for our analysis.

Interestingly, we observed that even in the absence of any promoter ( ‘empty vector’ strain), there was still a
degree of clearance under light exposure. But this effect was less pronounced than in Agrobacterium containing
phototoxic proteins. It suggests that the presence of phototoxic proteins can further reduce their viability.

![](https://static.igem.wiki/teams/4628/wiki/res/21.png)

Figure 30: Results of light experiments of Agrobacterium with KillerRed V.S. empty plasmid
(on a sunny day)

![](https://static.igem.wiki/teams/4628/wiki/res/22.png)

Figure 31: Results of light experiments of Agrobacterium with empty plasmid V.S. minisog (On
a cloudy day)

Furthermore, we conducted plate light experiments at a 1000x bacteria concentration. These experiments showed
that when the cumulative lux over time reached 3\*10^6 (30 min of light on a sunny day), the effect of bacterial
clearance could still be achieved.

![](https://static.igem.wiki/teams/4628/wiki/c1.png)

![](https://static.igem.wiki/teams/4628/wiki/c2.png)

![](https://static.igem.wiki/teams/4628/wiki/c3.png)

![](https://static.igem.wiki/teams/4628/wiki/c4.png)

Figure 32: 1000x concentration scavenging effect of KillerRed (dark control, 10 min, 20 min,
30 min))

## Further experiments

In our final experiment, we analyzed the promoter sequence of Agrobacterium tumefaciens and attempted to
enhance
it through directed evolution by introducing mutations. However, due to time constraints, this experiment was
not completed. In future experiments, we aim to develop a stronger constitutive promoter to enhance the safety
of our engineered bacteria.

Because of the weather, our light experiment could not be repeated enough. Future experiments are needed to
validate the significant killing effect of our phototoxic protein. In addition, we could try the promoter
Pvbp2,
to verify whether this part is indeed inducible by acetosyringone as a kill switch.

# Reference

[1] Zhang W, Thieme CJ, Kollwig G, Apelt F, Yang L, Winter N, Andresen N, Walther D, Kragler F. tRNA-Related
Sequences Trigger Systemic mRNA Transport in Plants. Plant Cell. 2016 Jun;28(6):1237-49.

[2] He Y, Zhang T, Sun H, Zhan H, Zhao Y. A reporter for noninvasively monitoring gene expression and plant
transformation. Hortic Res. 2020 Sep 19;7(1):152.

[3] Tyagi S. Imaging intracellular RNA distribution and dynamics in living cells. Nat Methods. 2009
May;6(5):331-8.

[4] Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM. Localization of ASH1 mRNA particles in
living yeast. Mol Cell. 1998 Oct;2(4):437-45.

[5] Lindbo JA. TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector. PLANT PHYSIOL.
2007;145(4):1232-40.

[6] Man M, Epel BL. Characterization of regulatory elements within the coat protein (CP) coding region of
Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP
subgenomic RNA promoter. J GEN VIROL. [Journal Article; Research Support, Non-U.S. Gov't]. 2004 2004-6-1;85(Pt
6):1727-38.

[7] Chujo T, Ishibashi K, Miyashita S, Ishikawa M. Functions of the 5'- and 3'-untranslated regions of
tobamovirus RNA. VIRUS RES. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2015 2015-8-3;206:82-9.

[8] Bendahmane M, Koo M, Karrer E, Beachy RN. Display of epitopes on the surface of tobacco mosaic virus:
impact of charge and isoelectric point of the epitope on virus-host interactions. J Mol Biol. 1999 Jul
2;290(1):9-20. doi: 10.1006/jmbi.1999.2860.

[9] Tamborski J, Krasileva KV. Evolution of Plant NLRs: From Natural History to Precise Modifications. Annu Rev
Plant Biol. 2020 Apr 29;71:355-378. doi: 10.1146/annurev-arplant-081519-035901.

[10] 徐丽萍. 基于荧光报告载体的农杆菌atu4860基因启动子调控机理研究[D].扬州大学,2017.

[11] Guo ML, Hou QM, Hew CL, et al. Agrobacterium VirD2-binding protein is involved in tumorigenesis and
redundantly encoded in conjugative transfer gene clusters[J]. Mol Plant Microbe Interact, 2007,
20(10):1201-1212.

[12] Denkovskienė E, Paškevičius Š, Werner S, Gleba Y, Ražanskienė A. Inducible Expression of Agrobacterium
Virulence Gene VirE2 for Stringent Regulation of T-DNA Transfer in Plant Transient Expression Systems. Mol Plant
Microbe Interact. 2015 Nov;28(11):1247-55. doi: 10.1094/MPMI-05-15-0102-R. Epub 2015 Oct 12. PMID: 26292850.

[13] Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T,
Inagaki F, Suzuki H, Nagai T. SuperNova, a monomeric photosensitizing fluorescent protein for
chromophore-assisted light inactivation. Sci Rep. 2013;3:2629. doi: 10.1038/srep02629. PMID: 24043132; PMCID:
PMC3775092.

[14] Lafaye C, Aumonier S, Torra J, Signor L, von Stetten D, Noirclerc-Savoye M, Shu X, Ruiz-González R,
Gotthard G, Royant A, Nonell S. Riboflavin-binding proteins for singlet oxygen production. Photochem Photobiol
Sci. 2022 Sep;21(9):1545-1555. doi: 10.1007/s43630-021-00156-1. Epub 2022 Jan 18. PMID: 35041199.

[15] Xia Y, Li K, Li J, Wang T, Gu L, Xun L. T5 exonuclease-dependent assembly offers a low-cost method for
efficient cloning and site-directed mutagenesis. Nucleic Acids Res. 2019 Feb 20;47(3):e15. doi:
10.1093/nar/gky1169. PMID: 30462336; PMCID: PMC6379645.

[16] de Oliveira ML, Febres VJ, Costa MG, Moore GA, Otoni WC. High-efficiency Agrobacterium-mediated
transformation of citrus via sonication and vacuum infiltration. Plant Cell

{% endblock %}