iGEM 2025 — Team ucas-china

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# Overview

To record the daily wetlab progress of our team and analyze the experimental results timely, we log the experimental
contents for every experiment, and finally organize them. The compiled notebook is as follows.

# Week 3, November 2024

The Initial Planning for 2025

* Promote our team across UCAS and begin recruitment efforts.
* Deliver introductory presentations to interested students.

# Week 4, November 2024

The Initial Planning for 2025

* Conduct recruitment interviews
* Finalize the selection of new members.

# Week 1, December 2024

The Initial Planning for 2025

* Organize Ice-breaking activities to integrate the new members into the team.

# Week 3, December 2024-Week 2, January 2025

Training for New Members

* Presented reviews of wikis from previous iGEM teams to familiarize new members with the competition.
* Provide theoretical and experimental wetlab training for new members.

# Week 1, March-Week 3, April 2025

Brainstorming

* Gather project ideas from winter brainstorming.
* Introduce the concept of a proteolysis-activated logic gate as a potential component of our project.

# Week 4, April-Week 2, May 2025

Further Brainstorming and Concept Development

* Selected tomato and *Pseudomonas syringae* pv. *tomato* (Pst) as the central elements of the
  project.

# Week 3, May-Week 2, June 2025

Pathway Design

* Define the overall framework and route for our project.
* Establish the experimental verification plan for the project.

# Week 3, June 2025

Pathway Design, Transformation, and Fluorescence Detection

* Conduct further background research on trehalase.
* Complete the initial design for most plasmids.
* Design primers for DNA cloning.
* Transform the constructed plasmids into E coli. BL21(DE3) strain competent cells.
* Carry out preliminary gene expression tests.

# Week 4, June 2025

Pathway Design and Transformation

* Perform additional background research on AvrRpt2.
* Transform constructed plasmids into BL21(DE3).
* Clone DNA fragments via PCR.

# Week 2, July 2025

Pathway Design and Plasmid Construction

* Conduct further background research on the second pathway.
* Transform constructed plasmids into the E coli. Top 10 and BL21(DE3) strains.
* Apply the overlap PCR technique to assemble DNA fragments.
* Perform homologous recombination for plasmid construction.

# Week 3, July 2025

Project Finalization, Plasmid Construction and Validation

* Finalize the design of the second pathway.
* Transform DNA fragments into Top 10 and BL21(DE3).
* Utilize overlap PCR and touchdown PCR techniques to construct plasmids.
* Send constructed plasmids for sequencing.

# Week 4, July 2025

Plasmid Redesign, Construction, and Protein Preliminary Expression

* Redesign the core plasmids of split\_GFP.
* Verify the transformation via PCR.
* Construct plasmids applying the overlap PCR technique.
* Induce gene expression with IPTG.

# Week 1, August 2025

Induction and Protein Purification

* Continue the induction.
* Verify expression by SDS-PAGE.
* Purify protein with Ni-NTA affinity chromatography.
* Construct plasmids via overlap PCR and homologous recombination.

# Week 2, August 2025

Bioluminescence Detection, Validation.

* Detect the bioluminescence signal from split\_Luc.
* Verify the protein expression by performing SDS-PAGE.

# Week 3, August 2025

Induction and Protein Purification

* Continue the induction of split\_GFP.
* Purify nTEVp utilizing Ni-NTA affinity chromatography.

# Week 4, August 2025

Plasmid Redesign, Construction, Induction, and Protein Purification.

* Redesign the insoluble proteins with an MBP-tag added.
* Construct the trehalase plasmid (TreA) by homologous recombination.
* Re-induce split\_GFP expression.
* Continue ongoing protein purification.

# Week 1, September 2025

Validation

* Verify the transformation by colony PCR.

# Week 4, September 2025

Plasmid Redesign, Reconstruction, and Validation

* Redesign the plasmids with AvrRpt2 cleavage sites.
* Verify TreA transformation by PCR.
* Reconstruct the plasmid with cTreA.

# Week 1, October 2025

Wiki Freeze, Presentation Video, and Protein Purification

* Utilize varied methods for the purification of proteins in the pellet.

# Full Records

For more detailed notebook description, please download the [PDF](https://static.igem.wiki/teams/5647/wetlab/2025-ucas-china-igem-wetlab-experiment-new-1.pdf) of
our lab record on Benchling.

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# Iteration 1

## Design

The chain exchange of CC domains is the core of our project, which requires different binding affinities of CC
domain pairs. Based on a previous study (Brechun, Arndt, & Woolley, 2019), we adopted CC domains WinZip B1
and WinZip A2, where WinZip B1/B1 is a medium-strength interacting pair and WinZip A2/B1 is a strongly
interacting pair.

We designated WinZip B1 as both the Target chain and Autoinhibitor chain, while WinZip A2 serves as the Displacer
chain (Fig.1). Thus, after the Linker between the two WinZip B1 domains is cleaved by proteases, WinZip A2 will
bind to B1. We applied this CC domain pair to our positive feedback loop. For further details, please refer to
our [Design](https://2025.igem.wiki/ucas-china/design).

![image-20251008004118724](https://static.igem.wiki/teams/5647/wetlab/eng-fig1.avif)

## Build

We first established a set of ordinary differential equations (ODEs) to model the positive feedback pathway,
simulating the dynamics of reconstituted TEV protease and relevant products before experimental validation.

## Test

We conduct a **response variable-time simulation** of our positive feedback loop (Fig.2). The
results indicate that our pathway achieved the anticipated positive feedback amplification effect, but the
amount of reconstituted TEV protease was **not as high as expected.**

![image-20251008004240812](https://static.igem.wiki/teams/5647/wetlab/eng-fig2-bnew.avif)

Fig.2 Response variable-time simulation of our positive feedback
loop.

## Learn

As both the Target chain and Autoinhibitor chain in our pathway are WinZip B1, during the chain-switching
reaction, WinZip A2 - acting as the Displacer - binds not only to the Target chain but also to the Autoinhibitor
chain. This led to approximately a 50% reduction in the amount of reconstituted TEV protease in our system.

# Iteration 2

## Design

To enhance the efficacy of the Input 1 component, specifically to reconstitute greater quantities of TEV
protease, we aimed to conduct directed evolution on the WinZip B1 target chain to improve its affinity for
WinZip A2.

## Build

Our Model analyzed the energy distribution between residues of WinZip A2 and WinZip B1 (Fig.3). Residues with
lower (more negative) ΔG values contribute more strongly to stabilizing the binding, while higher values
indicate weaker or destabilizing effects.

![image-20251008004414705](https://static.igem.wiki/teams/5647/wetlab/eng-fig3.avif)

Fig.3 Per-residue decomposition of ΔG calculated by MMPBSA using the
normal PB model，A for WinZip A2, B for WinZipB1.

As can be seen from Fig.3, residues Val1 and Glu30 exert a negative contribution towards the binding of the two
leucine zippers. We consider introducing a V1L mutation to enhance the hydrophobicity of its hydrophobic region,
in conjunction with an E30Q mutation to reduce electrostatic repulsion, thereby decreasing the binding energy.

## Test

Following directed evolution, we conducted **response variable-time simulations** under conditions
where the Target chain exhibited increased affinity toward the Displacer chain (Fig.4). It is evident that the
**total amount of reconstituted TEV protease is greater**.

 ![image-20251008004525387](https://static.igem.wiki/teams/5647/wetlab/eng-fig4.avif)

Fig.4 Response variable-time simulation of our positive feedback loop
with directed evolution.

## Learn

The results of our simulation confirmed that enhancing the binding affinity between WinZip A2 and the evolved
WinZip B1 variant effectively increased the amount of reconstituted TEV protease. Future work will focus on
experimental validation of the chain exchange efficiency.

# Iteration 1

## Design

To verify whether our split luciferase fragments could bind and restore activity in response to the interaction
of their fused Coiled-Coil domains (CC domain), AP4(C’)/P3(C), we designed two fusion proteins, nLuc\_AP4(C')
and P3(C)\_cLuc (Fig.5).

 ![image-20251008004658993](https://static.igem.wiki/teams/5647/wetlab/eng-fig5.avif)

Fig.5 Principle of the split luciferase complementation assay.

## Build

We commissioned a biological company to synthesize nLuc\_AP4(C')\_pET-15b and P3(C)\_cLuc\_pET-28a+ vectors, and
then performed double transformation of both plasmids into E.coli BL21 (DE3) competent cells. Later, selective
pressure with ampicillin and kanamycin was applied to ensure that only cells carrying both plasmids survived.
After that, Colony PCR was used to verify whether these two plasmids had been successfully transformed.

## Test

IPTG was added to induce the co-expression of nLuc\_AP4(C') and P3(C)\_cLuc, after that we expected to detect
luminescent signals at 590 nm using Bacterial Firefly Luciferase Reporter Gene Assay Kit (Beyotime, Bac-Lumi™).
However, we **failed to capture significant luminescence**, which might indicate poor intracellular
binding of nLuc\_AP4(C') and P3(C)\_cLuc.

## Learn

We were unable to determine whether the poor signal output was due to issues with the assay kit or the failure of
nLuc\_AP4(C') and P3(C)\_cLuc to reconstitute an active reconstituted luciferase in the cells. Therefore, we
constructed a plasmid expressing **intact luciferase as a positive control**—this not only verifies
the effectiveness of the luminescence detection method but also allows direct comparison of the activity between
reconstituted luciferase and intact luciferase.

# Iteration 2

## Design

We constructed a plasmid that expresses intact luciferase as a positive control based on known sequences of nLuc
and cLuc (Fig.6).

 ![e0e955d62e075978c36a4e6d9649f236](https://static.igem.wiki/teams/5647/wetlab/eng-fig6.avif)

Fig.6 Vector Luc\_pET-28a+.

## Build

We obtained sequences of nLuc and cLuc from nLuc\_AP4(C')\_pET-15b and P3(C)\_cLuc\_pET-28a+ by PCR and ligated
them to construct Luc\_pET-28a+ plasmid by homologous recombination. Then we transformed the plasmid into *E.
coli* BL21(DE3) competent cells, and the successful transformation was verified by colony PCR.

## Test

IPTG was used to induce the expression of intact luciferase, after that we expected to detect luminescent signals
at 590 nm using Bacterial Firefly Luciferase Reporter Gene Assay Kit (Beyotime, Bac-Lumi™). This time,
**significant luminescence was detected**, confirming the reliability of the assay kit (Fig.7).

 ![e0e955d62e075978c36a4e6d9649f236](https://static.igem.wiki/teams/5647/wetlab/eng-fig7.avif)

Fig.7 Luminescence of complete Luc.

## Learn

Our successful positive control ruled out issues with the assay kit, indicating that nLuc and cLuc may have
difficulty binding under the conditions of assay reagent or their complex may lack intracellular activity.

Since co-expression in cells makes it hard to control the relative expression levels of different proteins, and
in an attempt to eliminate interference from the complex intracellular environment on their binding, we decided
to purify these two fusion proteins (nLuc\_AP4(C') and P3(C)\_cLuc) for extracellular experiments.

In addition, although the protocol of the assay kit recommends incubating for 5 minutes after adding the assay
reagent before measuring luminescence, during communication with the BNU-China team, they reminded us that
luciferase luminescence decays rapidly. Therefore, in our subsequent experiments, luminescence measurements were
started immediately after the assay reagent was added and continued for 10 minutes.

# Iteration 3

## Design

Since the 6×His-tag is retained on our nLuc\_AP4(C')\_pET-15b and P3(C)\_cLuc\_pET-28a+ plasmids, we can purify
the two proteins using Ni-NTA column affinity chromatography.

## Build

The nLuc\_AP4(C')\_pET-15b and P3(C)\_cLuc\_pET-28a+ vectors were separately transformed into E.coli BL21(DE3)
competent cells, and protein expressions were induced with IPTG. The cells were lysed by ultrasonic cell
disruptor, and pure proteins were obtained via pre-packed Ni-NTA gravity columns with gradient imidazole
elution. These proteins were then dialyzed overnight against a buffer containing 250 mM NaCl and 20 mM Tris-HCl
(pH 7.5) to remove imidazole.

## Test

Purified nLuc\_AP4(C') and P3(C)\_cLuc proteins at different concentrations were added to a buffer containing
250 mM NaCl and 20 mM Tris-HCl (pH 7.5). After co-incubation for 1 hour, the Bacterial Firefly Luciferase
Reporter Gene Assay Kit (Beyotime, Bac-Lumi™) was used to measure the luminescent signals at 590 nm (Fig.8).

 ![e0e955d62e075978c36a4e6d9649f236](https://static.igem.wiki/teams/5647/wetlab/eng-fig8.avif)

Fig.8 The luminescence signal decays with time.

## Learn

Measurements over the time course showed that **the luminescent signal decayed very rapidly**, with
no significant luminescence observed at the 5-minute mark. This indicates that the earlier intracellular split
luciferase luminescence assay likely failed not because the split luciferase fragments failed to bind or the
reconstituted luciferase lacked intracellular activity, but because the luminescent signal had already decayed
to the baseline within 5 minutes of incubation.

Determination of the protein concentration gradient revealed that the protein concentration significantly
affected the initial luminescence intensity: the split luciferase fragments at a concentration of 10 μM
exhibited remarkably strong luminescence at 590 nm. This not only confirms that our reconstituted luciferase is
a reliable output in our cell-free system, but also demonstrates that **we can use split luciferase as a
characterization tool** to verify whether other split proteins in our system bind successfully.

# Iteration 1

## Design

We aimed to verify whether our trisplit GFP (comprising GFP1-9, GFP10, and GFP11) could be reconstituted through
rearrangement mediated by CC domains fused to GFP10 and GFP11. Initially, we design the GFP\_pET-28a+ vector to
co-express the three fusion proteins: GFP1-9, GFP10\_AP4(C'), and P3(C)\_GFP11 (Fig.9).

 ![12f385bd2a6d4396b08883c23c16c2b5](https://static.igem.wiki/teams/5647/wetlab/eng-fig9.avif)

Fig.9 Vector GFP\_pET-28a+.

## Build

The designed fragments were synthesized by a biotechnology company and assembled into a complete plasmid via
homologous recombination. The product was then transformed into *E. coli* TOP10 competent cells for
plasmid amplification and extraction.

## Test

We sent the open reading frame (ORF) region of the constructed plasmid to the biotechnology company for
sequencing. However, after several independent construction trials, sequencing results consistently showed that
**the segments between the double terminators (i.e., the sequence encoding GFP1-9) was lost**
(Fig.10).

 ![647d44edae9ae8de085d960980add137](https://static.igem.wiki/teams/5647/wetlab/eng-fig10.avif)

Fig.10 Sequencing results: missing segments encoding GFP1-9.

## Learn

We analyzed the cause of sequence loss. Notably, two identical double terminator sequences were designed on our
plasmid. Such long repetitive sequences tend to trigger homologous recombination during the replication in
*E. coli*, which results in the deletion of the segment of the intervening segment. Therefore, when
designing plasmids, the inclusion of multiple long repetitive sequences (such as double terminators) on a single
plasmid should be avoided. We need to design an additional plasmid to ensure the expression of GFP1-9.

We decided to retain the plasmid without sequence encoding GFP1-9 and renamed it
**GFP\_Final\_pET-28a+** (Fig.11).

 ![35847b1b5808a538bbc4127f3c745211](https://static.igem.wiki/teams/5647/wetlab/eng-fig11.avif)

Fig.11 Vector GFP\_Final\_pET-28a+.

# Iteration 2

## Design

To co-express GFP1-9 with GFP\_Final\_pET-28a+, we construct a separate plasmid GFP1-9\_pET-15b dedicated to GFP1-9
expression. GFP1-9\_pET-15b confers a different antibiotic resistance marker compared to GFP\_Final\_pET-28a+,
enabling the stable coexistence and co-expression of both plasmids in a single *E. coli* cell.

## Build

We commissioned the biotechnology company to synthesize the GFP1-9\_pET-15b plasmid. GFP\_Final\_pET-28a+ and
GFP1-9\_pET-15b are co-transformed into BL21(DE3) competent cells. To verify the successful transformation of
both plasmids, we conducted colony PCR on the resulting bacterial colonies.

## Test

The co-transformed *E. coli* cells were cultured in an ELISA reader with continuous shaking at 25°C,
induced with 0.3 mM IPTG and 0.1% rhamnose (Rha). The culture was monitored for 16 hours, with measurements of
fluorescence intensity and OD₆₀₀ taken every 10 minutes. After normalizing the fluorescence intensity to OD₆₀₀
(to account for cell density differences), we generated a curve graph illustrating the fluorescence intensity
change over time (Fig.12).

 ![image-20251008010755713](https://static.igem.wiki/teams/5647/wetlab/eng-fig12.avif)

Fig.12 Fluorescence Intensity of reconstituted GFP.

## Learn

Fluorescence intensity analysis revealed that a significant fluorescence signal was detected as early as 3 hours
after induction. This result confirms **the feasibility of using split GFP as the output module**
for our system.

In *E. coli* cells, the intracellular concentration of expressed proteins gradually increases from 0
during the induction process—a time-dependent accumulation inherent to *in vivo* protein expression. In
contrast, such a gradual expression process is unnecessary for *in vitro* experiments. Therefore, we
reasonably infer that **a shorter incubation period will be sufficient** to generate a detectable
fluorescence signal in *in vitro* assays. This experiment also demonstrated that the fluorescence
intensity of split GFP can serve as a reliable indicator to assess the binding of other split proteins.

To maximize the predictive accuracy of the Seq2Affinity-CC model for estimating the binding strength of
coiled-coil (CC) domains based on their amino acid sequences, we conducted an iterative optimization process
under the constraints of the available dataset.

# Iteration 1

## Design

Given the high-dimensional, small-sample-size characteristics of our dataset, we recognized that complex models
were prone to overfitting. We therefore screened a set of lightweight models suitable for such scenarios,
including random forests, sparse support vector machines (SVM), Lasso regression, and Ridge regression.

## Build

The dataset was split into training and test sets. Each regression model was built, trained, and evaluated by
calculating its R-squared (R²) score on the test set to assess performance. Among them, the random forest has
been fully tuned for parameters.

## Test

The comparative R² scores for all candidate models are shown in Fig. 1.

![](https://static.igem.wiki/teams/5647/model/engineering.avif)

Fig.13 The R² scores of different models.

## Learn

The results indicated that ridge and lasso regression outperformed random forests and sparse support vector
machine (SVM) for this specific dataset. The key reason is that lasso and ridge models incorporate
regularization, which reduces parameter dimensionality and effectively mitigates overfitting. (Principles are
provided in the [Model part].) Based on this finding, we selected Lasso and Ridge regression for further
optimization.

# Iteration 2

## Design

With the model types narrowed down, we pursued further improvement through careful hyperparameter tuning and
dataset refinement.

We also followed the suggestions from HP to explore transforming the regression model into a discriminative
(classification) model for potentially higher accuracy. Additionally, we considered incorporating non-binding
data into the dataset to expand its size and utility for the discriminative approach.

## Build

We implemented the following steps:

1. Re-examined the dataset to remove entries with high uncertainty due to low experimental accuracy.
2. Systematically screened optimal hyperparameters for the lasso and ridge models using grid search.
3. Implemented and tested a partial least squares (PLS) regression model and a discriminative model.

## Test

After dataset refinement, hyperparameter adjustment, and model development, we evaluated the models based on R²
scores for regression and classification accuracy for the discriminative model, followed by a comprehensive
comparison.

## Learn

The key findings were:

1. The lasso model achieved a higher predictive performance upper limit compared to the ridge model.
2. The PLS model performance was also inferior to that of the lasso model.
3. The discriminative model improved classification accuracy to some extent; however, its performance was still
   significantly constrained by the small dataset size, even after the inclusion of non-binding peptide pairs.

Consequently, we selected the lasso regression model as our final predictor, as it achieved a qualitative to
semi-quantitative prediction level suitable for our application.

In the process of improving the model, we realized that the superior performance of lasso regression in this
situation can be fundamentally attributed to its mechanism for handling high-dimensional, small-sample data:
Lasso tends to produce sparse solutions by driving some coefficients to zero, effectively performing variable
selection. This occurs because its constraint region has sharp corners (e.g., diamond-shaped in 2D), and the
optimal solution often lies at one of these corners. In contrast, ridge regression, with its smooth spherical
constraint region, does not force coefficients to zero but generally provides more stable estimates. For our
specific data characteristics, Lasso's feature selection capability simplifies the model more effectively,
enabling it to better identify and learn the key features within the dataset.

We also concluded that a substantial improvement in predictive performance is ultimately dependent on expanding
the dataset. As the dataset grows, our engineering framework will allow us to re-evaluate and select even more
suitable and powerful models.

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In the experimental stage of UCAS-China, we actively employed, developed, and refined a variety of protocols. Recognizing the value of a well-structured protocol collection to benefit all iGEM teams, we have written a total of fourteen protocols for the iGEM community.

# Protocols

1.
[PCR](https://static.igem.wiki/teams/5647/books/protocol1-pcr.pdf)

2.
[Agarose Gel Electrophoresis](https://static.igem.wiki/teams/5647/books/protocol2-agarose-gel-electrophoresis.pdf)

3.
[DNA Gel Extraction](https://static.igem.wiki/teams/5647/books/protocol3-dna-gel-extraction.pdf)

4.
[Uniclone One-Step Seamless Cloning](https://static.igem.wiki/teams/5647/books/protocol4-uniclone-one-step-seamless-cloning.pdf)

5.
[Chemical Transformation (Heat Shock)](https://static.igem.wiki/teams/5647/books/protocol5-chemical-transformation-heat-shock.pdf)

6.
[Colony PCR](https://static.igem.wiki/teams/5647/books/protocol6-colony-pcr.pdf)

7.
[Plasmid DNA Extraction and Purification](https://static.igem.wiki/teams/5647/books/protocol7-plasmid-dna-extraction-and-purification.pdf)

8.
[IPTG Induction Expression in 96-Well White Plate](https://static.igem.wiki/teams/5647/books/protocol8-iptg-induction-expression-in-96-well-white-plate.pdf)

9.
[IPTG Induction Expression for Protein Purification](https://static.igem.wiki/teams/5647/books/protocol9-iptg-induction-expression-for-protein-purification.pdf)

10.
[Overlap PCR](https://static.igem.wiki/teams/5647/books/protocol10-overlap-pcr.pdf)

11.
[Luciferase Bioluminescence Testing](https://static.igem.wiki/teams/5647/books/protocol11-luciferase-bioluminescence-testing.pdf)

12.
[Trehalase Activity Assay](https://static.igem.wiki/teams/5647/books/protocol12-trehalase-activity-assay.pdf)

13.
[Protein Purification](https://static.igem.wiki/teams/5647/books/protocol13-protein-purification.pdf)

14.
[SDS-PAGE (Coomassie Brilliant Blue Staining)](https://static.igem.wiki/teams/5647/books/protocol14-sds-page-coomassie-brilliant-blue-staining.pdf)

15.
[Native PAGE](https://static.igem.wiki/teams/5647/books/protocol15-native-page.pdf)

16.
[Protein Recovery from Inclusion Bodies](https://static.igem.wiki/teams/5647/books/protocol16-protein-recovery-from-inclusion-bodies.pdf)

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# Overview

After carefully designing and planning the experiment, we initiated the experimental part of our project. We
primarily focused on validating the activation of the downstream pathway through two inputs: the occurrence of
chain-exchange reactions in the CC domain and the efficiency of the positive feedback loop, as well as the
performance of the three outputs. We then collected our data and analyzed it according to the Engineering cycle (See
page [Engineering](https://2025.igem.wiki/ucas-china/results)) to improve our experiment.

Detailed information regarding how we implement it and the record kept in the wet lab can be found in the [Experiments](https://2025.igem.wiki/ucas-china/experiments) part and the [Notebook](https://2025.igem.wiki/ucas-china/notebook) part.

# Split Protein as Output

To evaluate whether CC-mediated molecular interactions could produce measurable functional signals, we designed three
CC-coupled split protein systems: split luciferase (Luc), split green fluorescent protein (GFP), and split trehalase
(TreA). We first validated the feasibility of using these split proteins as output modules, since their
reconstitution leads to luminescence, fluorescence, or enzymatic activity recovery, respectively. In each design,
the complementary CC domains AP4(C') and P3(C) were fused to the corresponding fragments of the split reporter
protein, enabling structural reassembly and activity restoration upon CC heterodimerization (Fig.1).

![image-20251004154809351](https://static.igem.wiki/teams/5647/results/res-fig1.avif)

Fig.1 CC-coupled split protein output systems. (a) Split
luciferase complementation. (b) Tripartite split GFP reconstitution. (c) Split trehalase complementation.

We next examined whether these CC-driven split protein systems could successfully reassemble and generate measurable
outputs in *E. coli* or in vitro. The performance of each system was characterized by monitoring its specific
signal - luminescence, fluorescence, or enzymatic activity - following CC-induced reconstitution.

## Split Luciferase

Both fusion proteins, nLuc\_AP4(C') ([BBa\_25DSNIG7](https://registry.igem.org/parts/bba-25dsnig7)) and
P3(C)\_cLuc ([BBa\_2527MQP2](https://registry.igem.org/parts/bba-2527mqp2)), were expressed in *E.
coli* BL21(DE3) and purified using Ni-NTA affinity chromatography. SDS-PAGE confirmed successful
purification (Figs.2, 3).

![image-20251007111816692](https://static.igem.wiki/teams/5647/results/res-fig2-bnew.avif)
![image-20251007111856576](https://static.igem.wiki/teams/5647/results/res-fig3-bnew.avif)

When equal molar amounts of nLuc\_AP4(C') and P3(C)\_cLuc were co-incubated and measured using Bac-Lumi™ luciferase
assay, a clear luminescent signal at around 590 nm was observed shortly after reagent addition (Fig.4). However, the
luminescence intensity decayed rapidly, returning to baseline within 5 minutes, in contrast to the 30-minute
stability expected from the manufacturer’s protocol. This may result from the lower structural stability of the
reconstituted split luciferase compared with the intact enzyme, as the complex likely dissociates after catalysis.
Nonetheless, the immediate luminescence upon reagent addition clearly demonstrated that our CC-mediated split
luciferase system successfully reconstituted and restored enzymatic activity.

![](https://static.igem.wiki/teams/5647/results/res-fig4.avif)

Fig.4 Luminescence of CC-coupled split luciferase in different
concentrations. Data were background-subtracted and the blank control therefore corresponds to a constant
baseline of zero.

## Split GFP

Similarly, two fusion proteins, GFP10\_AP4(C') ([BBa\_25EVV9VN](https://registry.igem.org/parts/bba-25evv9vn)) and P3(C)\_GFP11 ([BBa\_25EVV9VN](https://registry.igem.org/parts/bba-25evv9vn)), were co-expressed with GFP1-9 in *E.
coli* BL21(DE3).

Fluorescence at 528 nm (excitation 485 nm) was continuously monitored for 16 hours (Fig.5). A marked increase in
fluorescence was detected within 2 hours after induction, indicating successful reconstitution of the split GFP
complex mediated by the paired Coiled-Coil domains.

![](https://static.igem.wiki/teams/5647/results/res-fig5.avif)

Fig.5 Normalized fluorescence of CC-coupled tripartite split GFP. Data
were background-subtracted using the fluorescence and OD600 of blank LB medium.

Due to time constraints, we did not purify the proteins and repeat the experiment in a cell-free system. However,
considering that our construct responds directly to molecular interactions rather than protein accumulation, it is
expected to exhibit a detectable fluorescence signal within a shorter timeframe under cell-free conditions.

## Split TreA

Both fusion proteins, MBP\_nTreA\_AP4(C') ([BBa\_25MD5KSC](https://registry.igem.org/parts/bba-25md5ksc),
abbreviated as **nTreA**) and P3(C)\_cTreA ([BBa\_25V4VNKA](https://registry.igem.org/parts/bba-25v4vnka), abbreviated as **cTreA**),
were expressed in *E. coli* BL21(DE3) and purified using Ni-NTA affinity chromatography. SDS-PAGE confirmed
successful purification (Fig.6).

![image-20251007111816692](https://static.igem.wiki/teams/5647/results/res-fig6-bnew.avif)

Fig.6 SDS-PAGE analysis of cTreA (a) and nTreA (b) purification using
Ni-NTA affinity chromatography. Lane M: protein molecular weight marker; Lane FT: flow-through from Ni-NTA
column; Lane E1-E4: elution fractions with 20, 50, 100, and 200 mM imidazole, respectively.

We co-incubated equal concentration of nTreA and cTreA and employed the Trehalase (THL) Activity Assay Kit (BoxBio)
to detect in-vitro trehalase activity, characterised by OD505 (Fig.7). Compared with adding cTreA or nTreA only,
those samples both adding cTreA and nTreA exhibited significantly higher trehalase activity. This demonstrates that
our CC-mediated split trehalase was successfully reconstituted and restored to enzymatic activity.

![](https://static.igem.wiki/teams/5647/results/res-fig7-bbbnew.avif)

Fig.7 After adding the detection reagent, measure the absorbance at 505
nm. For the nTreA + cTreA group, nTreA and cTreA with both 0.5 mg/mL final concentration were added; For the
cTreA + cTreA and nTreA + nTreA groups, 1 mg/mL final concentration of the corresponding protein was added,
respectively; The negative control group received the negative control solution provided in the assay kit. All
OD505 readings were background-subtracted against the baseline absorbance of the buffer solution at 505 nm

# Cleavage Efficiency of AvrRpt2

According to previous reports (Chisholm et al., 2005), the AvrRpt2 protease specifically recognizes and cleaves its
native plant substrate RIN4 at a conserved heptapeptide motif (VPxFGxW). Chisholm et al. (2005) qualitatively tested
the cleavage efficacy of AvrRpt2 on a series of mutants based on the canonical RIN4 cleavage site (Fig.8), which
providing strong evidence that AvrRpt2 can indeed cleave the RIN4-derived sequence within our system.

![f6a7cafa6b7e907eeb4a9013cd99f782](https://static.igem.wiki/teams/5647/results/res-fig8.avif)

Fig.8 AvrRpt2-dependent cleavage of mutants based on the canonical RIN4
cleavage site. Putative targets do not accumulate in the presence of AvrRpt2. The indicated epitope-tagged
proteins were expressed in N. benthamiana leaves alone (-), with AvrRpt2:FLAG (lane A), or with catalytically
deficient mutants AvrRpt2:FLAG(C122A) (lane C). X, noninfiltrated leaf. Putative cleavage site amino acid
sequences are given in parentheses. Protein extracts were resolved by denaturing PAGE and used for immunoblot
analyses with αT7. Approximate positions of molecular mass standards are shown. (Chisholm *et al.*, 2005)

To investigate the cleavage efficiency of AvrRpt2, we selected a series of substrate variants (VPKFGNW, VPKFGDW,
VPAFGSW, VPAFGGW) based on the canonical RIN4 cleavage site, aiming to obtain variants with improved cleavage
efficiency. In order to quantitatively characterize the proteolytic activity of AvrRpt2, we employed the CC-coupled
Split Luciferase system previously validated in the Split Protein as Output section. In this system, different
AvrRpt2 cleavage sequences were inserted between the CC domain and the nLuc region of the split luciferase protein
nLuc\_AP4(C'). Only the uncleaved nLuc\_AP4(C') can reconstitute an active luciferase upon interaction with
P3(C)\_cLuc, leading to measurable luminescence (Fig.9).

![f6a7cafa6b7e907eeb4a9013cd99f782](https://static.igem.wiki/teams/5647/results/res-fig9.avif)

Fig.9 Verification of AvrRpt2 cutting efficiency. (a) Prior to AvrRpt2
cleavage, split luciferase exhibits normal luminescence. (b) After AvrRpt2 cleavage, split luciferase fails to
exhibit luminescence.

By co-incubating AvrRpt2 with nLuc\_AP4(C') constructs containing different cleavage sites and comparing the resulting
luminescence intensity with that of the control group (without AvrRpt2), we could quantify the relative cleavage
efficiency of AvrRpt2 toward each substrate variant.

AvrRpt2 was expressed in *E. coli* BL21(DE3) and purified using Ni-NTA affinity chromatography. SDS-PAGE
confirmed successful purification (Fig.8). P3(C)\_cLuc was successfully purified in the **Split Protein as
Output** section (Fig.10).

![image-20251007080921085](https://static.igem.wiki/teams/5647/results/res-fig10-bnew.avif)

Fig.10 SDS-PAGE analysis of AvrRpt2 purification using Ni-NTA affinity
chromatography. Lane designations are the same as in Fig.2.

As for the **nLuc\_AP4(C')\_pET-15b** construct containing the AvrRpt2 recognition site, colony PCR
results indicated that transformation into *E. coli* BL21(DE3) was unsuccessful. We plan to repeat the
transformation and subsequently express this protein in *E. coli* BL21(DE3). Since this construct differs
from the original **nLuc\_AP4(C')** only by the insertion of an AvrRpt2 cleavage site—and the latter was
successfully expressed in a soluble form—we believe that the modified protein can also be purified using a similar
approach.

In any case, although we have not yet been able to verify the cleavage efficiency of AvrRpt2 at our selected
recognition sites, the results reported by Chisholm *et al.* (2005) confirm that these sites are recognized
and cleaved by AvrRpt2.

# Interaction of COI1 and JAZ1

The **COI1** and **JAZ1** proteins constitute the second input pathway in our system.
According to previous studies (Sheard *et al.*, 2010), JAZ1 interacts with COI1 through a short peptide
segment (Glu200-Val220) and mediates the sensing of **Jasmonic Acid-Isoleucine (JA-Ile)**. In our
design, this peptide segment (ELPIARRASLHRFLEKRKDRV) was used to enable the specific interaction between JAZ1 and
COI1. **Coronatine (COR)** is a structural analogue of JA-Ile and can similarly activate the COI1-JAZ1
complex.

Thines et al. (2007) conducted in vitro pull-down assays (Fig.11a) using the functional c-Myc-tagged tomato COI1
(COI1-Myc) and full-length JAZ1, demonstrating that JA-Ile induces COI1-JAZ1 interaction in vitro. Also, Sheard et
al. (2010) validated via in vitro radioligand binding assays that our selected JAZ1 short peptide
(ELPIARRASLHRFLEKRKDRV) could bind to COI1 in the presence of COR(Fig.b). These results collectively indicate that
COR or JA-Ile can successfully mediate the binding between COI1 and the JAZ1 short peptide within our system design.

![afadc4d2845c198937959121f46789bd](https://static.igem.wiki/teams/5647/results/res-fig11.avif)

Fig.11 Previous studies on the interaction between COI1 and JAZ1. (a) Y2H
protein-protein interaction assay. Sl, Solanum lycopersicum; At, Arabidopsis thaliana. Blue colonies indicate
interaction, white colonies indicate no interaction. Colonies of a positive-control strain, pLexA-53/pB42AD-T,
are shown (top row). (b) Saturation binding of COI1-ASK1 and the JAZ1 +5 degron peptide, with a Kd of 108±29 nM.
All results are the mean ± S.E. of two to three experiments performed in duplicate. (Thines *et al.*, 2007;
Sheard *et al.*, 2010)

To verify whether COR or JA-Ile can act as input signals to activate our split protein system fused with COI1 and
JAZ1, and to determine their activation threshold, we employed the CC-coupled Split GFP system, previously validated
in the Split Protein as Output section (Fig.12). We constructed fusion proteins COI1-GFP11 and GFP10-JAZ1, and
co-incubated them with GFP1-9. By adding a series of JA-Ile concentrations and measuring fluorescence intensity, we
determined the minimal threshold concentration required for JA-Ile to activate the COI1-JAZ1 pair as a signal
switch. (Due to funding reasons, we only use JA-Ile in our experiments to validate the functionality of this
signalling module.)

![afadc4d2845c198937959121f46789bd](https://static.igem.wiki/teams/5647/results/res-fig12.avif)

Fig.12 Verification of COI1 and JAZ1 interaction.

Initially, we attempted to purify GFP10-JAZ1 directly using Ni-NTA affinity chromatography. However, due to its small
molecular weight (8.8 kDa), the protein was difficult to visualize on SDS-PAGE and could not be effectively
concentrated by ultrafiltration. Therefore, we constructed a fusion protein MBP-GFP10-JAZ1 ([BBa\_254RZXUU](https://registry.igem.org/parts/bba-254rzxuu)) containing a TEV protease cleavage site
between the MBP tag and GFP10. The MBP tag increased the molecular weight of the fusion protein to 49.5 kDa,
facilitating visualization and enrichment. MBP-GFP10-JAZ1 was expressed in *E. coli* BL21(DE3) and purified
via Ni-NTA affinity chromatography. SDS-PAGE analysis confirmed successful purification (Fig.13).

![afadc4d2845c198937959121f46789bd](https://static.igem.wiki/teams/5647/results/res-fig13-bnew.avif)

Fig.13 SDS-PAGE analysis of MBP-GFP10-JAZ1 purification using Ni-NTA
affinity chromatography. Lane M: protein molecular weight marker; Lane FT: flow-through from Ni-NTA column; Lane
E1-E4: elution fractions with 20, 50, 100, and 200 mM imidazole, respectively.

We also attempted to express COI1-GFP11 directly in *E. coli* BL21(DE3), but even under optimized induction
conditions (12 °C, 0.2 mM IPTG, 20 h), no soluble protein was detected in the supernatant (Fig.14). Therefore, we
constructed MBP-COI1-GFP11 to enhance the solubility of the COI1-GFP11 fusion protein through the MBP tag. However,
soluble MBP-COI1-GFP11 was still not obtained under the tested conditions (Fig.15). We plan to switch to a yeast
expression system or try the inclusion body refolding method to obtain soluble MBP-COI1-GFP11 proteins.

![afadc4d2845c198937959121f46789bd](https://static.igem.wiki/teams/5647/results/res-fig14-bbbnew.avif)

Fig.14 SDS-PAGE analysis of COI1-GFP11 purification using Ni-NTA affinity
chromatography. Lane M: protein molecular weight marker; Lane U: whole cell lysate before IPTG induction; Lane
I: whole cell lysate after IPTG induction; Lane P: insoluble fraction after sonication (pellet); Lane S: soluble
fraction after sonication (supernatant).

![afadc4d2845c198937959121f46789bd](https://static.igem.wiki/teams/5647/results/res-fig15-bbbnew.avif)

Fig.15 SDS-PAGE analysis of MBP-COI1-GFP11 purification using Ni-NTA
affinity chromatography. Lane designations are the same as in Fig.13. (a) Induction conditions: 30°C, 0.5 mM
IPTG, 200 rpm, 6 hours. (b) Induction conditions: Lane 2-5 22°C, 0.5 mM IPTG, 180 rpm, 16 hours; Lane 7-10 16°C,
0.3 mM IPTG, 160 rpm, 16 hours.

# CC domains

The **chain exchange reaction between CC domains** forms the core of our system design. To confirm the
occurrence of this reaction can occur and to evaluate its efficiency, we selected **CC domains A', B, and
A** as representative components. We employed the **CC-coupled Split GFP system**,
previously validated in our **Split Protein as Output** section, to characterize the chain exchange
reaction between these domains.

We designed two fusion proteins, **GFP10-A-TEVs-B** and **A'-GFP11**. In the presence of
**TEV protease**, the cleaved GFP10-A-TEVs-B is expected to undergo chain exchange with A'-GFP11,
enabling the reconstitution of an active Split GFP complex upon binding to **GFP1-9** (Fig.16).

![cb56ef92de2b3e9254029f81756187bc](https://static.igem.wiki/teams/5647/results/res-fig16.avif)

Fig.16 Verification of the chain exchange reaction between CC domains.

To construct the experimental system, the coding sequences of GFP10-A-TEVs-B, A'-GFP11, and GFP1-9 were cloned into
the pET-28a(+) vectors. The expression of GFP10-A-TEVs-B and A'-GFP11 was driven by an *rhaB*-inducible
promoter, while GFP1-9 was expressed under the constitutive J23119 promoter (Fig.17).

![81e4cace5bba0a9e1a24763c7a2cf472](https://static.igem.wiki/teams/5647/results/res-fig17.avif)

Fig.17 Plasmid map of A'\_B-A\_GFP\_pET-28a(+) construct.

However, the presence of multiple CC domains and repetitive sequences, such as double terminators, made this plasmid
construct was difficult to assemble successfully. In future experiments, we plan to express these proteins
separately using individual vectors, followed by in vitro assays after purification. Reflections and design
improvements related to this construct are discussed in detail in our Engineering section.

Although we were unable to successfully construct the A'\_B-A\_GFP\_pET-28a(+) vector, our design employed the same
logic gate configuration as that described in the literature (Fink *et al*., 2019). The binding strengths
between the CC domains we used were comparable to those of the CC domains they employed. They characterized the
rearrangement between the two CC domains in a system similar to ours, A'-B-A and C-D-E-C', using cleaved
luciferase and obtained significant luminescence results after 30 minutes (Fig.18).

![81e4cace5bba0a9e1a24763c7a2cf472](https://static.igem.wiki/teams/5647/results/res-fig18.avif)

Fig.18 Experimental analysis of the indicated SPOC logic function designs
in HEK293T cells with introduced genetic circuits. The design and expected response to all input combinations is
schematically shown below the graphs with experimental luciferase-based results. Input signals are combinations
of two orthogonal proteases, TEVp and PPVp, and the output signal is split luciferase activity. Values are the
means from two (a-b) cell cultures ±s.d. and are representative of two independent experiments. Significance was
tested by one-way analysis of variance (ANOVA) with Tukey’s comparison (values of confidence intervals, degrees
of freedom, F and P are indicated). (Fink *et al.*, 2019)

Given that our system employs an in vitro approach, eliminating the need for protein accumulation through cellular
expression, we anticipate that a shorter observation period will suffice to detect a pronounced luminescent signal.
Moreover, the signal intensity remains unaffected by interference from complex intracellular environments.

# Positive Feedback Loop

To verify whether our positive feedback pathway could amplify the AvrRpt2 signal and lower the limit of detection, we
designed a series of fusing protein (Fig.19), including cTEVp\*-B1-AvrRpt2s-TEVs-B1-nTEVp ([BBa\_25LMVZ4Y](https://registry.igem.org/parts/bba-25lmvz4y), abbreviated as nTEV), MBP-A'-cTEVp ([BBa\_258NBWZH](https://registry.igem.org/parts/bba-258nbwzh), abbreviated as cTEV) and
cXTEVp-B1-AvrRpt2s-B1-nTEVp ([BBa\_25LOPU94](https://registry.igem.org/parts/bba-25lopu94), abbreviated as
nonTEV). The cTEV and nTEV protein are designed to achieve positive feedback, while nonTEV, in which TEV protease
cleavage site was deleted, serves as a negative control lacking the function of positive feedback.

![b7c229e66e411787ce25f5a1509338f9](https://static.igem.wiki/teams/5647/results/res-fig19.avif)

Fig.19 Schematic diagram of the positive feedback loop section. (a)
Protein nTEV and cTEV exhibit positive feedback. (b) Protein nonTEV and cTEV fail to exhibit positive feedback

The fusion proteins nTEV, cTEV and nonTEV were expressed in *E. coli* BL21(DE3) and purified using Ni-NTA
affinity chromatography. SDS-PAGE confirmed successful purification (Figs.20a, b and c). We also purified AvrRpt2
protease (Fig.8) as the input of the positive feedback loop.

![image-20251007105055908](https://static.igem.wiki/teams/5647/results/res-fig20-bnew.avif)

Fig.20 SDS-PAGE analysis of nTEV (a), cTEV (b) and nonTEV (c)
purification using Ni-NTA affinity chromatography. Lane M: protein molecular weight marker; Lane E1-E4: elution
fractions with 20, 50, 100, and 200 mM imidazole, respectively.

We firstly co-incubated AvrRpt2 with nonTEV and nTEV at room temperature for two hours to preliminarily determine
whether our purified AvrRpt2 had cleavage activity, thereby enabling the initiation of a positive feedback reaction.
Theoretically, AvrRpt2-mediated cleavage of nTEV or nonTEV should yield two bands, each with a molecular weight of
approximately 20 kDa. However, SDS-PAGE analysis (Fig.21) did not reveal a distinct band around 20 kDa in the
co-incubated samples, indicating that AvrRpt2 did not perform effective cleavage to initiate the intended reaction.

![image-20251007105055908](https://static.igem.wiki/teams/5647/results/res-fig21-bnew.avif)

Fig.21 SDS-PAGE analysis of AvrRpt2 and nTEV/nonTEV co-incubation. The
molecular weight of AvrRpt2 is 30.3 kDa, while that of nTEV is 42.4 kDa and nonTEV is 41.2 kDa.

Through literature review (Chisholm et al., 2005; Jin, Wood, Wu, Xie, & Katagiri, 2003; Mudgett & Staskawicz,
1999), we found that the full-length AvrRpt2 (30 kDa) lacks cleavage activity. Upon entering plant cells, it is
activated by Host Cleavage Factor(s), causing AvrRpt2 to cleave off its own N-terminal 70 amino acids, thereby
generating an active AvrRpt2 with a molecular weight of approximately 25 kDa. We will subsequently attempt to
replicate the work of Jin, Wood, Wu, Xie, & Katagiri (2003) by activating AvrRpt2 using yeast extract and
employing the activated AvrRpt2 for further experiments.

# References

1. Brechun, K. E., Arndt, K. M., & Woolley, G. A. (2019). Selection of Protein-Protein Interactions of Desired
   Affinities with a Bandpass Circuit. *Journal of Molecular Biology, 431*(2), 391-400.
2. Chisholm, S. T., Dahlbeck, D., Krishnamurthy, N., Day, B., Sjolander, K., & Staskawicz, B. J. (2005).
   Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2. *Proc
   Natl Acad Sci U S A, 102*(6), 2087-2092.
3. Fink, T., Lonzarić, J., Praznik, A., Plaper, T., Merljak, E., Leben, K., ... Jerala, R. (2019). Design of fast
   proteolysis-based signaling and logic circuits in mammalian cells. *Nat Chem Biol, 15*(2), 115-122.
4. Jin, P., Wood, M. D., Wu, Y., Xie, Z., & Katagiri, F. (2003). Cleavage of the Pseudomonas syringae type III
   effector AvrRpt2 requires a host factor(s) common among eukaryotes and is important for AvrRpt2 localization in
   the host cell. *Plant Physiol, 133*(3), 1072-1082.
5. Mudgett, M. B., & Staskawicz, B. J. (1999). Characterization of the Pseudomonas syringae pv. tomato AvrRpt2
   protein: demonstration of secretion and processing during bacterial pathogenesis. *Molecular Microbiology,
   32*(5), 927-941.
6. Sheard, L. B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T. R., ... Zheng, N. (2010). Jasmonate
   perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. *Nature, 468*(7322), 400-405.

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