iGEM 2024 — Team zju-china

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{% block title %}Intergrated Human Practices{% endblock %}
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Let us go, eyes on the same sky, hearts beating the drums of the twilight

Let us go, we have not lost our memories, go to find the lake of life—

understand the problem

Design a good, safe, and responsible plan

Stakeholder communication

Close the loop

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Understand the Problem

The history of dyeing is incredibly ancient. Our ancesters used natural dyes from plants, animals, and
minerals. While modern dyeing industries have significantly improved efficiency and variety, they’ve
also caused serious environmental pollution and resource waste. The textile industry produces around 70
billion tons of toxic wastewater annually, with at least 50% of it being wasted. Much of this dye-filled
wastewater is discharged without proper treatment, sometimes even used directly for farmland irrigation,
leading to major environmental damage.

We were inspired by China's traditional tie-dye culture, a part of intangible cultural heritage. Culture
has drawn our attention to this interesting area, but also to the harm done to the environment by the
dyeing industry.In Chromobacterium violaceum, we discovered a unique pigment called violacein. It’s
slightly soluble in water, highly stable, and can be used for textile dyeing. Fabrics dyed with
violacein also have some antibacterial properties. Plus, violacein is a naturally occurring compound
that doesn’t negatively impact ecosystems, and it's widely used in fields like agriculture and food
processing. This led us to adopt it as our dye, and thus "Neovio Dye" was born, representing the future
of violacein dyeing.

Neovio Dye emerged during a brainstorming session where we aimed to recreate the same effects of
traditional tie-dyeing through synthetic biology. Our goal was to pass down this cultural heritage in a
new way, while also minimize environmental pollution. Initially, we tried to mimic the movements of
tie-dye artisans with motion-capture technology and recreate the process through hardware. After several
iterations, Neovio Dye evolved from traditional tie-dye, incorporating memory and random patterns to
achieve effects similar to modern digital printing, but in a much more fun and creative way.

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Design a good, safe, and responsible plan

From the very beginning, our team reached a consensus that we should listen to the world and not work
hard with our eyes covered. At first, we were not sure which stakeholders to contact, deliberate contact
would make the dialogue become a single output. After we had the initial idea, with the progress of the
project, we encountered many problems in theory, experiment, ethics, practice and other aspects, and we
were curious about the potential impact of each change in the project. At this point, it became clear
who to contact, and we believe that such contacts can successfully optimize the project and benefit both
parties.

According to the natural chronological order, we divided the intergrated human practice activities into
project design, experiment stage and keep in touch. in the keep in touch stage, our competition stage
has come to an end, but the story of Neovio Dye will not end. At this stage, we are still actively
contacting stakeholders, exploring the social responsibility and cultural mission of the project, and
making the final sprint for the experiment.

After each phase we had a big Gibbs review to make the next phase more smooth, and the cycle ran three
times in total. The Gibbs reflective cycle was developed by Graham Gibbs in 1988 to provide knots for
learning from experience, and it is ideally suited to our ongoing learning experience as we optimize our
projects. In the process of Evaluation, it is difficult to analyze the problem clearly by relying on
one's own strength. We consider integrating the process of contact with stakeholders into this step,
analyzing and solving problems and taking actions through self-analysis and external help, and
integrating them into the project.

![](https://static.igem.wiki/teams/5319/hp/ihp/1.png)

Figure 1 Gibbs reflective cycle.

We always adhere to the people-oriented, while keeping in mind the ethics, all human practice is carried
out with the consent of the individual concerned. Due to limited time, resources and funds, it is
difficult for us to meet all the requirements, so our projects prioritize certain requirements that meet
the values, so that we can provide the best and optimized solutions for our stakeholders, and the values
summarized after communication with stakeholders are as follows:

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Stakeholder communication

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Close the loop

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Overview

Yield Improvement Module

Cycle1 - purified protein
Cycle2 - phenotypic observation

Painting Module

Cycle1 - write in module based on split-cre-loxP
Cycle2 - Plasmid structure optimization
Cycle3 - Leakage expression risk exclusion

Readout plasmid construction for mutated HYER characterization

Cycle 1 - construction of readout plasmid
Cycle 2 - rectification of readout plasmid
Cycle 3 - purification and proof of fluorescence glowing of readout plasmid

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Overview

“Be brave when necessary, and be decisive when there is no turning back.”

-- Comment on Fan Zhendong's victory in Paris Olympic Games

“Be brave when necessary, and be decisive when there is no turning back.”This comment is a good reflection of
our mood in the process of project realization this year - actively changing the plan and thinking in difficult
times, and constantly learning lessons from setbacks.

The characteristics of bioengineering mainly include modularity and standardization, design and construction
based on engineering thinking, data-driven and computational modeling, and innovative ideas and reform schemes
in practice.

Unlike traditional engineering disciplines such as construction engineering and vehicle engineering,
bioengineering (synthetic biology) is full of instability and unpredictability in the process of practice, but
it is also full of the possibility of breakthrough. ZJU-China2024 is excited to be iGEMers and make our
contribution to the science of synthetic biology, which is full of possibilities. In this year's project, we
hope to show the richness of fashion through our creative engineering, while showcasing our unique engineering
design.

![](https://static.igem.wiki/teams/5319/wet-lab/engineering/1.png)

Fig 1

In the process of project implementation, we always adhere to the bioengineering iterative cycle of
brainstorming, experimentation, and repeated discussion of possibilities, constantly learn from setbacks, and
follow the "design-build-test-learn" cycle.

In the process of project implementation, we carried out various possibility prediction and experimental
exploration, including RNA secondary structure prediction, phenotype observation, protein expression detection,
luorescence expression and so on.

We construct protein-substrate binding models and microbial overall metabolism models through bioinformatics.
(For more information, see the [model page](https://2024.igem.wiki/zju-china/model))

We have also developed user-friendly software that allows users to design the fashion totem they want.
(For more information, see the [software
page](https://2024.igem.wiki/zju-china/software))

We standardized the components involved in the project to meet the needs of the iGEM community. You are very
welcome to use our section and contact us about how to use it. Any suggestions would be greatly appreciated.
To learn more, click [part](https://2024.igem.wiki/zju-china/part).

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Yield Improvement Module

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle1 - purified protein

### Design

We just want to explore and enhance the catalytic activity of the enzyme vioE, so we want to purify a single
vioE enzyme. We began to screen and experiment with different purification methods to select the most suitable
purification method for vioE.

### Build

In view of the wide application of his tag in protein purification experiments, we considered linking the vioE
gene to the LacI inducible promoter commonly used in E.coli, and isolating the pure vioE enzyme by his tag on
the advance marker after induction. At the same time, to verify the stability of our promoter, we attached sfGFP
to the back of the vioE enzyme.

![](https://static.igem.wiki/teams/5319/wet-lab/engineering/2.png)

Fig 2

### Test

Through the above method, we successfully induced the bacterial solution with green fluorescence using IPTG, and
verified that the promoter could be correctly expressed. Subsequently, we obtained the correct expression of
vioE enzyme by WB.

![](https://static.igem.wiki/teams/5319/wet-lab/engineering/3a.png)

Fig 3a IPTG(+/-) control green fluorescence

![](https://static.igem.wiki/teams/5319/wet-lab/engineering/3b.png)

Fig 3b coomassie blue staining results results From A1 to A5 is mixed protein obtained in turn by using washing liquid(buffer A).
From B1 to B8 is target protein obtained in turn by using elution buffer(buffer B).

### Study

The process of protein purification in the previous experiment was time-consuming and laborious. We hope to
evaluate vioE's function by directly observing the phenotype.

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle2 - phenotypic observation

### Design

A series of enzymes in the catalytic process of violacein were transferred into Escherichia coli to directly
observe the phenotype - the amount of pigment production.

### Build

We transferred a series of enzymes in the catalytic process of violacein into Escherichia coli, among which the
enzyme vioE contains different mutants, and directly observed the phenotype - the amount of pigment production
after induction. (See wiki [protocol page](https://2024.igem.wiki/zju-china/protocol)
for details.)

### Test

When OD values were measured after induction, we did observe large changes in enzyme activity brought about by
different vioE mutants. (See wiki [result
page](https://2024.igem.wiki/zju-china/results) for details.)

### Study

The importance of different purification methods for different cell products is well understood. Looking ahead,
we hope to develop methods for the extraction and measurement of most organic pigment synthases and their
products.

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Painting Module

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle1 - write in module based on split-cre-loxP

### Design

We select the division site and design two different vector plasmids to accommodate our split-cre-loxP.(more
detail of [design](https://2024.igem.wiki/zju-china/design) please click)

### Build

Using jump29 and jump42 as backbones, we constructed the Jump42-LoxP-TT-loxP-GFP
plasmid storing loxP sites and
the Jump29-LACuV5-creN-creC plasmid for split cre.

### Test

After molecular cloning, and gene sequencing, finally we got the right plasmid after many failures.

![](https://static.igem.wiki/teams/5319/wet-lab/engineering/4.png)

Fig 4 failure sequencing results
 Due to the proximity of VVD domains, sequencing results
often result in one domain being ignored, making
plasmid validation difficult to sustain

### Learn

However, such a design makes us find that the repetition of VVD at close distances causes great trouble in
sequencing or molecular cloning, and poses serious challenges for subsequent functional verification

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle2 - Plasmid structure optimization

### Design

We designed another two plasmids which two elements of the cre enzyme on two vectors with different replicons
and resistance (pJUMP42-lac promoter-creC-promoter-loxP-TT-loxP-GFP and pET15b-lacUV5-creN) is induced by IPTG
via Lac opreaon, and LacI expression was compensated by pET15 vector.

### Build

We successfully constructed the relevant plasmids, and the sequencing results indicated this.

### Test

We verified the feasibility by means of co-translocation of E.coli and colony PCR, which will also become the
basic paradigm for our subsequent functional characterization, details can be seen on the result page.

### Learn

We were able to observe phenotypes at the genetic level, however, there were still considerable intensity of
leakage expression and unstable expression of splitting enzyme, which prompted further modification of plasmid
structure

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle3 - Leakage expression risk exclusion

### Design

We want to modify the distance from RBS to the start codon to make it easier to express the enzyme. In the
meantime, the cap binging site in front of the GFP component promoter was deleted for less leakage expression.

On the other hand, considering that our modified enzyme works directly on DNA, we designed a PCR-based editing
efficiency detection method to avoid the influence of fluorescence leakage expression.

### Build

We successfully constructed the relevant plasmids with recombination, and the feasibility of the new method is
simply tested.

### Test

We cultured E.coli after co-transfer, and colony PCR verified the leakage level with low cutting activity, based
on which we further performed light-induced characterization to measure its cutting efficiency. (more detail of
[design](https://2024.igem.wiki/zju-china/design) please click)

### Learn

We successfully constructed a tool that can be photoinduced to edit genes, but only in response to one signal,
and it inspires us to explore tool kits that can respond orthogonally to multiple signals.

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Readout plasmid construction for mutated HYER characterization

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle 1-construction of readout plasmid

### Design

To test if mutated HYER could block the expression of genes downstream, we put one loxp site with TRS/RS
sequence on both sides between promoter and RBS, controlling the expression of sfGFP.

### Build

Through homologous recombination, we inserted the TRS/RS-loxp-TRS/RS module into desired place.

### Test

We checked the agar plate but found out that colonies were white.

### Learn

After sequencing, we knew that there were a few bases missing(including ATG) upstream of sfGFP sequence.

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle 2 - rectification of readout plasmid

### Design

We planned to insert the missing bases in two ways: do overlap PCR, or add those bases in a new primer and get
the right DNA fragment with recognition site for another homologous recombination.

### Build

As described above, we carried out overlap PCR and homologous recombination with fragment from regular PCR.

### Test

Gene sequencing showed that the plasmid was right this time. Theoretically, the plasmid might appear green
without any intervention. Actually, we picked green colonies on the agar plate and got the right sequence. But
after first readout-HYER co-transformation test, we found out that in control group(just readout plasmid), only
from those white colonies could we find right sequence.

### Learn

We thought it was indicated that the plasmid wasn’t pure from the beginning(probably because there could be
multiple plasmids in one bacterium though the copy number of our readout plasmid was small), so we had to dilute
the bacteria solution and culture them on agar plate again to get single colony that seemed to be white.

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Cycle 3 - purification and proof of fluorescence glowing of readout plasmid

### Design

Since there're different copies of plasmids in our bacteria and the right plasmid tended to make colonies white
instead of green, we decided to isolate those white colonies for plasmid purification.

### Build

We repeated plate smearing with diluted bacteria solution, and picked relatively white colonies for purified
readout plasmids and green colonies as control group of wrong plasmids.

### Test

Via gene sequencing, we confirmed that the white colonies produced the right plasmids, and proved sfGFP was
expressed by using fluorescence microplate reader.

### Learn

Though the colonies still appeared to be low in fluorescence intensity, the bacteria solution indeed glowed
fluorescent green under fluorescence microplate reader, so the readout plasmid was practicable for next step. We
thought sfGFP might not work well as a direct fluorescence readout for our experiment because of low efficiency
of the ribozyme and the fact that amount and characterization performance of sfGFP was not ideal, so we finally
turn to qRT-PCR instead of fluorescence microplate reader as the final readou

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Reference

[1] Light-Inducible Recombinases for Bacterial OptogeneticsMichael B. Sheets, Wilson W. Wong, and Mary J.
DunlopACS Synthetic Biology 2020 9 (2), 227-235 DOI: 10.1021/acssynbio.9b00395

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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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](https://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)

{% endblock %}

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{% block title %}Results{% endblock %}
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Yield Improvement

Construction of producing strain
Semirational design of enzymes
RNA skeleton assists phase separation to improve yeild
Metabolic modification of E. coli

Communication module

characterization of receptors sensitivity
Construction and verification of producer
Functional verification of signal transduction

Painting module

Controlled pigment synthesis

Blocking module

Acquire wild type and mutated HYER
Construct sfGFP readout plasmid for function characterization
Confirm transcription level with qRT-PCRR

The module of biosafety

Construction of plasmid vectors
Illumination experiments

Reference

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Yield improvement module

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Construction of producing strain

In order to enable Escherichia coli to better express purple bacillin synthesis genes[1], we performed
codon optimization on these genes and synthesized purple bacillin synthesis genes suitable for
expression in Escherichia coli. Using homologous recombination technology, we constructed plasmids
containing five enzymes for the synthesis of purple bacillin.

![](https://static.igem.wiki/teams/5319/wet-lab/results/1.jpg)

Figure 1 Construction of two plasmids containing vioA, vioB, vioC, vioD, vioE. a.The plasmid
containing vioA, vioB, vioD; b..The plasmid containint vioC, vioE

Two plasmids above were transferred into Escherichia coli K12 by heat shock conversion, and the results
showed that the colonys turned out to be purple after 12 hours(Fig 2A).
In order to understand the growth status of E. coli after expressing heterologous protein, we measured
its growth curve. It was found that the high expression of these five proteins inhibited the growth of
E. coli to some extent (Fig. 2B).

![](https://static.igem.wiki/teams/5319/wet-lab/results/2.jpg)

Figure 2 Phenotypic observation of Escherichia coli carrying five enzymes. a.The color of the
strain with the five enzymes; b.The growthcurve of the strain with the five enzymes compared with
the wild-type,

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Semirational design of enzymes

Our team identified the conserved domain of the vioE gene family and we found at least four conserved
domains on the vioE sequence derived from Purplebacterium(Fig. 3 a). The docking simulation between vioE
and substrate molecules is also an important basis for us to determine the amino acid mutation site
library. It can be seen from the docking details(Fig. 3 b) that ASP at positions 27 and 28 provides
charge forces for substrate catalysis, and PRO at position 52 provides rigid forces for the stability of
the substrate pocket. Based on the above considerations, we obtained a library of amino acid mutation
sites with high transformation potential(Fig. 3 c)

![](https://static.igem.wiki/teams/5319/wet-lab/results/3.jpg)

Figure 3. Screening of amino acid mutation sites. a. Identification of the homologous domain of
vioE from multiple species and construction of an evolutionary tree reveal the conservation of the
modified site; b. Detailed diagram of the docking between vioE and the substrate, showing directly
interacting amino acid residues and hydrogen bonds; c. Final determination Amino acid mutation site
library.

Considering the catalytic mechanism of vioE[2], we selected two sites with high design potential from
the point mutation site library: 151Asp and 187Phe. And saturated point mutation primers were designed
based on the pJUMP29-1A plasmid (Fig 4 a):
Mu-F: TCCGAGCACCCTGTATCTGNNKGCAGCGTCCGGCGAACC
Mu-R: CGTTAACGNNKAGCGGCAAACACAGTCCGGGATCTCC

By ligating the product obtained by PCR with this pair of primers to the original plasmid, we
successfully constructed a saturated mutation library at the 151Asp and 187Phe sites (Figure 4 a, b, c).
We introduced the CE plasmid and ABD plasmid with the saturated mutation library into E. coli K12
(Figure 4 d). Starting from the growth of the first strain, we used time-lapse photography to
continuously capture images for 7 hours, recording the previous The growth process of 42 single
colonies.

![](https://static.igem.wiki/teams/5319/wet-lab/results/4.jpg)

Figure 4. Construction of protein mutant library. a. Plasmid map of the double-point saturation
mutation library; b. Point mutation followed by amplification primers and sequences; c. Point
mutation sequencing results, with red boxes marking the successfully mutated protein coding sites at
position 151 and position 187; d. The plasmid of the mutant library was successfully transferred
into E. coli and grown. Produce violacein.

Subsequently, we transferred it to a 1.5 ml EP tube containing 1 ml of liquid LB medium for preliminary
incubation. After 5 hours of incubation, measure the OD600 of the bacterial solution. We transfer the
incubated bacterial solution to a shaking tube containing 5 ml of LB medium. After 13 hours of
fermentation, dissolve it with methanol and incubate it for 5 hours. We measure the purple color in the
solution. Concentration of bacteriocin - OD578. Repeating this three times, we obtained the following
results (Figure5 a).

Through heterogeneous ensemble learning, we constructed the enzyme activity from the enzyme molecular
structure property data (including but not limited to molecular properties and energy landscape data)
obtained from the two-site saturation mutation library (in this experiment, we used the crude protein of
violacein. Yield represents the causal relationship diagram of enzyme activity) contribution. Then a
baseline is constructed by rearranging the graph relationship, and the graph model constructed in the
previous step is tested; the data is recombined based on the model that least violates the local markov
condition in the second step, and multiple rounds of single Bayesian sampling was performed to generate
the expectation-multifactor distribution map (Figure 5 b).

Based on this relationship, we were able to screen out the sample group with the greatest increase in
enzyme activity from a larger virtual mutant library. In the predicted region (pocket size 600Å±100Å or
1200±100Å, channel length 6±2Å), it is most likely to obtain mutants with higher enzyme activity (Figure
5 b), so we conducted further experimental verification and repeated it twice The measurement results
are shown in Figure 5 c. The correctness of the software was confirmed by the experimental results. We
obtained mutants that increased the enzyme activity by up to 32% in the predicted region (pocket size
600±100Å or 1200±100Å, channel length 6±2Å).

![](https://static.igem.wiki/teams/5319/wet-lab/results/5.jpg)

Figure 5. vioE semi-rational design. a. Double-point mutation library enzyme activity data; b.
Bayesian sampling process of the enzyme activity prediction model; c. Larger virtual Samples
screened from the mutant library were re-verified for yield.

We performed MD simulations[3] on Glu-His mutants to elucidate the molecular mechanism of increased
enzyme activity. Compared with WT-ligand, the mutant significantly enlarged the substrate binding pocket
(Fig.6 a,b). The larger substrate-binding pocket reduces the steric hindrance for ligand binding to the
enzyme, thus facilitating the catalytic reaction. The Rg value of the mutant complex was significantly
greater than the Rg value of the wild-type (WT) protein, indicating that after the substrate was docked
with the mutant protein, the protein chain expanded and the enzyme activity increased relative to the WT
protein (Fig.6c, d).

Calculation of the number of hydrogen bonds showed that the mutant complex formed more hydrogen bonds
than the WT protein, thereby increasing the rate of the catalytic reaction (Figure 6e, f). Furthermore,
we noticed an altered charge distribution in the mutant substrate binding pocket (Fig.6g, h). This
results in a reduction in the number of positive charges in the active center and a shift in charge
effect to neutrality. Improve the enzyme activity of vioE by eliminating the influence of electrostatic
interactions.

![](https://static.igem.wiki/teams/5319/wet-lab/results/6.jpg)

Figure 6. Structural basis for the altered activity of the mutant. a, b. Analysis of Substrate
Pocket Volume Differences; c、d. Calculating the number of hydrogen bonds; e, f. Rg calculation;
g、h. Substrate pocket surface charge changes

![](https://static.igem.wiki/teams/5319/general/star-24.png)

## RNA skeleton assists phase separation to improve yeild

We plan to co-localize vioABCDE with the help of specific interaction between RNA hairpin boxB and
λ-peptide. To achieve this effect, we have to add λ-peptide onto each of the five enzymes, which raises
the risk that the activity of enzymes may be influenced.

### Structure prediction

Firstly, we predict the structure of vioABCDE with or without λ-peptide at N-terminal with the help of
Alphafold2 as shown in figure 1. Then we overlap corresponding proteins respectively to see if the
structure of the enzyme itself is damaged with λ-peptide.

From the overlap results, we find out that though there’re still some changes in the structure of vioB
and vioC, most domains don’t seem to shift much.

![](https://static.igem.wiki/teams/5319/wet-lab/results/7.png)

Figure 7. structure overlap of vioABCDE with and without λ-peptide

The sequence of RNA scaffold from the paper “Spatial engineering of E.coli with addressable
phase-separated RNAs”[4]. To test if different number of boxB will work in different efficiency, we only
retain two boxB with BcuI restriction site in between for further tests. By the way, we visualize the
interaction of RNA scaffold and λ-peptide-sfGFP with Alphafold3. Figure 8 shows the interaction
prediction of λ-peptide-sfGFP with a 74-nt RNA sequence with one boxB, indicating they’re jointed
together by λ-peptide and a RNA hairpin. Figure 9 shows the interaction prediction of λ-peptide-sfGFP
with the whole 47CAG-2boxB RNA scaffold. However, the predicted structure of whole RNA scaffold is low
in credibility.

![](https://static.igem.wiki/teams/5319/wet-lab/results/8.jpg)

Figure 8. interaction prediction of λ-peptide-sfGFP with a one-boxB RNA sequence

![](https://static.igem.wiki/teams/5319/wet-lab/results/9.jpg)

Figure 9. interaction prediction of λ-peptide-sfGFP with the whole RNA scaffold

### plasmid constraction

We finally added λ peptide and GS linker to the front of every enzyme, and we proved that the added λ
peptide and GS linker won’t infulent their function and the strain with these enzymes that has been
modified could turn to purple as expected(Fig 10).

![](https://static.igem.wiki/teams/5319/wet-lab/results/10.jpg)

Figure 10. Strain with modified vioA/B/C/D/E

Unfortunately, although we initially obtained a long fragment of multi-copy boxb through RCA reaction
and overlap PCR(Fig.11), due to time reasons, we failed to finally construct an rna skeleton expression
plasmid with multi-copy boxb so we couldn’t test the function of RNA skeleton.

![](https://static.igem.wiki/teams/5319/wet-lab/results/11.jpg)

Figure 11. gel image of multiple-boxB fragment yet to insert built with different methods

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Metabolic modification of E. coli

Thanks to the help of modeling, we constructed a whole-genome model[4] of E. coli that introduced the
heterologous synthesis pathway of violacein, and screened out the appropriate modified gene through the
metabolic flux model - the gene tnaA encoding tryptophanase (responsible for l -tryptophan degradation)
and sdaA involved in serine degradation (See [modeling page](https://2024.igem.wiki/zju-china/model)).

We chose to use λRed recombination technology to complete the target gene replacement of chassis
microorganisms (Fig. 12 a). For this purpose, we selected 500 bp upstream and downstream sequences of
the target genes (tnaA and sdaA) as the homology arms of the knockout fragment, and selected FRT
sequences at both ends. The KanR and CAT genes were used as resistance selection markers, and overlap
PCR was used to connect the upstream and downstream homology arms and the selection genes to obtain a
complete gene knockout fragment (Fig. 12 b). Then, electroporation was used to transform the gene
knockout fragment into arabinose-induced E. coli K12-PKD46 electroporation competent cells. After
incubation and growth, two single-gene knockout strains were finally obtained. We verified the success
of the knockout by designing specific primers in the internal and external homology arm segments of the
target gene (Fig. 12 c).

![](https://static.igem.wiki/teams/5319/wet-lab/results/12.jpg)

Figure 12. Verification of metabolic engineering and yield improvement of E. coli K12. a.
Indicates the relative position of the target gene and the gene knocked out by homologous
recombination; b. Indicates the PCR verification of the gene knockout E. coli colony

In order to verify the impact of this metabolic modification on the biological performance of the host bacteria, we measured the growth curve of the gene knockout strain and compared it with the wild type. We discovered that the maximum growth amount and growth rate of the modified strain decreased. After that, we verified the ability of the metabolically engineered strain to produce violacein. Finally, although the mutant's growth was somewhat affected, it can be seen that the violacein production of the △tnaA strain is significantly increased by 48% compared with the wild type, and the violacein production of the △sdaA strain is significantly increased by 23%.

![](https://static.igem.wiki/teams/5319/wet-lab/results/13.jpg)

Figure 13. Growth curve of metabolically engineered strain measured for yield verification. a.
Growth curves of two gene knockout types; b. Determination of violacein production of two gene
knockout types.

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Communication module

After reading a literature[4], we found that the production of final color can be changed by modify the
expression of vioC and vioE. While vioA, vioD, vioB and vioE are expressing normaly without vioC, the
produced pigment will be green one(oxychromoviridans). Nevertheless, if the five genes are expressing
togather, the pigment will be the purple one(violacein). Therefore, we hope to use two orthogonal quorum
sensing systems to regulate the expression of vioC and vioE respectively to get different colors.

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## characterization of receptors sensitivity

We first constructed the promoters corresponding to RhlR[5] and nahR[6] in front of sfGFP, which are
induced by C4-HSL and salicylic acid respectively. Meanwhile, we constructed a plasmid containing
corresponding receptors.

![](https://static.igem.wiki/teams/5319/wet-lab/results/14.jpg)

Figure 14. Plasmid containing RhlR and nahR

In order to verify the induction ability of salicylic acid and C4-HSL on the promoters we used, and to
avoid the situation that excessive concentration of salicylic acid would lead to poor bacterial growth
in subsequent experiments, we used the fluorescence microplate to detect the fluorescence intensity
under different signaling molecule concentrations. The results showed that the fluorescence intensity increased gradually with the salicylic acid concentration increasing, and then decreased because the salicylic acid concentration is too high, which inhibits the growth of bacteria. And the
higher the concentration of C4-HSL, the higher the fluorescence intensity. And we found that salicylic
acid promoter has certain leakage expression. (fig 15)

![](https://static.igem.wiki/teams/5319/wet-lab/results/fig-15.png)

figure 15. RhlR and nahR sensitivity detection. a.nahR sensitivity
detection.
; b.RhlR sensitivity detection

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Construction and verification of producer

Since the AHL synthetase may produce different AHL molecules in different species of bacteria, we chose
the C4-HSL synthetase(pagI) of Pantobacter, which is closely related to Escherichia coli. We also found
out pchBA can control sal synthesis in E.coli. By genes synthesis and homologous recombination, We
constructed plasmids respectively contain AHL synthetase and salicylic acid synthetase gene.

![](https://static.igem.wiki/teams/5319/wet-lab/results/16.jpg)

Figure 16. Two plasmids containing PagI and pchBA respectively.

The control strains containing pagI gene were fermented and extracted to obtain methanol solution
containing AHL molecules, and the AHL molecular characteristics were determined by mass spectrometry. As
can be seen in the figure below, heterogenous expression of pagI in Escherichia coli can produce a
common characteristic peak of AHL molecules (m/z=102), and this AHL molecule peaks at 4.3min to 4.6min.
As shown in Figure B, further mass spectrometry showed that the mass charge ratio of the AHL molecule in
cationic mass spectrometry was 172. Therefore, it can be shown that this gene can heterogeneously
produce AHL molecules in Escherichia coli, and the AHL molecules are C4-HSL and it is exactly C4-HSL
molecules.(Fig. 17)

![](https://static.igem.wiki/teams/5319/wet-lab/results/17.jpg)

Figure 17. Mass spactrum of AHL produced by PagI. a. Graph extracted out from m/z=102.055. b.
Graph showing the different m/z peaks that appear at 4.5 minutes, the pink lines represent the map
of strains with pagI, and the blue lines represent the map of wild-type E. coli

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Functional verification of signal transduction

In order to further verify that the signaling molecules can be accepted by the corresponding receptor
strains after diffusion outside the cell, and that the two quorum sensing systems do not interfere with
each other, we separately point different receptor strains under different control strains, and the
results show that our two quorum sensing systems have strong orthogonality. The signaling molecules
produced by the control strain can spread outside the cell and be sensed by the corresponding receptor
strain, but due to the limited concentration of the generated signaling molecules, the receptor strain
that is far away from the control strain will have difficulty in accepting the signaling
molecules(Fig.18a,b). And we further verify that these two quorum sensing systems do not interact with
each other(Fig. 18c,d).

![](https://static.igem.wiki/teams/5319/wet-lab/results/fig-18.png)

Figure 18. Function and independence verification of two quorum sensing system

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Painting module

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Controlled pigment synthesis

In order to control the formation of different color pigments, we modified the vioC-vioE plasmid,
changing the promoter before vioC to P\_RhlR and the promoter before vioE to P\_nahR, and inserted a
strong terminator between the expression modules of vioC and vioE to avoid the interaction between them.

![](https://static.igem.wiki/teams/5319/wet-lab/results/19.jpg)

Figure 19. Plasmid of vioC and vioE whose promoter have been changed to P\_RhlR and P\_nahR
respectively

To verify the regulation of the produced pigment color by the presence of salicylic acid and C4-HSL, we
transferred the plasmids with vioA, vioD, vioB, double receptor plasmid and the above plasmids into E.
coli K12. They were treated with salicylic acid induction and co-induction of salicylic acid and C4-AHL
by adding corresponding signal producing strains, and were fermented for 24 hours. The results showed
that the strains can be induced by the control strains and can produce different color according to the
the kind of signal sended by controller strains(Fig.20).

![](https://static.igem.wiki/teams/5319/wet-lab/results/20.jpg)

Figure 20. Bacterial fluid color induced by different control strains

Firstly, we constructed the split-cre-loxP system plasmid, this part contains pJUMP42-lac promoter-creC-promoter-loxP-TT-loxP-GFP (figure21.a)and pet15b-lacUV5-creN (figure21.b), For better comparison, we also constructed plasmids containing complete cre, pet15b-lacUV5-cre(figure18.c) and corresponding pJUMP42-promoter-loxP-TT-loxP-GFP(figure21.d).[7]

![](https://static.igem.wiki/teams/5319/wet-lab/results/figure21.png)

Figure 21.the plasmid design of memeory system

In our design, after co-transiting Escherichia coli K12, iptg was used to induce the expression of mitosome, and we can see it in Coomassie bright blue stain. After protein successfully induced, through colony PCR, we successfully observed that the combination of creN and creC showed activity, resulting in the same strip cutting as the positive control group, that is, the complete cre enzyme expression group, and a relatively complete editing level after a lone time induced culture, while the control group showed that no single split cre element could do this. However, we observed the weak cleavage activity in the presence of creC alone in the cutting activity of single creC components was observed in the experiment of culture for two days after one induction (figure22.b), but this phenomenon was not observed in subsequent repetitions, which may be a phenomenon of activity leakage with a low probability

![](https://static.igem.wiki/teams/5319/wet-lab/results/figure22.jpg)

Figure 22.Verification of split-cre-loxP gene editing activity only when two components are present at the same time

Further, considering the weak cleavage activity in the presence of creC alone, we tried to explore the influence of different cleavage sites on the activity after cleavage enzyme polymerization, aiming to develop a split-cre-loxP system with lower background leakage level and higher cleavage activity.We selected a total of 3 candidates through comprehensive literature reports [1][2][3]and software algorithm prediction[4], and observed that the division at 43AA had the best division efficiency and activity

![](https://static.igem.wiki/teams/5319/wet-lab/results/23333.png)

Figure 23.Verification of gene editing efficiency at different division sites
a. Simple information and screening basis of candidate fission sites
b. Coomassie bright blue staining successfully induced the expression of various components of mitase
\*:creC
\*\*:creN
\*\*\*:cre
\*\*\*\*:GFP
c. d. Two efficiency tests at three split sites in different repeat experiments
e. cleavage efficiency test histogram

Next, we characterized split-cre-loxP. By changing the influence of different induction time and lighting time, we obtained the experimental results as shown in the figure below, which further proved its feasibility, indicating that 2-4 hours of light time may be the best time for light induction, while too long time of light induction may lead to the stagnation of cutting activity, it may be because of the inhibition of protein synthesis, the degradation of fission enzymes, and the failure of IPTG..

![](https://static.igem.wiki/teams/5319/wet-lab/results/figure24.jpg)

Figure 24.Blue light induction duration experiment

On the basic split-cre-loxP, we try to extend the orthogonality to include the orthogonality of loxP sites in order to realize the regulation of orthogonal multiplex signals

![](https://static.igem.wiki/teams/5319/wet-lab/results/figure25.jpg)

Figure 25.simply test of orthogonal loxp site

in the read in part, we select a set of toehold switches characterized by iGEM to verify its specific ability to read data. At first, we tried to apply RNA trigger externally to observe the color change of single colonies, but the expensive price of RNA synthesis and high usage prevented us from making many attempts and did not get good results. We turned to the construction of the expression of trigger RNA in bacteria and ultimately simply obtained the activation of green fluorescence, and plan to design more complete phenotypic experiments in the future

![](https://static.igem.wiki/teams/5319/wet-lab/results/figure26.jpg)

Figure 26.simply test of toehold switch .The left to right co-rotation combinations are toe1，toe1+trigger3，toe1+trigger1，toe3，toe3+trigger1， toe3+trigger3
(BBa\_K2621011,BBa\_K2621011+BBa\_K2621016,BBa\_K2621011+BBa\_K2621014,BBa\_K2621013,BBa\_K2621013+BBa\_K2621014,BBa\_K2621013+BBa\_K2621016)

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Blocking module

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Acquire wild type and mutated HYER

Firstly, we acquire sequence of original HYER-1 from the paper which identified it. Based on its
structure analysis, we focus on 3 sites: catalytic triad, J2/3 and 2nt bulge. After literature work, we
decide to make 3 mutations: G567A[10], G338A[11], 584\_585del[12].

To visualize structure of mutated HYER more clearly in case it loses basic conformation, we simulate
their folding with DNAMAN in comparison with original HYER(Fig.27), and assume that they fold roughly in
the same way.

![](https://static.igem.wiki/teams/5319/wet-lab/results/27.jpg)

Figure 27. predicted structure of HYER and mutated HYER, from DNAMAN

We use point mutation kit to build mutated HYER. Since the recognition site of HYER widely exists in
vectors, we don’t put a promoter upstream of HYER in the beginning, and finish point mutation at this
stage. Later, the promoter is added. We choose lacUV5, for its transcription initiation site is
relatively clear(6-8 bp downstream from the Pribnow box).

![](https://static.igem.wiki/teams/5319/wet-lab/results/28.jpg)

Figure 28. lacUV5 upstream of HYER, recognition site for transcription initiation is shown in
green

To make HYER’s recognition more specific, we add 14-nt recognition site referring to the article
identifying it. Probably because of its catalytic activity, we only get two mutated HYER in this
way(plasmids shown below).

![](https://static.igem.wiki/teams/5319/wet-lab/results/29.jpg)

Figure 29. the general view of lacUV5-J2/3-RS and lacUV5-2nt-RS

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Construct sfGFP readout plasmid for function characterization

To test if mutated HYER works as we want, we construct readout plasmids containing one loxp site with
TRS/RS on both sides, simulating controlled sequence with recognizable sites. The bacteria containing
readout plasmids appear green without potential blocking effects of HYER.

For tests of blocking function, TRS/RS sequence are added to the sequence between promoter and RBS as
shown in figure 30.

![](https://static.igem.wiki/teams/5319/wet-lab/results/30.jpg)

 Figure 30. readout plasmid(left) and the recognized blocking part(right)

We’ve planned to characterize the blocking activity by quantifying the intensity of sfGFP. After
co-transformation with mutated HYER with/without RS, we find that colonies from both control group and
co-transformation group appear white instead of green, and none of the several modifications in readout
plasmid work it out. This may be due to the addition of recognition sites too close to RBS, resulting in
the formation of toe like structures that inhibit the translation of GFP.

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Confirm transcription level with qRT-PCR

We co-transform readout plasmid and each of the two plasmids with constitutive expression of mutated
HYER(J2/3 mutation and 2nt bulge mutation). After ~24h of culturing on agar plate(Amp+ Spec+ for
co-transformation, Spec+ for readout only), they’re kept in -70℃ fridge temporarily and refreshed when
we’re ready to test the expression of sfGFP.

For qRT-PCR, we prepare 4 groups: control group with readout plasmid only(negative control), readout
plasmid with RS-inserted J2/3 mutated HYER, readout plasmid with RS-inserted 2nt bulge mutated HYER, and
readout plasmid with 2nt bulge mutated HYER(conditional negative control).

For HYER expression detection, it’s clear that this RNA is expressed in all three co-transformation
groups, as shown in figure 31.

![](https://static.igem.wiki/teams/5319/wet-lab/results/31.jpg)

Figure. 31 qRT-PCR results of HYER expression

Since there’re some accidents in readout plasmid building, and we’re
out of time to use the right plasmid for formal repetition experiments to check the transcription level
of sfGFP, we currently lack the data of sfGFP mRNA expression.

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# The module of biosafety

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Construction of plasmid vectors

The plasmid we chose was pJUMP29, all of our vectors were accomplished using Gibson Assembly.

![](https://static.igem.wiki/teams/5319/wet-lab/results/32.jpg)

Figure32 .The plasmid for biosafety

![](https://static.igem.wiki/teams/5319/general/vector-583.svg)

## Illumination experiments

We chose KillerRed that had been used in the iGEM Parts ( BBa\_K4628047) to commit suicide. To test
whether the killerre system is still applicable in E. coli, we did a simple and interesting
light-induced experiment.

Under the same bacterial concentration, we performed white light irradiation on four groups with the
same bacterial patterns for different durations. It can be seen that there is a clear trend of bacterial
clearance after 3 hours of irradiation.

![](https://static.igem.wiki/teams/5319/wet-lab/results/33.jpg)

Figure.33  scavenging effect of KillerRed. From left to right are the dark control group, the
1-hour light exposure group, the 2-hour light exposure group, and the 3-hour light exposure group.

![](https://static.igem.wiki/teams/5319/general/star-24.png)

# Reference

[1]Kang MS, Han SS, Kim MY, Kim BY, Huh JP, Kim HS, Lee JH. High-level expression in Corynebacterium
glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production. Appl
Microbiol Biotechnol. 2014,98:4379–87.

[2]Ryan KS, Balibar CJ, Turo KE, Walsh CT, Drennan CL. The violacein biosynthetic enzyme VioE shares
a fold with lipoprotein transporter proteins. J Biol Chem. 2008 Mar 7;283(10):6467-75.

[3]Collier TA, Piggot TJ, Allison JR. Molecular Dynamics Simulation of Proteins. Methods Mol Biol.
2020;2073:311-327. doi: 10.1007/978-1-4939-9869-2\_17. PMID: 31612449.

[4]Guo, H., Ryan, J.C., Mallet, A., Song, X., Pabst, V., Decrulle, A.L., & Lindner, A.B. Spatial
engineering of E. coli with addressable phase-separated RNAs. bioRxiv.2020;185(20):2823

[5]Kang MS, Han SS, Kim MY, Kim BY, Huh JP, Kim HS, Lee JH. High-level expression in Corynebacterium
glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production. Appl
Microbiol Biotechnol. 2014;98:4379–87.

[6]Jin, X., Gao, Y., Chen, X., Wang, S., Qi, Q., & Liang, Q. The construction of the self-induced
sal system and its application in salicylic acid production. Molecules, 2023;28(23):7825.

[7]Light-Inducible Recombinases for Bacterial Optogenetics Michael B. Sheets, Wilson W. Wong, and
Mary J. DunlopACS Synthetic Biology, 2020;9 (2): 227-235.

[8] Weinberg, B.H., Cho, J.H., Agarwal, Y. et al. High-performance chemical- and light-inducible
recombinases in mammalian cells and mice. Nat Commun 2019;10:4845.

[9] Wu, J., Wang, M., Yang, X. et al. A non-invasive far-red light-induced split-Cre recombinase
system for controllable genome engineering in mice. Nature Communication,2020; 11:3708.

[10]Dagliyan, Onur, Andrey Krokhotin, Irem Ozkan-Dagliyan, Alexander Deiters, Channing J. Der, Klaus
M. Hahn, and Nikolay V. Dokholyan. "Computational design of chemogenetic and optogenetic split
proteins." Nature Communications, 2018;9(1): 4042

[11]Chan RT, Robart AR, Rajashankar KR, Pyle AM, Toor N. Crystal structure of a group II intron in
the pre-catalytic state. Nat Struct Mol Biol. 2012;19(5):555-7. .

[12]Jeong W, Kang C. Start site selection at lacUV5 promoter affected by the sequence context around
the initiation sites. Nucleic Acids Res. 1994 11;22(22):4667-72.

[13]Liu ZX, Zhang S, Zhu HZ, Chen ZH, Yang Y, Li LQ, Lei Y, Liu Y, Li DY, Sun A, Li CP, Tan SQ, Wang
GL, Shen JY, Jin S, Gao C, Liu JG. Hydrolytic endonucleolytic ribozyme (HYER) is programmable for
sequence-specific DNA cleavage. Science. 2024;383(6682):eadh4859.

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