iGEM 2025 — Team um-macau

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[Weekly Progress](#1)
[Detailed Progress](#2)

## Wet Lab

##### **Weekly Progress**

##### **Detailed Progress**

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[Overview](#over)
[DBTL Cycle 1](#c1)

[Design #1](#d1)
[Build #1](#b1)
[Test #1](#t1)
[Learn #1](#l1)

[DBTL Cycle 2](#c2)

[Design #2](#d2)
[Build #2](#b2)
[Test #2](#t2)
[Learn #2](#l2)

[DBTL Cycle 3](#c3)

[Design #3](#d3)
[Build #3](#b3)
[Test #3](#t3)

[Summary](#sum)

## Overview

| Part | Name | Cloning Target | Amplicon (bp) | Primers |
| --- | --- | --- | --- | --- |
| BBa\_25G76T6V | pET28a-FNR-NSP4-K-Hecate | Hecate fused with modified NSP4-K secretion signal | 189 | **Forward:** CCTCTAGAAATAATTTTG  **Reverse:** TAGCAGCGGATCCTTACAGCGCTTTTTTCAGCGC |
| BBa\_25UVLJOJ | pET28a-FNR-NSP4-K-Melittin | Melittin fused with modified NSP4-K secretion signal | 189 | **Forward:** CCTCTAGAAATAATTTTG  **Reverse:** TAGCAGCGGATCCTAAACGCAAACGCCAGCAGTAA |
| BBa\_25HS5FHK | pET-T7-CYP450-Flag | The pET backbone with T7 promoter system | 5614 | **Forward:** TAAGGATCCGCTGCTAACAAAG  **Reverse:** CAAAATTATTTCTAGAG |

## DBTL Cycle 1: Diagnosis of Cloning PCR Failure

##### **Design #1:**

To evaluate the expression efficiency of the FNR and T7 promoters for the genes NSP4-K-Hecate and NSP4-K-Melittin, novel recombinant plasmids demonstrated pET-T7-NSP4-K-Hecate (Fig. 1a) and pET-T7-NSP4-K-Melittin (Fig. 1b), were designed. The pET-T7-CYP450-Flag plasmid was selected as the backbone. The entire plasmid, with the exception of the CYP450-Flag gene, was amplified so as to generate a linearized vector possessing homologous ends designed for seamless ligation with the inserts.

The coding sequences for NSP4-K-Hecate and NSP4-K-Melittin were amplified from pET28a-FNR-NSP4-K-Hecate and pET28a-FNR-NSP4-K-Melittin, respectively.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/hecate-and-melittin.avif)

Figure 1: **The illustration of our desired recombinant plasmid. a) Hecate gene in the pET-T7 backbone. b) Melittin gene in the pET-T7 backbone**

##### **Build #1:**

The initial stage started with PCR amplification to generate the Hecate and Melittin inserts, as well as to linearize the pET-T7 vector backbone for subsequent Gibson Assembly, where the inserts will replace the original CYP450-Flag coding sequence in the plasmid pET-T7-CYP450-Flag.

##### **Test #1:**

Analysis of these PCR products by gel electrophoresis demonstrated that amplification for all three fragments—the Hecate insert, the Melittin insert, and the vector backbone—was unsuccessful, as no bands of the expected sizes were observed. Specifically, neither the Melittin nor Hecate inserts produced detectable bands, indicating complete amplification failure. For the pET-T7 backbone, several non-specific bands appeared at approximately 8 kb, 3 kb, and 2 kb, but there was no band at the expected ~5.6 kb size corresponding to the linearized vector.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/eng-2.webp)

Figure 2: **The DNA gel electrophoresis for the Cloning PCR result of Hecate insert (2), Melittin insert (3) and Vector (5)**

##### **Learn #1:**

The PCR failure may be attributed to suboptimal primer design, as there is a significant difference in melting temperatures (Tm) between the forward and reverse primers. This discrepancy complicates the selection of an appropriate annealing temperature; therefore, we have decided to redesign the primers in the next cycle.

## DBTL Cycle 2: Optimization and Targeted Troubleshooting

##### **Design #2**

Based on the lessons from Cycle 1, we initiated a comprehensive redesign. New primers are designed for the Hecate insert, Melittin insert, and pET-T7 backbone from scratch using Snapgene software to optimize binding characteristics, hope to obtain successful PCR amplification of all fragments.

The newly designed primers:

| Template Plasmid | Primer Name | Primer Sequence |
| --- | --- | --- |
| pET28a-FNR-NSP4-K-Hecate | Gibson-NSP4-K-F1 | TGAGCGGATAACAATTCCCC TTTGTTTAACTTTAAGAAGGAGATATACCA |
| Gibson-Hecate-R1 | GCTTTGTTAGCAGCGGATCC GCATTACAGCGCTTT |
| pET28a-FNR-NSP4-K-Melittin | Gibson-NSP4-K-F1 | TGAGCGGATAACAATTCCCC TTTGTTTAACTTTAAGAAGGAGATATACCA |
| Gibson-Melittin-R1 | GCTTTGTTAGCAGCGGATCC TTACTGCTGGCGTTTGCGT |
| pET-T7-CYP450-Flag | Vector-F | GGATCCGCTGCTAACAAAGC |
| Vector-R | GGGGAATTGTTATCCGCTCAC |

Note: The highlighted in red are the overhangs.

##### **Build #2**

Subsequent to the redesign of the primers, we undertook several iterative rounds of PCR optimization in an effort to amplify the three target fragments. Despite the implementation of newly designed primers and verified templates, standard PCR protocols remained unsuccessful. Consequently, we systematically modified key PCR parameters. Annealing temperature was optimized by performing temperature gradient PCR, testing a range of temperatures surrounding the calculated Tm of the new primers to identify the optimal condition. Additionally, the annealing time was significantly increased from the conventional 30 seconds to 1 minute to promote more complete and specific primer-template hybridization.

##### **Test #2**

After adjusting the PCR protocol and the primer, we finally got clear bands for the Hecate insert and Vector, which was confirmed using gel electrophoresis. In the Fig. 3a, we can see bands at the correct position for the Hecate insert fragment (189bp), and in the Fig. 3b, the Vector fragment (5614bp) compared to the original plasmid. We cut these bands and performed gel purification. In an effort to resolve the persistent PCR failure for the Melittin insert, the original pET28a-FNR-NSP4-K-Melittin template plasmid was sequenced; sequencing revealed that the clone obtained from the company was incorrect and did not contain the intended insert.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/eng-3.webp)

Figure 3: **The DNA gel electrophoresis of the Hecate and Melittin inserts and the vector.**

##### **Learn #2:**

Agarose gel electrophoresis confirmed successful PCR amplification of both the Hecate insert and the pET-T7 backbone; however, all attempts to amplify the Melittin insert consistently failed. Consequently, Gibson Assembly was employed to synthesize the Hecate construct (Fig. 1a). As repeated failures were observed for the Melittin insert, we hypothesized that the issue may reside with the original plasmid template. Therefore, we proceeded to re-extract the plasmid and have submitted it for sequencing analysis.

## DBTL Cycle 3: Assembly and Strategic Reassessment

##### **Design #3**

Sequencing analysis from the previous cycle confirmed a problem with the original clone obtained from the commercial supplier. As a result, we reordered the Melittin plasmid (pET28a-FNR-NSP4-K-Melittin). Consequently, progress on the Melittin recombinant constructs was temporarily halted, while synthesis of the Hecate construct continued without interruption.

##### **Build #3**

Following gel purification, we successfully obtained the DNA fragments required for subsequent Gibson assembly. The assembled products will be verified by agarose gel electrophoresis, after which they will be transformed into BL21 competent cells for plasmid replication. The extracted plasmids will then be submitted for sequencing to confirm the assembly accuracy.

##### **Test #3**

In Fig. 4, DNA gel electrophoresis analysis of the assembled product revealed a faint band at an unexpected position. Despite the suboptimal gel result, the band was gel-purified, transformed into BL21 cells, subjected to plasmid miniprep, and subsequently sequenced. Sequencing results indicated that the assembly had not succeeded, as the recombinant plasmid was not correctly formed.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/eng-4.webp)

Figure 4: **The DNA gel electrophoresis of the Gibson assembly of the Hecate construct. No.2 stands for the Hecate insert, and No.5 stands for the pET-T7 Vector.**

## Summary and Conclusion

This Design-Build-Test-Learn (DBTL) cycle exemplifies a common challenge in synthetic biology: the iterative and often unpredictable nature of genetic construct development. We aimed to construct two recombinant plasmids to assess promoter strength. However, initial difficulties in fragment amplification via cloning PCR necessitated improvements in primer design, such as altering binding sites, increasing GC content for greater stability, and incorporating longer overlapping regions. This process was time-intensive and ultimately led to the strategic decision to halt the project, thereby enabling the reallocation of resources to other critical components of our iGEM project. The experience gained in troubleshooting PCR, performing Gibson Assembly, and managing the project is invaluable and provides a robust foundation for future teams.

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[Protocols](#Protocols)

[Protocol1](#Protocol1)
[Protocol2](#Protocol2)

[HQ](#hq)

[Aim](#aim1)
[Experimental Design](#exp1)
[Results](#res1)
[Discussion](#dis1)
[References](#ref1)

[EcTI](#ecti)

[Aim](#aim2)
[Experimental Design](#exp2)
[Results](#res2)
[Discussion](#dis2)
[References](#ref2)

[Hecate & Melittin](#melittin)

[Aim](#aim3)
[Experimental Design](#exp3)
[Results](#res3)
[Discussion](#dis3)
[References](#ref3)

## Protocols

##### **General Protocol**

##### **Experimental Protocol**

## HQ

##### **Aim**

The primary objective of this study is to elucidate the catalytic role of cytochrome
P450 (CYP450) in the enzymatic hydroxylation of phenol to hydroxyquinone (HQ),
a key chemical implicated in termite attraction mechanisms. Specifically, this work aims
to compare the expression efficiency, catalytic activity, and substrate conversion kinetics
between the wild-type CYP450 and its engineered double mutant (A83F/A329F). Both enzymes were
heterologously expressed in *Escherichia coli* BL21 (DE3) using T7 promoter–driven
plasmid constructs. The study employs a multi-layered analytical approach: (1) Fluorescence
microscopy for monitoring mCherry-tagged protein localization, (2) Western blotting for validation
of FLAG-tagged protein expression, and (3) spectrophotometric assays for quantification of
HQ formation over time — to determine whether the double mutation enhances enzymatic performance and
contributes to more efficient HQ biosynthesis.

##### **Experimental Design**

**1. Plasmid Construction and Preparation**

Wild-type and double-mutant (A83F/A329F) CYP450 genes were cloned into T7 promoter–based
plasmids, each fused with either FLAG or mCherry tags for protein detection: T7-CYP450-flag [(BBa\_25HS5FHK)](https://registry.igem.org/parts/bba-25hs5fhk),
T7-CYP450-mCherry [(BBa\_25H2URHN)](https://registry.igem.org/parts/bba-25h2urhn),
T7-CYP450DM-Flag [(BBa\_25MIYL3S)](https://registry.igem.org/parts/bba-25miyl3s) and
T7-CYP450DM-mCherry [(BBa\_25MRBB84)](https://registry.igem.org/parts/bba-25mrbb84). Control plasmids
without CYP450 insert were used as negative references. All constructs were custom synthesized
(VectorBuilder) and purified using a Plasmid Midi Preparation Kit (Beyotime D0020). DNA purity
and concentration were assessed by UV spectrophotometry at 260/280 nm, with A260/A280 ratios
maintained around 1.8 and A260/A230 > 2.0. Purified plasmids were stored at −20 °C until further use.

**2. *E. coli* Transformation and Protein Expression**

Competent *E. coli* (DE3) cells were transformed with the respective plasmids using
a standard heat-shock method. Following recovery in antibiotic-free LB medium, transformants
were selected on antibiotic-containing agar plates and incubated overnight at 37 °C. Single
colonies were inoculated into liquid LB medium and grown to mid-log phase (OD600 = 0.5–0.8)
before induction with 0.5 mM IPTG for 4–6 h at 37 °C. Cells expressing mCherry-tagged constructs
were directly subjected to fluorescence microscopy, whereas those carrying FLAG-tagged constructs
were harvested for protein extraction and Western blot analysis.

**3. Fluorescence Detection**

To verify successful expression of mCherry-tagged CYP450 fusions, induced and uninduced cultures
were examined under a fluorescence microscope (excitation: 540–580 nm; emission: 592–668 nm)
using consistent exposure parameters. Controls included untransformed
*E. coli* BL21 cells and PBS/LB only background slides. Both bright-field (TL-DIC)
and fluorescence images were captured to correlate cellular morphology with protein-localized
fluorescence.

**4. Western Blot Analysis**

Protein expression of FLAG-tagged CYP450 variants was analyzed by Western blotting following
standard protocols. Briefly, IPTG-induced
*E. coli* BL21 (DE3) cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl pH 8.0,
150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with phosphatase and
protease inhibitors (Roche). Lysates were centrifuged at 16,000 × g for 20 min at 4 °C, and
protein concentrations were determined by BCA assay.

Equal amounts of protein (20 µg) were denatured in 2× Laemmli buffer, separated by SDS–PAGE,
and transferred onto PVDF membranes in Tris/Glycine buffer containing 20% methanol. Membranes
were blocked with no fat milk in distilled water and incubated with anti-FLAG primary antibodies
overnight at 4 °C, followed by rabbit-derived secondary antibody for 2 h at room temperature.
Protein bands were visualized using enhanced chemiluminescence (ECL).

**5. HQ Production Experiment**

Cell suspensions were prepared by washing induced *E. coli* BL21 cultures with 100 mM
potassium phosphate buffer (pH 8.0). Reactions (4 mL per tube) were supplemented with glucose
(0.05 g mL⁻¹) and phenol (10 mM) and incubated at 30 °C and 220 rpm. Samples (150 µL) were
withdrawn at different time points (0, 0.5, 1, 2, 2.5, 3, and 4 h), and enzyme activity was
quenched by methanol addition. After centrifugation, HQ concentrations in the supernatants were
determined spectrophotometrically at 290 nm based on a pre-established standard curve. Kinetic
parameters were analyzed via nonlinear regression to evaluate the catalytic efficiency of wild-type
and mutant enzymes.

##### **Results**

**Plasmid Construction and Preparation**

To investigate the role of CYP450-mediated hydroxyquinone (HQ) synthesis in termite attraction,
CYP450 and its variant (a double mutation at position A83F and A329F) were cloned into distinct
plasmid backbones, alongside control plasmids for expression validation. All plasmids were
extracted using a standard plasmid isolation kit (Plasmid Midi Preparation Kit for All Purpose,
Beyotime D0020), and the DNA concentration were quantified by UV spectrophotometry. The
concentrations of the extracted plasmids were as follows:

| Plasmid Name | Concentration (ng/μL) |
| --- | --- |
| T7-CYP450-flag | 65.1 |
| T7-CYP450 DM-flag | 46.1 |
| T7-CYP450-mCherry | 47.4 |
| T7-CYP450 DM-mCherry | 145.9 |

**Protein Expression Validation**

**Fluorescence Results**

**Experimental Controls for Fluorescence Background Validation**

Fluorescence microscopy was used for detecting mCherry, a red fluorescent protein,
with uniform settings (excitation: 540–580 nm, dichroic mirror: 585 nm, emission:
592–668 nm), and transmitted light-differential interference contrast (TL-DIC)
microscopy to visualize. To rule out background interference, two fundamental control
groups (PBS buffer control and *E. coli* BL21 control) were established. The *E. coli*
BL21 (no plasmid) sample **(Fig. 1a)** served as a host-specific control for T7-driven
constructs (all transformed into BL21); the TL-DIC channel revealed well-defined cells,
verifying viability, whereas the fluorescence channel showed no detectable red
fluorescence, eliminating BL21 as a background source.

**Fluorescence Microscopy for mCherry-Tagged Constructs**

Fluorescence imaging was performed to validate the expression of mCherry-tagged protein
using the following section: excitation wavelength 540–580 nm, and emission wavelength
592–668 nm). All *E. coli* BL21 samples, transformed with different constructs:
T7-CYP450-mCherry T7-CYP450 DM-mCherry and T7-CYP450-mCherry (wild-type) and with or
without IPTG induction, were imaged in both TL-DIC and fluorescence channels to correlate
cell morphology with fluorescence signals. For the T7-CYP450-mCherry and T7-CYP450 DM-mCherry
constructs transformed into BL21, cultures were subjected to IPTG induction prior to fluorescence
microscopy analysis. PTG induction entailed growing cultures to mid-logarithmic phase
(OD600 ≈ 0.6), adding IPTG to a final concentration of 0.5 mM, and
incubating at 37°C for 4 h to induce protein expression. In the IPTG-induced T7-CYP450-mCherry
sample **(Fig. 1b)**, the TL-DIC channel revealed healthy BL21 cells, confirming that observed
fluorescence was not attributable to debris, whereas the fluorescence channel exhibited strong
red fluorescence, conformed the expression of the wild-type fusion protein. Similarly, for the
IPTG-induced T7-CYP450 double-mutant-mCherry sample **(Fig. 1c)**, the TL-DIC channel confirmed
viable cells, and the fluorescence channel displayed intense red fluorescence, aligning with
higher expression levels observed in flag-tagged mutants. For the negative controls of the
bacteria without IPTG induction, both the T7-CYP450-mCherry **(Fig. 1d)** and T7-CYP450 DM- mCherry
**(Fig. 1e)** samples showed healthy cells in the TL-DIC channel, but no detectable red fluorescence
in the fluorescence channel, thereby validating the IPTG dependency of the T7 promoter protein
expression.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/mcherry.avif)

Figure 1: **IPTG-Induced Expression of T7-CYP450-mCherry and CYP450-DM-mCherry Fusion
Proteins in *E. coli* BL21.** ***a*** *E. coli* BL21 (no plasmid control).
***b*** T7-CYP450-mCherry with IPTG-induction. ***c*** T7-CYP450 DM-mCherry with
IPTG-induction. ***d*** T7-CYP450-mCherry negative control (without IPTG induction).
***e*** T7-CYP450 DM-mCherry negative control (without IPTG induction).

**Western Blot Analysis**

Western blot analysis was employed to validate the expression of flag-tagged CYP450
constructs. After SDS-PAGE and transferred the protein to the PDVF membranes.
Membranes were probed with anti-flag primary antibodies, followed by visualization
through ECL chemiluminescence. For the T7-CYP450-flag constructs transformed into
*E. coli* BL21, a distinct band at approximately 115 kDa was detected in the IPTG-
induced T7-CYP450-flag sample using anti-flag antibodies **(Fig. 2a)**, confirming the
expression of the wild-type CYP450 enzyme. The expression levels are in similar level
after 4 hours IPTG induction, with the strongest expression at 6 hours. For the IPTG-
induced T7-CYP450 DM-flag sample **(Fig. 2b)**, a pronounced band at ~115 kDa was
observed after 6 hours of IPTG induction, which is similar to the wild-type protein.

**a**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/hq2a.webp)

**b**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/hq2b.webp)

*Figure 2: Detection was performed using anti-flag primary antibody (specifically targeting
the flag tag fused to CYP450 variants) and a rabbit-derived secondary antibody (for
signal amplification), with visualization via chemiluminescence. **a** T7-CYP450-flag
(wild type; ~115 kDa band in IPTG-induced BL21). **b**T7-CYP450 DM-flag (intense
~115 kDa band in IPTG-induced BL21), indicating higher expression of the mutant.*

**Note:** "Mock" in the blot represents the empty vector control (i.e., *E. coli* BL21
harboring only the empty plasmid backbone, without the T7-CYP450-flag insert), used
to rule out non-specific binding of antibodies to the vector or host strain’s intrinsic
proteins.

**CYP450 Function and Hydroxyquinone (HQ) Detection**

**Standard Curve for HQ Quantification**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/hq3.webp)

*Figure 3: **Standard curve for hydroxyquinone (HQ) quantification at 290 nm.***

To enable accurate quantification of hydroxyquinone (HQ) production, the maximum
absorption wavelength (λmax) of HQ was determined to be 290 nm through full-spectrum
scanning. A standard calibration curve was subsequently constructed by plotting HQ
concentrations (mg/mL) against corresponding absorbance values at 290 nm. Linear
regression analysis produced the equation Y = 4.200X + 1.286 (where Y denotes
absorbance and X represents HQ concentration), accompanied by a high coefficient of
determination (R2 = 0.9936), signifying robust linearity. Further statistical evaluation
affirmed the significance of the non-zero slope (F = 312.8, P = 0.0032), with the 95%
confidence interval for the slope (3.178–5.222) excluding zero. These metrics
collectively validate the standard curve as a dependable method for HQ quantification
in ensuing bacterial culture analyses **(Fig. 3)**.

**Time-course of HQ Production**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/hq4.webp)

*Figure 4: **Time-course of phenol-to-HQ conversion by CYP450 variants.***

To assess the functional activity of CYP450 variants in converting phenol (substrate for
CYP450-mediated hydroxylation) to HQ, bacterial cultures expressing either the wild-
type (T7-CYP450-flag) or double-mutant (T7-CYP450 DM-flag) constructs were
supplemented with phenol and incubated under shaking conditions for 4 hours. Samples
(150 μL) were collected at 30-minute intervals, centrifuged, and the pellets resuspended
in potassium phosphate buffer for spectrophotometric measurement of HQ
accumulation at 290 nm. This wavelength was selected according to the HQ
characteristic λmax attributable to conjugated double bonds and hydroxyl groups,
facilitating precise quantification.

The double-mutant strain exhibited a distinct kinetic pattern: HQ levels remained low
for the first ~2.5 hours, followed by a sharp increase after 3 hours, reaching
approximately 0.25 mg mL-1 at 4 hours. In contrast, the wild-type enzyme showed only
a gradual increase, with HQ concentration remaining below 0.05 mg mL-1. Nonlinear
regression confirmed a strong fit for the mutant (R2 = 0.9959) but a weaker one for the
wild type (R2 = 0.8575), reflecting greater catalytic efficiency.

Statistical analysis supported these observations: although the mean difference between
the two curves was not significant (unpaired t-test, p = 0.0615), an F-test revealed a
highly significant difference in reaction variance (F(8, 8) = 35.04, p < 0.0001),
indicating markedly different kinetic behaviors. The mutant displayed a more
cooperative and consistent catalytic trajectory, achieving significantly higher HQ
accumulation by the end of the assay.

These results collectively demonstrate that the A83F/A329F double mutation
substantially enhances catalytic performance and reaction stability in CYP450-
mediated phenol hydroxylation.

**4-hour Endpoint Analysis of HQ Concentration**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/hq5.webp)

*Figure 5: **4-hour endpoint analysis of HQ production***

At the 4-hour endpoint, HQ concentrations were quantified across experimental groups
to evaluate catalytic performance (**Fig. 5**). The control strain (BL21 without plasmid)
produced 0.029 mg mL⁻¹ HQ, confirming negligible endogenous activity. The wild-
type T7-CYP450-flag yielded 0.043 mg mL⁻¹, while the double-mutant T7-CYP450
DM-flag reached 0.269 mg mL-1—representing ~6.2-fold and ~9.2-fold increases over the wild-
type and control, respectively.

A one-way ANOVA revealed a highly significant difference among the three groups
(F(2,6) = 1882, p < 0.0001, R² = 0.9984), with Tukey’s post-hoc test confirming that
the double-mutant group was significantly higher than both the wild-type and control
(p < 0.05). Brown–Forsythe (p = 0.4823) and Bartlett’s (p = 0.349) tests verified
homogeneity of variances, validating the ANOVA assumptions.

Collectively, these results demonstrate that the A83F/A329F double mutation
substantially enhances CYP450’s catalytic efficiency in phenol hydroxylation,
confirming the superior functionality of the engineered variant.

##### **Discussion**

This study examined the cytochrome P450 from Bacillus megaterium (CYP102A1,
P450-BM3) and its engineered double mutant (A83F/A329F) for the bioconversion of
phenol to hydroquinone (HQ). Both constructs were expressed in *E. coli* BL21(DE3),
and the mutant produced significantly higher HQ levels (0.269 mg mL-1) after 4 hours
compared with the wild type (0.043 mg mL-1), indicating improved catalytic efficiency
resulting from rational residue substitutions.

**Expression stability revealed by Western blot**

Western blotting revealed that the double mutant accumulated rapidly during the first 6
hours of IPTG induction but declined after overnight expression. This suggests that the
mutant folds efficiently in the early phase but becomes unstable over extended
induction, likely due to aggregation or proteolytic degradation. Aromatic substitutions
such as phenylalanine can enhance folding through hydrophobic interactions but may
also expose aggregation-prone surfaces [1].

Overall, these results suggest a trade-off between high short-term expression and
reduced long-term stability in the mutant—a phenomenon frequently observed in
enzyme engineering, particularly among hyperactive P450 variants [2][3]. Indeed, in CYP450 BM3-based biocatalysts, highly active mutants
have been reported to lose activity rapidly during prolonged reactions, reflecting a
general stability–activity trade-off [4].

**Catalytic performance and possible mechanisms**

The improved HQ formation by the mutant may arise from multiple, non-exclusive
factors. On one hand, the higher HQ yield observed for the mutant may result from
increased levels of soluble, catalytically competent enzyme at 6 h, providing more
active protein for the reaction. On the other hand, the A83F/A329F substitutions could
intrinsically enhance catalytic efficiency—by influencing substrate orientation or
electron transfer within the active site—similar to the effects reported for the
A82F/A328F variant of P450-BM3 [5].

While this mechanistic rationale may also apply to A83F/A329F, direct kinetic evidence
(e.g., kcat, Km, and coupling efficiency) will be required to disentangle contributions
from increased enzyme abundance versus intrinsic catalytic enhancement.

**Physiological relevance and potential applications**

HQ acts as a phagostimulating and aggregation cue in termites, effective at nanogram
surface loads (ng cm-2), while higher concentrations can reduce attractiveness [6]. The HQ level achieved in this study (~0.269 mg mL-1) exceeds
typical behavioral thresholds, suggesting that dilution or controlled-release formulation
would be required for practical application. Nevertheless, this system provides a green
and biosafe route for HQ synthesis suitable for pheromone-based termite management.

**Implications for the “Attraction–Killing” termite control system**

The target species, Coptotermes formosanus Shiraki, uses HQ and 2-phenoxyethanol
as foraging and recruitment cues [7, 8]. HQ
produced via CYP450 catalysis could be incorporated into toxic baits to attract termites
while ensuring biosafety, as only purified HQ—not engineered bacteria—is used.
However, the optimal concentration range must be determined to avoid overdosage or
conversion of HQ to quinones, which may reduce attractiveness [6].

Stabilization methods such as microencapsulation, slow-release polymers, or
antioxidants can further improve field stability and minimize environmental impact.

**Limitations and future outlook**

While the A83F/A329F variant demonstrated higher short-term expression and
improved catalytic performance, its reduced stability upon extended induction indicates
that future optimization is needed. Approaches such as co-expression with chaperones,
modulation of induction temperature, or directed evolution targeting structural rigidity
may further enhance enzyme robustness. Additionally, expanding substrate scope
studies will clarify whether this mutation generally improves aromatic hydroxylation
or is specific to phenol. Integrating computational modeling with experimental
screening could provide deeper mechanistic insights and guide the next generation of
CYP450 engineering.

##### **References**

1. [1] Bertelmann, C., & Buhler, B. (2024). Strategies found not to be suitable for stabilizing high steroid hydroxylation activities of CYP450 BM3-based whole-cell biocatalysts. *PLoS One, 19*(9), e0309965.
   https://doi.org/10.1371/journal.pone.0309965
2. [2] Cornelius, M. L., & Grace, J. K. (1997). Effect of termite soldiers on the foraging behavior of Coptotermes formosanus (Isoptera: Rhinotermitidae) in the presence of predatory ants. *Sociobiology, 29*(3), 247-253.
   ://WOS:A1997WR54200003
3. [3] Diallo, S., Kasparova, K., Sulc, J., Johny, J., Krivanek, J., Nebesarova, J., Sillam-Dusses, D., Kyjakova, P., Vondrasek, J., Machara, A., Luksan, O., Grosse-Wilde, E., & Hanus, R. (2025). Identification of the trail-following pheromone receptor in termites. *Elife, 13*.
   https://doi.org/10.7554/eLife.101814
4. [4] Dodhia, V. R., Fantuzzi, A., & Gilardi, G. (2006). Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego. *J Biol Inorg Chem, 11*(7), 903-916.
   https://doi.org/10.1007/s00775-006-0144-3
5. [5] Reinhard, J., Lacey, M. J., Ibarra, F., Schroeder, F. C., Kaib, M., & Lenz, M. (2002). Hydroquinone: a general phagostimulating pheromone in termites. *J Chem Ecol, 28*(1), 1-14. https://doi.org/10.1023/a:1013554100310
6. [6] Stimple, S. D., Smith, M. D., & Tessier, P. M. (2020). Directed evolution methods for overcoming trade-offs between protein activity and stability. *Aiche Journal, 66*(3). https://doi.org/ARTN e1681410.1002/aic.16814
7. [7] Teufl, M., Zajc, C. U., & Traxlmayr, M. W. (2022). Engineering Strategies to Overcome the Stability-Function Trade-Off in Proteins. *ACS Synth Biol, 11*(3), 1030-1039. https://doi.org/10.1021/acssynbio.1c00512
8. [8] Zhou, H. Y., Wang, B. J., Wang, F., Yu, X. J., Ma, L. X., Li, A. T., & Reetz, M. T. (2019). Chemo- and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase. *Angewandte Chemie-International Edition, 58*(3), 764-768. https://doi.org/10.1002/anie.201812093

## EcTI

##### **Aim**

From a biological perspective, *Enterolobium Contortisiliquum* Trypsin Inhibitor
(EcTI) is a plant-derived trypsin inhibitor. Its mechanism of action involves targeting
and inhibiting the activity of trypsin, a key serine protease. EcTI is capable of binding
to the active site of trypsin, a property that makes it a potential tool in termite control
research. This is because trypsin plays a crucial role in termites’ ability to hydrolyze
dietary proteins into absorbable nutrients. Focusing on EcTI, this project aims to
verify its inhibitory effect on trypsin related to termites and lay the foundation for its
application in termite control. The practical application potential of EcTI lies in its
specific mechanism of action targeting digestive enzymes. Unlike broad-spectrum
toxic agents, EcTI targets trypsin, a core component of the termite digestive system.
This means it may interfere with the physiological functions of termites in a more
targeted manner [1]. The purpose of studying EcTI in this project is
not only to confirm its in vitro activity of inhibiting trypsin, but also to establish a
theoretical basis for its application in termite control.

##### **Experimental Design**

**2. Plasmid Preparation and Validation**

To investigate the role of *Enterolobium Contortisiliquum* Trypsin Inhibitor (EcTI) in
inhibiting the trypsin activity in the termite killing process, the EcTI-His tag sequence
[(BBa\_25N3Q748)](https://registry.igem.org/parts/bba-25n3q748) was cloned into certain plasmid backbones with J23100
promoter [(BBa\_J23100)](https://registry.igem.org/parts/bba-j23100) and ampicillin resistance gene to harvest EcTI. The
plasmid was custom-synthesized by VectorBuilder
and extracted using a standard plasmid isolation kit
(Medium-Speed Plasmid Extraction Kit (Universal Type), Beyotime D0020).
The DNA concentration was quantified via UV spectrophotometry (dsDNA setting) to
ensure suitability for downstream experiments.

The concentration of the extracted plasmid is listed as follows:

* J23100-EcTI [(BBa\_251SK4RC)](https://registry.igem.org/parts/bba-251sk4rc): 207.3 ng/μL

**3. Plasmid Construction and Expression Validation**

To provide an oxidative environment more conducive to the formation of disulfide
bonds of EcTI, we chose to express the constructed plasmid in the Shuffle T7 strain
(NEB lab) [2]. For complex proteins that require the formation of disulfide bonds (such
as enzymes or secreted proteins containing multiple cysteines), the environment in
Shuffle T7 can significantly reduce misfolded folding and increase the yield of active
proteins. While J23100 acts as a strong constitutive promoter [3], its continuous and
efficient drive of gene expression may lead to excessive protein accumulation and
increased folding pressure. Shuffle T7 precisely alleviates this problem.

**4. Preparation of bacterial supernatant**

* Control group: Shuffle T7 strain without plasmid culturing overnight
* EcTI group: Shuffle T7 carrying the J2310019-EcTI-His tag plasmid culturing overnight

The bacterial culture was collected after overnight incubation
and then centrifuged and washed with PBS buffer to remove LB Broth. Subsequently, the bacteria were
subjected to ultrasonic lysis.

**5. EcTI Function Analysis**

After obtaining the EcTI extract from the Shuffle T7 strain, a trypsin activity assay
using the Colorimetric Trypsin Activity Assay Kit (Beyotime P0324S) was conducted
to analyze the inhibition effect of our product. The kit involves using a substrate
labeled with pNA. Trypsin hydrolyzes this substrate to release free pNA, whose
concentration can be detected by measuring absorbance at a specific wavelength. The
change in pNA concentration reflects trypsin activity, and by comparing pNA
concentration changes, the inhibition effect of EcTI on trypsin activity can be
assessed.

Both the control group and EcTI group extraction were diluted 10-fold using Assay
Buffer (P0324S-1) for further detection. The EcTI group was also diluted 1000-fold
and 100,000-fold, as the trypsin activity was unknown.

##### **Results**

To quantify the amount of pNA produced in the reaction system, a pNA concentration
standard curve for the trypsin activity detection is constructed **(Fig. 1)**. The
absorbance at 405 nm (OD405) of each well was measured. Linear regression was
performed with the pNA concentration (nmol) as the abscissa and the OD405 value as
the ordinate, yielding the equation Y = 0.02249X + 0.01803 (where Y represents the
OD405 value and X represents the pNA concentration). In subsequent steps, the
OD405 value of the sample was substituted into this equation to calculate the pNA
production, and then the trypsin activity was computed according to the activity
formula provided in the kit.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/ecti-1.webp)

*Figure 1: **pNA concentration (nmol) Standard curve***

As shown in **Fig. 2a**, the pNA concentration change of the control group (Control
(D10) 10-fold dilution) rose gradually over time, reaching nearly 6 nmol at 60
minutes and suggests the trypsin activity. In contrast, the pNA concentration change
is relatively low with different dilution EcTI treatment (EcTI (D10) 10-fold
dilution, EcTI (D1000) 1000-fold, EcTI (D100000) 100000-fold). The data suggested
the reactions were suppressed when compared to the control group. **Fig. 2b** demonstrated that the trypsin
activity of the control group (Control (D10) 10-fold dilution) was about 6 nmol/hour,
while the trypsin activities of different dilution EcTI treatment groups were
significantly lower than that of the control group and a dose-dependent inhibition effect was found,
which proved EcTI could effectively inhibit trypsin activity.

**a**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/ecti-2.webp)

**b**

![](https://static.igem.wiki/teams/5698/wiki/wetlab/ecti-3.webp)

*Figure 2: **Inhibitory effect of EcTI extract to trypsin activity.***
(a) Time-course change of pNA concentration (nmol) with EcTI group and control group
(b) Enzyme activity of trypsin with different dilution factors of EcTI group and control group

To assess EcTI's inhibitory effect on trypsin activity, one-way ANOVA and Tukey's
multiple comparisons were performed. ANOVA showed a significant overall group
difference (F = 6.460, P = 0.0049), and the Brown-Forsythe test confirmed
homogeneous variances (P = 0.2474). Tukey's test revealed that compared to the
Control Plasmid group, remaining EcTI-treated groups (D10, D1000, and D100000)
had significantly lower trypsin activity (P < 0.05 for D1000 and D100000; P < 0.01
for D10), indicating potent inhibition. Even at a high dilution (D100000), EcTI was
effective.

##### **Discussion**

Mechanistically, the inhibitory effect of EcTI on trypsin conforms to the functional
characteristics of plant-derived trypsin inhibitors (PTIs). Trypsin is a key serine
protease in the termite digestive system, responsible for hydrolyzing dietary proteins
into absorbable peptides and amino acids. By binding to the active site of trypsin,
EcTI blocks its catalytic activity and disrupts the protein digestion process in termites.
This mechanism suggests that long-term exposure to EcTI may lead to nutrient
deficiency, growth retardation, or even death in termites, providing a theoretical basis
for its application in termite control.

The current experiments have only verified the trypsin-inhibitory activity of EcTI in
vitro (using bacterial extracts). However, the termite digestive system is a complex
microenvironment involving multiple influencing factors. The termite midgut is
alkaline (pH 8.5-10.0), while the in vitro assay was conducted in a standard buffer.
The stability and inhibitory activity of EcTI may change under alkaline conditions;
therefore, subsequent experiments need to simulate the pH of the termite midgut to
evaluate its actual effect. In addition, termites secrete other proteases (such as
chymotrypsin and elastase) and carbohydrate enzymes. EcTI may interact with these
enzymes (e.g., being degraded by other proteases), reducing its effective
concentration in the gut. Future studies should analyze the stability of EcTI in termite
gut homogenates and its cross-inhibitory effect on other proteases.

##### **References**

1. [1] Ferreira, R. D., Napoleo, T. H., Silva-Lucca, R. A., Silva, M. C. C., Paiva, P. M. G., & Oliva, M. L. V. (2019). The effects of serine protease inhibitor on the survival of the termite Nastitermes corniger, and its use as affinity adsorbent to purify termite proteases. *Pest Management Science*, *75*(3), 632-638. https://doi.org/10.1002/ps.5154
2. [2] Bayer, C. N. (2022). *Engineering bacterial genomes throughout the central dogma of molecular biology*.
   Technical University of Denmark. https://backend.orbit.dtu.dk/ws/portalfiles/portal/311756128/
   PhD\_Thesis\_Carolyn\_Nicole\_Bayer.pdf
3. [3] Ting, W. W., Huang, C. Y., Wu, P. Y., Huang, S. F., Lin, H. Y., Li, S. F., Chang, J. S., & Ng, I. S. (2021). Whole-cell biocatalyst for cadaverine production using stable, constitutive and high expression of lysine decarboxylase in recombinant Escherichia coli W3110. *Enzyme and Microbial Technology*, *148*, 109811. https://doi.org/10.1016/j.enzmictec.2021.109811

## Hecate & Melittin

##### **Aim**

Peptides, by biological definition, are short chains of amino acids that are
fundamental to many physiological processes. Their relatively small size, compared to
larger proteins, makes them versatile tools in therapeutic and research applications.
This project focuses on two specific lytic peptides—Hecate and Melittin—which
possess the ability to disrupt cell membranes. Hecate is a synthesized lytic peptide
known for its antimicrobial effects. In contrast, Melittin is a natural lytic peptide and
the primary toxic component in European honeybee venom, also renowned for its
potent, non-specific ability to rupture cell membranes. Our project will utilize the core
lytic function in a general manner to conduct a preliminary toxicity assessment on
termites.

The real-world applications of these peptides highlight their distinct mechanisms.
Hecate is typically designed for targeted disruption, making it a valuable asset in pre-
clinical studies for diseases like cancer [1]. Melittin, however, acts as a
powerful, broad-spectrum blowtorch against cells. The purpose of employing these
lytic peptides in our project is not to directly kill termites. Instead, we
aim to leverage their membrane-disrupting properties to target and impair the
symbiotic protozoa that reside within the termite's gut. These protozoa are essential
for the termite's ability to digest cellulose, its main food source. By disrupting this
critical symbiosis, we can explore a novel approach to termite control.

**Plasmid construction**

We constructed the plasmids with pET28a backbone, using the inducible anaerobic
promoter FNR promoter to control the expression in low oxygen conditions where
glucose is present; and added the kanamycin resistance gene for later transformation
screening. Our Hecate and Melittin sequences are fused with a secretion signal NSP4
at the C-terminus of the secretion peptide.

Important to state that NSP4 secretion signal was modified:

NSP4 Amino Acid Sequence Modification

| Stage | Sequence | Note |
| --- | --- | --- |
| Original NSP4 | MKKITAAAGLLLLAAQPAMA | Ends with A |
| Modified NSP4 | MKKITAAAGLLLLAAQPAMK | Last A changed to K |
| Finalized NSP4-K | MKKITAAAGLLLLAAQPAMKK | One more K is added |

This modification introduces a trypsin cleavage site, enabling targeted separation of
the peptide from the secretion signal if needed. It also supports inducible gene
expression and secretion of the peptide. Pure peptide can be obtained by enzymatic
removal of the signal sequence. The plasmid was synthesized by VectorBuilder and DNA extracted using the Medium-Speed Plasmid Extraction Kit (Beyotime D0020),
with concentration measured by UV spectrophotometry at 260 and 280 nm.

Totally, three plasmids were designed:

**(1) pET28a-FNR-mCherry:** Serve as the control to verify the expression of genes
under anaerobic expression that controlled by FNR promoter.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/lh-1.webp)

*Figure 1: **The illustration of pET28a-FNR-mCherry***

**(2) pET28a-FNR-Hecate:** The plasmid encoded the NSP4-K-Hecate sequence,
and the expression is controlled by the FNR promoter.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/lh-2.webp)

*Figure 2: **The illustration of pET28a-FNR-NSP4-K-Hecate***

**(3) pET28a-FNR-Melittin:** This plasmid expresses the NSP4-K-Melittin peptide
using the FNR promoter.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/lh-3.webp)

*Figure 3: **The illustration of pET28a-FNR-NSP4-K-Melittin***

After extracting the plasmids, we introduced them into competent cell *E. coli*
MG1655 cells, which are compatible with the FNR promoter. The transformed cells were
plated on LB agar containing kanamycin and incubated overnight at 37°C. Single
colonies were then picked and cultured in liquid LB broth with kanamycin overnight.
The bacteria were either harvested for DNA extraction or used to make frozen stocks
by adding glycerol to reach a final concentration of 50%. The stocks were stored at -80°C.

After plasmid DNA extraction, the concentration of the plasmids is as follows:

Table 1: The Plasmid Name and Concentrations

| Plasmid Name | Concentration |
| --- | --- |
| pET28a-FNR-mCherry | 36 ng/μL |
| pET28a-FNR-NSP4-K-Hecate | 11.2 ng/μL |
| pET28a-FNR-NSP4-K-Melittin | 23.8 ng/μL |

##### **Experimental Design**

**1. Preparation of the mCherry plasmid carrying *Escherichia coli* MG1655**

The plasmid is introduced into MG1655 competent cells by the heat shock method.
After transformation, the cells are plated on LB agar containing kanamycin using the
streak plate technique and incubated overnight at 37°C. Only cells that have
successfully taken up the plasmid will survive and grow on the selective medium.

The use of the mCherry reporter gene offers the advantage of enabling detection in
live bacteria. This approach eliminates the need to extract protein or perform
additional analyses, such as Western blotting, to confirm protein expression.

**2. Preparation of bacterial supernatant:**

**2.1 Culture the bacteria**

* Control: MG1655 strain without plasmid
* Hecate: MG1655 strain carrying the FNR-Hecate
* Melittin: MG1655 strain carrying the FNR-Melittin

The bacteria are cultured in liquid LB broth medium supplemented with glucose at a
final concentration of 1 mg/mL. The cultures are grown in tubes placed on a shaker
and incubated at 37°C under aerobic conditions until the OD600 reaches 0.4–0.6. At
this point, the tube lids are sealed tightly to initiate anaerobic conditions and induce
gene expression via activation of the FNR promoter.

**2.2 Induce expression in anaerobic condition and collect the supernatant**

When the OD600 of the cultures reaches 0.4–0.6, the tube lids are sealed tightly to
induce gene expression by activating the anaerobic FNR promoter. As the oxygen is
gradually consumed, samples are collected at various time points: 4 hours, 6 hours,
and overnight (O/N). After collection, cultures are centrifuged at 5,000 rpm for 5
minutes. The supernatant is then transferred to clean Eppendorf tubes and stored at -
80°C.

**3. Preparation of the pure peptides:**

In addition to extracting peptides from the bacterial expression system, we also
obtained a small amount (1 mg) of each pure synthetic peptide from a commercial
supplier (China Peptide) for comparison with the extracted samples

**3.1 Stock solutions**

* Control: Pure DMSO
* Hecate: Pure DMSO is added to 1mg of peptide to make up 1mg/mL stock solution.
* Melittin: Pure DMSO is added to 1mg of peptide to make up 1mg/mL stock solution.

The solutions of pure peptides are aliquoted into 100 μL portions and stored at –80°C.
Since repeated freeze-thaw cycles can degrade the peptides, aliquoting into separate
tubes allows individual portions to be used as needed, minimizing damage to the
remaining samples.

**4. Toxin assay**

We performed testing on *E. coli* to confirm the functionality of lytic peptides, Hecate
and Melittin, which are known to have antimicrobial effects that suppress bacterial
growth. The general procedure involved mixing both the bacteria-synthesized
peptides and pure peptides with a culture of the *E. coli* strain DH5α. Bacterial growth
was then monitored by measuring the optical density at 600 nm (OD600) over a 12-
hour period, with an additional data point collected at 24 hours.

Second, we assessed the effect of the lytic peptides on the multicellular organism *C. elegans*.
This worm is a suitable model for preliminary toxicity assessment due to the
ease of observing morphological and behavioral changes. We first tested the peptides'
effect on motility by exposing the worms to the peptides and counting the number of
thrashes within a 30-second interval in M9 buffer. Furthermore, we used the act-
5p::GFP strain to enable direct observation of the gut morphology following peptide
exposure.

The detailed protocols for these experiments are provided in the [LAB PROTOCOL](https://static.igem.wiki/teams/5698/wiki/wetlab/experimental-protocol4.pdf).

##### **Results**

**1. The FNR mediated protein expression by fluorescence detection of mCherry under anaerobic conditions**

We introduced the pET28a-FNR-mCherry plasmid into *E. coli* strain MG1655
competent cell and induced gene expression. The result of functional analysis of the
FNR promoter demonstrated successful, though non-specific, induction of mCherry
expression. While the expected anaerobic induction was observed, fluorescence was
also detected in transformed cells under aerobic conditions. The absence of
autofluorescence in the plasmid-free MG1655 control confirms that the signal was
plasmid-derived.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/lh-4.webp)

*Figure 4: **The gene expression of the mCherry reporter protein after introducing the pET28a-FNRmCherry
plasmid into E. coli strain MG1655.***

**2. Pure Peptides have Antimicrobial Effect on *Escherichia coli* strain DH5α in
the presence of DMSO**

Our results identify the most effective antibacterial concentrations for each peptide:
0.025 μg/μL for Hecate and both 0.025 and 0.05 μg/μL for Melittin **(Table 2 and 3)**. However,
interpreting this activity required careful consideration of the DMSO solvent. We confirmed that DMSO itself exerts a time-dependent inhibitory effect, which is most
pronounced at a 5% concentration.

Therefore, to isolate the specific contribution of the peptides, we directly compared
each peptide treatment to its respective DMSO vehicle control. This analysis,
supported by two-way ANOVA and Area Under the Curve (AUC) comparisons,
clearly demonstrates that the antibacterial effect in the 1.25% and 2.5% DMSO
conditions is significantly stronger when the peptide is present. This confirms that the
observed growth inhibition is a direct result of peptide activity and not merely an
artifact of the solvent.

Table 2: AUC Measurements and Percent Inhibition of Hecate and Melittin with
Varying DMSO Concentrations

| Treatment | Description | OD600 | SD | Inhibition Rate |
| --- | --- | --- | --- | --- |
| Blank | No Treatment | 0.4456 | 0.002376 | 0% |
| Kana | Positive control | 0.2078 | 0.005093 | 100% |
| 5% DMSO | DMSO only | 0.3663 | 0.002194 | 33.3% |
| 2.5% DMSO | DMSO only | 0.4220 | 0.002241 | 9.9% |
| 1.25% DMSO | DMSO only | 0.4427 | 0.004446 | 1.2% |
| 5% DMSO + H | DMSO + Hecate | 0.3991 | 0.006559 | 19.5% |
| 2.5% DMSO + H | DMSO + Hecate | 0.2609 | 0.005410 | **77.6%** |
| 1.25% DMSO + H | DMSO + Hecate | 0.3595 | 0.003503 | 36.1% |
| 5% DMSO + M | DMSO + Melittin | 0.2997 | 0.006343 | 61.3% |
| 2.5% DMSO + M | DMSO + Melittin | 0.2993 | 0.002194 | **61.4%** |
| 1.25% DMSO + M | DMSO + Melittin | 0.3633 | 0.003382 | 34.6% |

**Footnote:** The inhibition percentage is calculated using the following equation, assuming that Kana
AUC is the maximum inhibition percentage that can be achieved: % Inhibition = [(Blank AUC -
Sample AUC) / (Blank AUC - Kana AUC)] × 100

Table 3: The Respective Peptide Concentrations in each Treatment group

| Group | DMSO (%) | Hecate (μg/μL) |
| --- | --- | --- |
| 1.25% DMSO | 1.25 | 0 |
| 2.5% DMSO | 2.5 | 0 |
| 5% DMSO | 5.0 | 0 |
| 1.25% DMSO + H | 1.25 | 0.0125 |
| 2.5% DMSO + H | 2.5 | 0.025 |
| 5% DMSO + H | 5.0 | 0.05 |
| 1.25% DMSO + H | 1.25 | 0.0125 |
| 2.5% DMSO + H | 2.5 | 0.025 |
| 5% DMSO + H | 5.0 | 0.05 |

**2.1 The Effect of DMSO**

To ensure that the solvent DMSO did not confound our results, we evaluated its
impact on bacterial growth. As shown in **Figure 5**, a high concentration of DMSO
(5%) significantly inhibited the growth of DH5α compared to the untreated control
(Blank). In contrast, lower concentrations (2.5% and 1.25%) had no observable effect
on growth, as the optical density was comparable to the Blank control. As expected,
the kanamycin (Kana) control completely suppressed growth.

A two-way ANOVA revealed that both time and Treatment were significant factors
influencing the outcome, but their interaction was the most critical (p < 0.0001 for all
factors). This significant Time x Treatment interaction indicates that the effect of the
DMSO and Kanamycin depended heavily on the time point measured, rather than
having a consistent effect over the entire experiment. Time accounted for the largest
proportion of variance in the model, suggesting the measured variable changes
considerably throughout the duration of the experiment, regardless of Treatment.

![](https://static.igem.wiki/teams/5698/wiki/dmso.avif)

*Figure 5: The effect of DMSO on the growth of DH5α*

Table 4: Two-way ANOVA results for the effects of DMSO and Kana Treatment.

| Source of Variation | % of Total Variation | P Value |
| --- | --- | --- |
| Time | 70.63 | < 0.0001 |
| Treatment | 23.29 | < 0.0001 |
| Time × Treatment | 5.56 | < 0.0001 |

**Footnote:** Data are from a two-way repeated measures ANOVA with Geisser-Greenhouse correction.
The Treatment factor (4 degrees of freedom) compares the Blank, Kana, 5% DMSO, 2.5% DMSO, and
1.25% DMSO groups (n=12 per group).

**2.2 The Effect of Pure Hecate (dissolved in DMSO)**

Hecate exhibited potent, concentration-dependent antibacterial activity against DH5α
**(Fig. 6a)**. The antibacterial effect was attributable to the peptide itself, as growth in all
Hecate-treated groups was significantly reduced compared to their corresponding
vehicle controls (DMSO alone at the same concentration) and the blank control **(Figs.
6b-d)**. The intermediate Hecate concentration (0.025 μg/μL) showed efficacy
comparable to the kanamycin control.

The growth inhibition caused by Hecate was highly dependent on both the peptide
concentration and the duration of exposure. A two-way ANOVA confirmed that Time
was the dominant source of variation, but a highly significant Time x Treatment
interaction (p < 0.0001) was the most critical finding **(Table 5)**. This indicates that the
differences between the Hecate Treatment groups and their effects on bacterial growth
became more pronounced and distinct as the experiment progressed, rather than being
constant over time.

![](https://static.igem.wiki/teams/5698/wiki/hecate.avif)

*Figure 6: **Concentration-dependent antibacterial activity of Hecate against *E. coli* DH5α. (a)** Bacterial growth curves
treated with varying concentrations of Hecate. **(b-d)** Comparison of Hecate activity against vehicle controls at specific DMSO
concentrations. The final test concentrations for each group are listed in Table 2. The "Blank" group represents untreated
cells, and "Kana" (kanamycin) serves as the positive antibiotic control.*

Table 5: Two-way ANOVA results for the effects of Hecate Treatment.

| Source of Variation | % of Total Variation | P Value |
| --- | --- | --- |
| Time | 58.66% | < 0.0001 |
| Hecate Treatment | 18.87% | < 0.0001 |
| Time × Treatment | 21.43% | < 0.0001 |

**Footnote:** Data are from a two-way repeated measures ANOVA with Geisser-Greenhouse correction.
The Treatment factor (4 degrees of freedom) compares the Blank, 5% DMSO+M, 2.5% DMSO+M,
and 1.25% DMSO+M groups (n=12 per group).

**2.3 The Effect of Pure Melittin (dissolved in DMSO)**

Melittin exhibited strong, dose-dependent antibacterial activity against DH5α,
with higher concentrations (0.025 and 0.05 μg/μL) showing a similar and significant
inhibitory effect after 4 hours of Treatment ,**(Fig. 7a)**. This effect is directly attributable
to melittin, as growth in all melittin-treated groups was substantially reduced compared to their
corresponding vehicle controls containing DMSO alone **(Figs. 7b-d)**. The effect of the Melittin
Treatment was highly dynamic over time. A two-way ANOVA revealed a highly significant Time x Treatment
interaction (p < 0.0001), which accounted for a substantial portion of the total variance ,**(Table 6)**.
This indicates that the differences in antibacterial efficacy between the groups were not constant but changed significantly throughout the experiment.

![](https://static.igem.wiki/teams/5698/wiki/melittin.avif)

*Figure 7: **Concentration-dependent antibacterial activity of Melittin
against E. coli DH5α. (a)** Bacterial growth curves treated with varying
concentrations of Melittin. **(b-d)** Comparison of Melittin activity against
vehicle controls at specific DMSO concentrations. The final test
concentrations for each group are listed in Table 2. The "Blank" group represents
untreated cells, and "Kana" (kanamycin) serves as the positive antibiotic control.*

Table 6: Two-way ANOVA results for the effects of Melittin Treatment.

| Source of Variation | % of Total Variation | P Value |
| --- | --- | --- |
| Time | 58.66 | < 0.0001 |
| Treatment | 18.87 | < 0.0001 |
| Time × Treatment | 21.43 | < 0.0001 |

**Footnote:** Data are from a two-way repeated measures ANOVA with Geisser-Greenhouse correction. The Treatment factor (4 degrees of freedom) compares the Blank, 5% DMSO+M, 2.5% DMSO+M, and 1.25% DMSO+M groups (n=12 per group).

**3. Bacterial Supernatant Shows No Acute Toxicity to *C. elegans* After Two-Hour Exposure**

To assess the effects of toxins on *C. elegans*, we exposed the worms to bacterial
supernatant for 2 hours. Hecate and Melittin peptides, which primarily disrupt cell
membranes, are ingested into the gut; therefore, we hypothesized they could
potentially harm the worm’s gut. However, under brightfield microscopy, no clear
phenotypic changes were observed. Observations using Green-Fluorescent Protein
(GFP) fluorescence revealed the gut remained linear and smooth, without twists,
indicating the worms were healthy after 2 hours of toxin exposure. This suggests these
peptides may exhibit selectivity for certain cell membranes, as eukaryotic membranes
differ from protozoan membranes.

![](https://static.igem.wiki/teams/5698/wiki/c-elegans.avif)

*Figure 8 **The stage L4 Caenorhabitis elegans strain act-5p::GFP**, which contains GFP gene in the gut
cells, observed under the Nikon SMZ18 stereomicroscope at 90× magnification, to take the brightfield
images and combined with Nikon Intensilight C-HGFI to observe the GFP signal and photograph.
Note: The images taken by the software only presents in white and black.*

**4. Hecate Bacteria Supernatant Shows Impact on *Caenorhabditis elegans* Thrashing Ability**

While bacterially-synthesized Hecate and Melittin were not lethal to *C. elegans*,
Hecate specifically induced a significant paralytic effect. Treatment with Hecate
resulted in a statistically significant reduction in thrashing rate compared to the
control (p=0.0124), whereas Melittin had no significant effect on motility (p >
0.9999). This suggests that Hecate's designed affinity for eukaryotic membranes may
allow it to disrupt neuromuscular function in the nematode more effectively than the
general cytolysin Melittin.

![](https://static.igem.wiki/teams/5698/wiki/wetlab/lh-8.webp)

*Figure 9: Scatter bar graph comparing the effect of feeding bacteria with Hecate or Melittin
expression plasmids on C. elegans.*

**Table 7:** The statistics of Bonferroni's post-hoc analysis for reference.

| Comparison | Mean Difference | 95% CI | P-value |
| --- | --- | --- | --- |
| **Control vs. Hecate** | 16.05 | 3.26 to 28.84 | 0.012 |
| **Control vs. Melittin** | 2.21 | -11.88 to 16.30 | >0.99 |

**Footnote:** ns = not significant; \* = statistically significant (p < 0.05 after Bonferroni adjustment for 2
comparisons).

##### **Discussion**

**1. The Non-Specific Gene Regulation of the FNR promoter**

The FNR promoter, traditionally characterized as an anaerobic promoter in prior
studies and documented in the iGEM registry, demonstrated unexpected gene
expression activation under both aerobic and anaerobic conditions in our experiment.
This observation suggests that the FNR promoter may not be exclusively anaerobic. A
related finding was reported by the iGEM team [NCKU Tainan 2019](https://2019.igem.org/Team%3ANCKU_Tainan), which
characterized the same FNR promoter part [2]. They noted significantly higher fluorescence signals in aerobic conditions
compared to anaerobic ones, though their analysis focused on GFP expression, while
our study utilized the mCherry protein.

Further investigation revealed a potential issue with the mCherry reporter gene, which
may obscure promoter regulation. The codon-optimized mCherry exhibits constitutive
background fluorescence due to aberrant translation initiation. This phenomenon
arises from an internal alternative translation initiation site (ATIS) at the 10th
methionine residue (M10). An upstream Shine-Dalgarno-like sequence enhances
ribosome recruitment to this site, resulting in the production of a truncated protein
isoform that retains functional chromophore properties and fluoresces red [3]. Consequently, significant background fluorescence arises even
under repressive conditions, complicating the interpretation of promoter activity,
particularly for the oxygen-sensitive FNR promoter in aerobic environments.

Looking for future research, the immediate priority is to refine the genetic system by
validating the FNR promoter with an alternative reporter like GFP and exploring other
tightly regulated inducible promoters to ensure precise control.

**2. The Better Antimicrobial Efficacy of Lytic Peptide Hecate**

A comparative analysis of the 2.5% DMSO and 1.25% DMSO conditions, as
presented in **Table 2**, reveals that the inhibition percentage in the Hecate Treatment
group consistently surpasses that observed in the Melittin group. These findings
indicate that Hecate demonstrates an enhanced antimicrobial efficacy relative to
Melittin, especially in the 2.5%DMSO condition.

It is well-established that DMSO functions as a commonly used vehicle solvent and
possesses inherent cytotoxic properties [4].

Consequently, rigorous experimental controls were implemented to ensure that the
measured inhibitory effects primarily originate from the peptides, rather than from
DMSO itself. Parallel vehicle control groups were included to facilitate accurate
assessment. The data unequivocally demonstrate that the observed inhibition cannot
be attributed solely to DMSO. Although DMSO displays some degree of inhibitory
activity, which appears to be potentiated in the presence of lytic peptides, this trend is
clearly evidenced in **Table 2**. Notably, at a concentration of 1.25% DMSO, the solvent
alone exerts minimal inhibition on *E. coli*. However, pronounced inhibitory effects are
observed upon administration of Hecate and Melittin, highlighting the significant
contribution of the peptides to the overall antimicrobial activity.

Experimental data show that Melittin remains stable at both 5% and 2.5% DMSO,
while Hecate’s stability decreases dramatically under higher DMSO concentrations.
At 5% in water, DMSO acts as a mild denaturant by competing for and breaking the
hydrogen bonds that stabilize peptide secondary structures [5],
particularly alpha-helices, through strong hydrogen bonding with the peptide
backbone. Melittin resists DMSO denaturation due to its relatively stable, cooperative
hydrogen-bond network, a strong hydrophobic core that makes it harder for DMSO to
disrupt, and clustering of positive charges away from the helix core [6], reducing solvent accessibility to charged residues. In contrast, Hecate’s weaker
hydrogen bonding, high charge density (lysine-rich sequence), and insufficient
hydrophobic core can make it more prone to DMSO attack. These features
collectively destabilize the peptide and allow easier access for DMSO to disrupt its
structure.

*In vivo* testing on *E. coli*, Heacte demonstrated superior antimicrobial activity over
Melittin when delivered in a 1.25% and 2.5% DMSO solvent, highlighting how a
peptide's inherent lytic potential is critically modulated by its stability in the delivery
formulation.

**3. Hecate and Melittin Show No Acute Toxicity in the Non-Target Organism *Caenorhabditis elegans***

In our experiment, *Caenorhabditis elegans* were exposed to both purified lytic
peptides and peptides synthesized by bacteria. The nematodes were fully immersed in
a liquid medium containing the respective peptides; however, no mortality was
observed. We hypothesize that this outcome is primarily due to the protective cuticle
of *C. elegans*, a well-documented, rigid barrier that protects against diverse
environmental toxins and pathogens []. This robust
exoskeleton likely prevents the penetration of lytic peptides like Hecate and Melittin,
which are known to exert their effects through direct interaction with cell membranes [8, 9]. This feature could account for the observed
resistance to Hecate and Melittin.

Alternatively, lytic peptides may only exert their action upon ingestion, which occurs
through the rhythmic pharyngeal pumping of the medium into the digestive tract.
Nevertheless, microscopic examination of gut tissue revealed no visible cellular
damage; the intestinal linings appeared smooth and undistorted. These findings
support the hypothesis that Hecate and Melittin may selectively target unicellular
organisms, consistent with our objective to avoid toxic effects on non-target
organisms.

**4. The Greater Impact of Bacterially Synthesized Hecate on *C. elegans***

The observation that bacterial supernatant containing Hecate markedly impairs *C. elegans*
thrashing motility more effectively than Melittin might indicate the difference
in the interactions of these antimicrobial peptides with a complex multicellular
organism.

Although Hecate was derived from melittin, key amino acid substitutions have
rendered it a more potent disruptor of *C. elegans* motility in our assay. While both
peptides are membranolytic, we hypothesize that Hecate's uniformly cationic and
highly amphipathic alpha-helical structure facilitates a more potent interaction with
the anionic phospholipids prevalent in neuronal and muscle cell membranes. This
targeted disruption of the neuromuscular system would manifest as a rapid and severe
loss of motility. Alternatively, the difference may be kinetic; Hecate may induce a
pronounced paralytic state at sub-lethal concentrations more effectively than melittin,
which may cause more immediate generalized lysis. This aligns with Hecate's
designed mechanism of carpet-like membrane disruption, which could be more
effective on the complex multicellular systems of a nematode compared to melittin's
toroidal pore formation.

In future research, to fully leverage the intriguing bioactivity of Hecate, its
mechanism should be investigated in *C. elegans* using fluorescently tagged tissues to
confirm the site of action and distinguish between paralysis and lethality.

**5. Key Conclusions**

The FNR promoter showed activity in both aerobic and anaerobic conditions, likely
due to mCherry reporter background fluorescence, necessitating alternative promoters.
Hecate exhibited superior antibacterial activity against *E. coli*, especially at lower
DMSO concentrations, but is less stable at higher DMSO. Neither peptide showed
acute toxicity to *C. elegans*, likely due to the nematode cuticle, indicating selective
toxicity. Hecate uniquely induced non-lethal paralysis in *C. elegans*, suggesting
specific neuromuscular disruption not seen with Melittin.

##### **References**

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