iGEM 2025 — Team hzau-china


# Engineering

![headflower](https://static.igem.wiki/teams/5918/main-pic/headflower.webp)

[Circuit Engineering](#Part_1)
[Global Regulation Module](#Part_2)
[BioPROTAC](#Part_3)
[Anti-VEGF Nanobody](#Part_4)
[ROS Sensing](#Part_5)
[OMV Delivery Module](#Part_6)
[PelB-Di-melittin](#Part_7)
[Suicide and Medicine-Food Collaboration](#Part_8)
[Future work](#Part_9)
[References](#Part_10)

# Circuit Engineering

## Cycle 1

In our initial circuit, we selected *Clostridium butyricum* as the
chassis organism because it produces butyric acid, which helps restore
the normal microbial community structure in the perianal region.
For the functional modules, we initially considered using an anti-VEGF
single-chain variable fragment (scFv) to inhibit angiogenesis.
To facilitate the proper folding of the scFv, we also planned to overexpress
the GroEL/GroES chaperones in the chassis bacteria.
In the anti-inflammatory module, we employed a ROS-activated promoter to
drive the expression of the anti-inflammatory cytokine IL-10.
For biosafety, this version of the circuit utilized an HrpR/S AND gate,
ensuring that the engineered bacteria would only survive under hypoxic
conditions and at sites of inflammation.

However, a limitation of this design was that both IL-10 and the scFv are not ideally expressed
in
prokaryotic systems. Furthermore, *Clostridium butyricumis* is notoriously
difficult to genetically manipulate.

![cycle1](https://static.igem.wiki/teams/5918/bioprotacwet/cycle1.webp)
Figure 1. The circuit diagram initially designed during brainstorming
sessions, using *Clostridium butyricum* as the chassis organism.

## Cycle 2

In the second version, we switched the chassis organism to *Escherichia coli* Nissle 1917
(EcN). This probiotic strain can naturally migrate towards sites of inflammation, which aids the
function of the anti-inflammatory pathway and helps optimize the gut microbiota. To enhance
therapeutic efficacy, we focused on the HIF-1α-mediated angiogenesis pathway under hypoxia and
adopted the Pepper-tCMA system to target and degrade HIF-1α during hypoxia. We intended to use
the basic helix-loop-helix (bHLH) domain to enable our tCMA element to bind HIF-1α.
Simultaneously, we replaced the original scFv and IL-10 with a nanobody and an engineered
melittin peptide, respectively, as these are more amenable to expression in prokaryotes.

To improve the intracellular delivery of the Pepper-tCMA system, we planned to package it
MS2-based virus-like particles (VLPs) and utilize a suicide system to release the assembled
VLPs. The VLP surface was designed to display the wound-homing peptide CAR for targeting
neo-vascular sites.

At the regulatory level, we introduced an adenosylcobalamin (vitamin B12)-responsive
riboswitch, AdoCbl, to create an activation circuit triggered by vascular rupture (a hallmark of
advanced hemorrhoids). In this version, we planned to create two separate circuit variants for
early-stage and late-stage treatment to broaden the therapeutic applicability.

However, this design had drawbacks: the tCMA system lacked sufficient specificity, its large
molecular size prevented efficient packaging VLPs, and the switching mechanism between
early and late-stage treatments was somewhat cumbersome. This led to further iterations.

![](https://static.igem.wiki/teams/5918/bioprotacwet/cycle2.webp)

Figure 2. Circuit diagram of cycle 2 for treating hemorrhoids in both early and late stages,
utilizing EcN as the chassis organism.

(A) Early stage treatment circuit, VEGF-scFv was replaced with nanobodies, and
IL-10 was replaced with engineered melittin.
(B) Late stage treatment circuit, a riboswitch is employed to sense hemorrhage
and activate the expression of a downstream circuit. This circuit utilizes a Pepper-tCMA
system packaged in virus-like particles (VLPs) to target HIF-1α for degradation.​

## Cycle 3

To address the delivery and specificity issues of tCMA, we rationally designed a bioPROTAC
(VHH-VHL) based on the nanobody VHH212 (specific for HIF-1α) and the VHL subunit of the E3
ubiquitin ligase complex. This molecule mediates the ubiquitination and degradation of HIF-1α
under hypoxic conditions. We replaced VLPs with bacterial outer membrane vesicles (OMVs) for
delivery, engineering them for targeted delivery by displaying the OmpA-CAR fusion on their
surface.

Inspired by the traditional Chinese medicine concept of "medicinal adjuvants" (药引, a guiding drug that enhances
the
efficacy of main ingredients), we proposed a "Medicine-Food Collaboration" paradigm using a
negative riboswitch and toxin protein. This riboswitch responds to a specific metabolite found
in certain foods. In the absence of the ligand, the engineered bacteria express a toxin protein,
leading to self-elimination and stopping the treatment. This paradigm aims to help patients
develop healthier dietary habits alongside the therapy.

To address the potential risks of engineered bacterial over-proliferation *in vivo* and associated
issues such as therapeutic module expression fluctuations and toxicity, we designed an
intelligent global regulatory system based on a LuxR/TetR dual-factor cascade. This system
employs a quorum-sensing mechanism to monitor bacterial density and utilizes a negative feedback
loop to regulate the expression of downstream genes, thereby achieving precise control.

![](https://static.igem.wiki/teams/5918/bioprotacwet/cycle3.webp)

Figure 3. Circuit diagram of Cycle 3, featuring OMV-delivered bioPROTAC and the Medicine-Food
Collaboration paradigm.

(A) Anti-angiogenesis Module, including bioPROTAC, OmpA-CAR and anti-VEGF
nanobody;
(B) Medicine-Food Collaboration Module, consisting of a negative riboswitch and
toxin protein;
(C) Global Control Module, a module with a quorum sensing mechanism for the
negative regulation of the functional module.

## Cycle 4

With the progress of Human Practices, we aimed to expand the application scenarios of HemorrEaser,
potentially creating new treatment options for specific groups like pregnant women.
Consequently, we designed the anti-angiogenesis module as the core of an oral medication,
integrating it with the "Medicine-Food Collaboration" paradigm to combine pharmaceutical
treatment with dietary health management. To alleviate the pain and inflammation associated with
hemorrhoids, we developed the anti-inflammatory module as a separate topical drug. In the oral administration mode, we plan to process the engineered bacteria into lyophilized powder that retains viability, delivering it via enteric-coated capsules to the small intestine for targeted therapeutic action. ​​In the topical administration mode, the engineered bacteria are formulated into an ointment along with TPP, which serves as a survival signal. This topical
formulation incorporates simple suicide elements to ensure biosafety.

![OD](https://static.igem.wiki/teams/5918/bioprotacwet/2rou.webp)
Figure 4. Two Administration Routes: oral and topical, targeting angiogenesis
and inflammation.

## Cycle 5

Based on the survey results of stakeholders from our Human Practices work, we incorporated an
"expiry-date" circuit the Medicine-Food Collaboration module. This circuit delays the
bacterial suicide mechanism, making it more convenient for patients to take the oral formulation
of HemorrEaser. You can refer to the [Design](https://2025.igem.wiki/hzau-china/design)
for details of the final circuit.

![](https://static.igem.wiki/teams/5918/bioprotacwet/final-circuits.webp)

Figure 5. Circuit diagram of HemorrEaser in final.

(A) Oral Formulation, added "expiry-date" circuit to the Medicine-Food
Collaboration module;
(B) Topical Formulation.

# Global regulation module

## Cycle 1

##### Design

We selected *E. coli* Nissle 1917 (EcN) as the chassis strain for our engineered bacteria.
Our design phase focused on creating a LuxR/TetR-based dual-factor cascade system, autonomously
control
gene expression in response to bacterial population density, address the risk of uncontrolled
proliferation and
overtreatment.
As shown in Figure 6., the core circuit was designed as follows: constitutive expression of
*luxI* and
*luxR* ensures low-level production of the AHL signal. As the cell density increases,
accumulated AHL
activates LuxR, which then binds to the Plux to drive the expression of the TetR
repressor. TetR
subsequently binds to the Ptet to inhibit the expression of any downstream gene. To
macroscopically
validate the functionality of this circuit, we chose the AmCyan fluorescent protein as a
reporter
gene downstream
of Ptet in a pET-28b(+) backbone, enabling visual assessment of circuit
functionality.

![回路图](https://static.igem.wiki/teams/5918/quorum-sensing/global-regulation.webp)
Figure 6. Circuit diagram of the global regulation module.

##### Build

As shown in Figure 7.A, the *amCyan* gene was placed downstream of the Ptet. After the
construct
was successfully introduced *E. coli* BL21(DE3), colony PCR was performed, and as shown in
Figure 7.B,
the bands matched the expected size. Sequencing analysis further confirmed that the recombinant
plasmid
was consistent with the design as shown in Figure 7.C.

![](https://static.igem.wiki/teams/5918/srcgraph/qsvec.webp)

Figure 1. pET-28b(+)-*luxI*-*luxR*-*tetR*-*amCyan* plasmid construction.

(A)The constructed plasmid map;
(B)Gel electrophoretic map for colony PCR;
(C)Alignment map of sequencing results vs. expected plasmid sequence.

The finalized plasmid was then transformed the *E. coli* BL21(DE3) expression host.

##### Test

The first functional test employed our designed macroscopic observation protocol. Transformed
*E. coli*
BL21(DE3) cells were cultured in M9 minimal medium supplemented with 50 μg/mL kanamycin. After
48
hours of
incubation in a white light environment, we observed distinct phenotypic differences: the
experimental group
exhibited blue-green fluorescence visible to the naked eye under ambient light, with the
fluorescence
intensity gradually increasing over time, while the empty-plasmid control group showed no
observable
fluorescence as shown in Figure 10.

![Fluorescence intensity](https://static.igem.wiki/teams/5918/quorum-sensing/qs-fluorescence.webp)
Figure 8. Fluorescence intensity over time.

This qualitative result provided crucial initial evidence that our genetic circuit was
functioning
as
intended, with *amCyan* expression successfully triggered through the density-sensing
cascade.

##### Learn

While the macroscopic assay provided preliminary confirmation of functional fluorescence
expression
and
suggested the presence of characteristic kinetic profiles, visual observation alone was
insufficient
to definitively determine key circuit parameters such as activation kinetics, threshold density,
or
feedback strength. ​​Furthermore, it remained unclear whether the circuit could maintain
downstream
promoter activity within a stable range rather than merely providing transient suppression. We
learned
that a quantitative approach was essential to understand the precise temporal relationship
between
population growth and circuit output. This insight led to the redesign of our testing strategy,
moving
from qualitative observation to quantitative, time-resolved measurement.

## Cycle 2

##### Design

To address the limitations of the macroscopic assay, we designed a quantitative experiment using
a
microplate reader. This approach enabled precise quantification of both bacterial growth and
gene
expression dynamics, allowing us to fit the one-step growth curve and the relative fluorescence
unit (fluorescence intensity/OD600) profile. Consequently, the Ptet repression kinetics could
be characterized through temporal changes in normalized fluorescence intensity. The redefined
test
protocol involved monitoring the culture in real-time, with simultaneous measurement of optical
density
(OD600) and fluorescence intensity (AU, Ex/Em: 453/486 nm) every 20 minutes over a
5.5-hour
growth period. This approach allowed us to generate high-resolution, kinetic data for both
growth
and
circuit output, enabling the calculation of relative fluorescence units (fluorescence intensity/OD600)
to
normalize
expression to cell density.

##### Test

The kinetic data obtained from the microplate reader revealed a characteristic progression of
the
relative
fluorescence. The OD600 data exhibited a standard one-step growth curve, while the
expression
profile showed a rapid increase in fluorescence intensity/OD600, which peaked and then declined to a
steady
state.
The kinetic profile obtained was accurately fitted by an ODE model (R2 = 0.9078),
providing a
quantitative representation of the quorum-sensing feedback system, as shown in Figures 11 and 12.

![growth curve](https://static.igem.wiki/teams/5918/quorum-sensing/growth-curve-fit.webp)
Figure 9. Measured OD600 values and the corresponding logistic fit
of
the
one-step growth curve(R2 = 0.9943).
![fitted curve](https://static.igem.wiki/teams/5918/quorum-sensing/output.webp)
Figure 10. Measured relative fluorescence Units (RFU) and the corresponding
fitted
curve
of the global regulation circuit dynamics(R2= 0.9078).

The data clearly demonstrated that the feedback inhibition circuit rapidly reached a peak
expression
level
and subsequently maintained long-term stability in output, thereby achieving effective negative
feedback
control over the downstream therapeutic circuit.

##### Learn

Experimental data provide clear validation of the circuit's activation characteristics. The
quantitative
retesting phase was crucial for ODE-based model fitting. RFU intensity peaked at approximately
30
minutes, while the OD600 at this timepoint was only 0.15, indicating that basal
expression
from
the
constitutive promoter enables preliminary AHL accumulation. This allows partial LuxR-mediated
transcriptional
activation to occur prior to the population reaching high density. Subsequently, RFU levels
decreased rapidly.
The high-fitting model further visually demonstrates this "fast-activation/early-repression"
kinetic
pattern,
confirming the success of our circuit design. Notably, the system completed a full
activation-repression
cycle before the culture entered the stationary phase, demonstrating its ability to preemptively
regulate
gene expression in response to minor population changes and achieve autonomous control before
potential
overproliferation risks emerge. The integration of quantitative kinetic data with the molecular
mechanism
ultimately validates the reliability of this quorum sensing feedback system as the global
regulation
module
in the project.

# BioPROTAC

## Cycle 1

##### Design

The primary goal of our design was to specifically target and degrade the HIF-1α protein to block its
downstream inflammatory signaling cascade, thereby reducing the abnormal expression of target genes
and mitigating inflammation in hemorrhoidal lesions. This strategy was initially conceptualized to
leverage the chaperone-mediated autophagy (CMA) pathway.

##### Build

Specifically, we utilized RFdiffusion to design a novel protein that, upon specific binding to
HIF-1α, undergoes a conformational switch that exposes the KFERQ degradation tag, consequently
inducing its CMA-mediated degradation.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/tcma.webp)
Figure 11. Schematic diagram of pre- and postamCyaninteraction conformational states
in protein-protein interactions.

(A) Pre-binding; (B) Post-binding, light blue indicates HIF-1α.

##### Test

Intriguingly, during the design process, we discovered that HIF-1α carries an autophagic degradation
sequence (KFERQ-like motif), suggesting that the CMA pathway is an intrinsic component of its
endogenous degradation machinery. Further literature review indicates that under normoxia, HIF-1α is
tightly controlled and primarily degraded by the ubiquitin-proteasome system (UPS). Under hypoxia,
however, UPS function is impaired, leading cells to primarily depend on the autophagy pathway for
HIF-1α homeostasis. Crucially, the suboptimal efficiency of CMA degradation allows HIF-1α to
readily accumulate and nuclear-translocate, activating the hypoxia signaling pathway and sustaining
its high steady-state levels.

##### Learn

Following discussions with Professor Zhang Xiaoyan and further research, we concluded that merely
augmenting CMA activity would be insufficient to robustly decrease HIF-1α's steady-state level.
Consequently, we pivoted our strategy to the ubiquitin ligase system. We now propose to construct a
bioPROTAC molecule to mediate the selective ubiquitination and subsequent proteasomal
degradation of HIF-1α, anticipating a significantly more efficient suppressive outcome. You can see
details in [Human
Practices](https://2025.igem.wiki/hzau-china/human-practices)

## Iteration of BioPROTAC

##### Linker

During the design of bioPROTAC, the linker's conformation optimization underwent multiple rounds of
experimentation. Initially, to prevent structural interference between VHH212 and pVHL, we attempted
a fully rigid α-helical linker (EAAAK)n to ensure spatial separation of the two ends. However,
excessive rigidity was found to restrict the overall molecular flexibility, hindering the
conformational adaptation of the ubiquitinating complex. Subsequently, we tested a fully flexible
linker (GGGSS), which offered greater spatial freedom but increased interference between the two
protein structures and reduced stability. Balancing the trade-offs between rigidity and flexibility,
we ultimately adopted a hybrid linker design: the sequence EAAAKGPGSGGS. The EAAAK segment forms an
α-helix, providing initial structural orientation for the amCyan while avoiding interference between
structural components. The terminal GPGSGGS segment offers a flexible tail, enabling the two
terminal proteins to maintain spatial separation while allowing necessary rotation. This iterative
optimization ensures the amCyan preserves the structural integrity of both terminal proteins while
providing sufficient flexibility to support bioPROTAC functionality.

##### Nuclear Localization Signal

Under hyp
oxic conditions, the primary functional sites of HIF-1α and its transcriptional regulatory
functions are concentrated within the cell nucleus. Therefore, the efficiency of nuclear delivery
for bioPROTAC is crucial for achieving effective targeted degradation. Initially, we did not
incorporate a nuclear localization signal (NLS) tag the bioPROTAC structure, as the fusion
protein's molecular weight of approximately 33 kDa falls below the passive diffusion threshold
(around 40 kDa) for the nuclear pore complex (NPC). Theoretically, this allowed passive
diffusion the nucleus to bind with nuclear HIF-1α. However, considering that passive diffusion
is typically slow and exhibits significant concentration gradient dependence, under cellular hypoxia
stress conditions, viscosity differences between the nuclear and cytoplasmic environments and
protein interactions may further limit the molecular nuclear transport rate, thereby affecting
degradation efficiency.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/nls.webp)
Figure 12. Schematic of the trans-nuclear labeling mechanism.Figure is created with bioGDP(https://biogdp.com/).

Based on this, we introduced the classic nuclear localization sequence (NLS) PKKKRKV at the
C-terminus of bioPROTAC to enhance its nuclear entry efficiency via importin-mediated active
transport. The incorporation of NLS not only significantly enhances the nuclear enrichment and
stability of bioPROTAC but also accelerates its spatial interaction with HIF-1α and the subsequent
ubiquitination induction process. Through this enhancement, we aim to overcome the rate-limiting
constraints of passive diffusion, ensuring bioPROTAC efficiently localizes to the nucleus under
hypoxic conditions. This enables precise recognition and degradation of HIF-1α, maximizing its
functional efficacy.

##### Ubiquitinylation Site

Initially, we hypothesized that bioPROTAC could promote modification of HIF-1α at its VHL-mediated
classical ubiquitination sites (K532, K538, and K547). However, molecular docking and complex
modeling revealed that these lysine residues are partially shielded and significantly less
accessible in the spatial conformation formed by the bioPROTAC complex with Elongin B and Elongin C.
Further analysis reveals that the long helical backbone of Cullin-2 struggles to bend sufficiently
in this conformation to guide ubiquitin molecules to these shielded sites, thereby limiting
ubiquitin accessibility and the feasibility of covalent transfer geometry.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/ubsite.webp)
Figure 13. Schematic diagram of ubiquitin site masking.

Through literature review, we learned that the application of PROTAC technology often leads to
alterations in the ubiquitinylation sites of target proteins. As Crowe et al. pointed out, the
introduction of PROTAC may cause relocation of ubiquitinylation sites by reshaping the spatial
conformation of the POI-PROTAC-E3 ternary complex. This primarily stems from changes in spatial
conformation and increased steric hindrance, making it difficult for the E2-E3 complex to
effectively recognize the original ubiquitinylation site. Therefore, we urgently need to explore new
target residues with ubiquitinylation potential to ensure the degradation process proceeds smoothly.

The essence of the ubiquitination reaction is a nucleophilic addition process: the ε-amino group of
lysine (Lys) on the substrate protein undergoes deprotonation to convert into the -NH₂ form,
acquiring strong nucleophilicity. This enables it to attack the thioester bond in the ubiquitin-E2
complex, cleaving it and forming a new isopeptide bond, thereby achieving the transfer of the
ubiquitin molecule. Based on this, we first employed [GPS-Uber](https://gpsuber.biocuckoo.cn/index.php)
to predict potential ubiquitinylation sites on the
substrate protein, yielding multiple candidate lysine residues. Subsequently, we employed
[PROPKA 3](https://github.com/jensengroup/propka) to calculate the pKa values of these sites, evaluating
their deprotonation capacity under physiological conditions to infer their potential
ubiquitinylation activity.

The results indicate that Lys11 exhibits superior deprotonation capability, with a predicted pKa
value of 6.96—significantly lower than the typical value of approximately 10.5 for lysine residues.
This indicates that under physiological pH (7.4) conditions, the ε-amino group of Lys11 is
predominantly deprotonated (-NH₂), thereby exhibiting enhanced nucleophilicity and favoring attack
on the thioester bond to form an isopeptide bond. Structural analysis further supports this
conclusion:

1. Lys11 exhibits a surface exposure of up to 98%, indicating that this residue is spatially
   accessible and readily recognized by the E2-E3 complex;
2. Its desolvation energy of -3.46 indicates that this site resides in a low-dielectric-constant
   environment, which helps lower the pKa and stabilize the deprotonated state;
3. This residue does not form significant hydrogen bonds, thereby avoiding potential stabilization
   effects that could restrict the active site.
4. Weak electrostatic interactions with Arg89 and Lys65 (-0.01 and -0.07) provide additional local
   shielding effects, further promoting the deprotonation process.

Subsequently, we employed HADDOCK and HDOCK for molecular docking and complex modeling of
the complete substrate-E3-E2 ubiquitinylation system. The results indicate that the spatial distance
between the reaction center of Lys11 and the Gly76 residue of the ubiquitin molecule is
approximately 10.6 Å, satisfying the geometric requirements for effective nucleophilic attack. This
suggests that this site possesses potential reactivity and can be validated as a novel
ubiquitinylation target.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/complex.webp)
Figure 14. Schematic diagram of the ubiquitinylation system complex mediated by
bioPROTAC.

Finally, we performed a 100 ns molecular dynamics simulation of the substrate-E3-E2-ubiquitin system
using Amber. From the simulation trajectory, we extracted a plausible attack conformation,
i.e., the pre-reactive conformation. Building upon this, we selected the reaction core region for
quantum mechanical calculations using Gaussian 16 to determine the energy barrier for
nucleophilic attack. The results indicate that the transition state energy barrier for this reaction
is approximately 8.2 kcal/mol, falling within a reasonable range and further supporting the
feasibility of the Lys11 ubiquitinylation site.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/eng.webp)
Figure 15. Energy barrier diagram of the reaction core region.

## Cycle 3

##### Design

In the design phase of the *in vitro* ubiquitination assay, we initially planned to use full-length
HIF-1α as the substrate. The protein was intended to be recombinantly expressed and purified using a
prokaryotic expression system (*E. coli* BL21(DE3)) to simulate ubiquitination under physiological
conditions.

![OD](https://static.igem.wiki/teams/5918/bioprotacwet/pet-28b-hif-1.webp)
Figure 16. Expression vector with full-length HIF-1α.

##### Build

We successfully constructed the recombinant plasmid pET-28b(+)-HIF-1α. Positive clones were
preliminarily screened by colony PCR, and sequencing confirmed the correct insertion, indicating
successful construction of the HIF-1α expression vector (Figure 17).

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/bacpp.webp)
Figure 17. Colony PCR confirmed the correct
insertion.

##### Test

However, when the expression products were analyzed by 8% SDS-PAGE and Western blot, no clear band
corresponding to HIF-1α was observed at the expected molecular weight. This suggested that
full-length HIF-1α was either expressed at low levels or unstable in this system, making effective
purification difficult.

##### Learn

Literature review revealed that the N-terminal region (amino acids 1-365) of HIF-1α contains both the
VHH212 nanobody binding domain and key ubiquitination sites, while the C-terminal portion consists
mainly of flexible loop regions with minimal impact on core structure and function. Therefore, we
proposed a truncated expression strategy to improve soluble expression levels.

##### Redesign

Based on this analysis, we redesigned the experimental approach, replacing the substrate in the ubiquitination assay with a truncated version of HIF-1α(1-365aa) to enhance protein expressibility and experimental feasibility.

![OD](https://static.igem.wiki/teams/5918/bioprotacwet/pet28b-hif-1a-small.webp)
Figure 18. Expression vector with truncated HIF-1α.

##### Build

We successfully constructed the expression vector for truncated HIF-1α. Western blot analysis
confirmed its high-level expression in BL21(DE3), and high-purity target protein was obtained
through purification.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/expall.webp)
Figure 19. ​​Expression validation of individual proteins used in the *in vitro* ubiquitination system.

​​(A) Western blot for VHH-VHL and EloC. Lane 1: VHL-VHH-His, 33.6 kDa; Lane 2: EloC-His, 13.0 kDa;(B) Western blot for Rbx1 and EloB. Lane 1: HIF-1α-His,94.0 kDa (no band observed); Lane 2: Rbx1-His, 11.8 kDa; Lane 3: EloB-His, 12.1 kDa;(C) Western blot for HIF-1α (truncated)-His, 44.3 kDa;(D) 12% SDS-PAGE for Cullin2-His, 88.8 kDa.

##### Test

In the reconstituted ubiquitination reaction system, incubation of VHH-VHL, truncated HIF-1α, E3
ligase complex components (EloB, EloC, Rbx1, Cullin-2), and the ubiquitination system (UBA1, E2,
ubiquitin) resulted in a distinct upward shift of the HIF-1α band on Western blot, confirming the
occurrence of specific polyubiquitination.

![待放回路图](https://static.igem.wiki/teams/5918/bioprotacwet/ub3.avif)
Figure 20. *In vitro* ubiquitination assay result.

​​Western blot analysis of *in vitro* ubiquitination reactions at 2 h, 4 h, and 6 h.​​ Reactions without UBC8 and UBA1 served as negative controls; reactions without VHH-VHL served as blank controls. All control reactions were terminated at 6 h; HIF-1α(truncated)-Ubn, above 44.3 kDa.

##### Discussion

By integrating computational rational design with multidimensional experimental validation, the
engineered bioPROTAC molecule VHH-VHL effectively recruits the ubiquitination pathway to achieve
targeted protein degradation while maintaining its VHH212 module's high-affinity binding to HIF-1α.
These findings demonstrate that VHH-VHL effectively mediates the ubiquitin-proteasome degradation
pathway of HIF-1α under hypoxic conditions *in vivo*, providing a molecular basis and therapeutic
potential for inhibiting hypoxia-induced pathological angiogenesis in hemorrhoidal tissues. Next, we
will validate the ubiquitination effect in mammalian cells.

# Anti-VEGF Nanobody

## Cycle 1

##### Design

To enable efficient expression of an anti-VEGF therapeutic in engineered bacteria, we
adopted a rational design strategy. Unlike conventional IgG antibodies, nanobodies are
intrinsically compatible with prokaryotic expression. We therefore selected a tandem
bivalent nanobody construct that exhibits superior pharmacokinetic stability compared with
the monovalent form. An N-terminal PelB signal sequence was included to direct the protein
to the periplasm, and a C-terminal hexahistidine tag was added to enable purification by Ni-NTA affinity chromatography.

##### Build

The cassette was cloned pET-28b(+) under the control of the lac operon, and the
resulting plasmid was transformed *E. coli* SHuffle T7 Express.

![待放回路图](https://static.igem.wiki/teams/5918/antivegf/bivalent.webp)
Figure 21. Flowchart for constructing Anti-VEGF tandem bivalent nanobody sequence
into the pET-28b(+) plasmid.

Agarose gel electrophoresis of the PCR products from the colonies demonstrated that
we successfully constructed the expression vector.

![](https://static.igem.wiki/teams/5918/antivegf/pcr.webp)

Figure 22. Colony PCR for fragments containing ati-VEGF tandem bivalent nanobody.

M: DL2000 DNA Marker; lane 1-8: Eight single colonies transformed with the recombinant
plasmid.

##### Test

In the induction expression experiment, when the culture reached an OD600 of 0.5, expression
was induced with 0.5 mM IPTG for 16 h at 16 °C. Cells were harvested, periplasmic proteins
extracted, and the extract purified by Ni²⁺-affinity chromatography. SDS-PAGE showed no
detectable band at the expected ~30 kDa.

##### Learn

Further literature survey and sequence inspection led us to hypothesize that due to the
identical repetitive segments in tandem bivalent repeat sequences, erroneous recognition
and pairing may occur during homologous recombination. This likely hinders the proper expression
of tandem bivalent nanobodies in SHuffle strains.

## Cycle 2

##### Design

Professor Xu Shutong pointed out that our tandem sequence repeats were excessively high, which
significantly increased the probability of sequence mismatches, making it difficult for the
antibody protein to express correctly. Subsequent literature review revealed no significant
difference in binding affinity between tandem bivalent nanobodies and monovalent nanobodies
against VEGF. To address issues encountered in tandem bispecific nanobody expression, we
redesigned the antibody sequence, retaining the N-terminal pelB tag and histidine tag while
replacing the tandem bivalent nanobody sequence with a monovalent nanobody sequence.

![待放回路图](https://static.igem.wiki/teams/5918/antivegf/.webp)
Figure 23. Anti-VEGF monovalent nanobody.

##### Build

The tandem bivalent sequence was converted to a monovalent form and constructed into the pET-28b(+)
plasmid via homologous recombination, with expression controlled by the lac operon.

##### Test

Through literature analysis and numerical modeling predictions and analysis, we concluded that the
pharmacokinetic stability of monovalent nanobodies is reduced compared to tandem bivalent
nanobodies.

##### Learn

To improve the pharmacokinetic stability of anti-VEGF nanobodies, we decided to explore methods
to enhance the solubility and stability of monovalent nanobodies.

## Cycle 3

##### Design

To optimize the anti-VEGF nanobody and offset the stability loss resulting from the
tandem-to-monomeric
conversion, we inserted the 33-residue P17 peptide between the PelB signal and the N-terminus of the
monovalent nanobody. Studies indicate that the introduction of this P17 short peptide effectively
enhances
the antibody's stability and solubility.

##### Build

We constructed the optimized monovalent nanobody sequence containing the P17 fragment into the
pET-28b(+) plasmid via homologous recombination, maintaining expression regulation by the lac
operon.
After transformation into *E. coli* SHuffle, sequencing verified the correct insert.

![待放回路图](https://static.igem.wiki/teams/5918/antivegf/monovalent.webp)
Figure 24. Flowchart for constructing P17-Anti-VEGF monovalent nanobody sequence
into the pET-28b(+) plasmid.

##### Test

*E. coli* SHuffle carrying the recombinant plasmid was grown at 37 °C, induced with 0.5 mM IPTG
at
16°C for 16 h, and periplasmic extracts were purified. SDS-PAGE and Western blot of total and
purified
fractions showed a single band at the expected 17.7 kDa, confirming expression.

![](https://static.igem.wiki/teams/5918/antivegf/sds-and-wb.webp)

Figure 25. SDS-PAGE and Western Blot analysis of purified samples.

(A) 15% SDS-PAGE of unpurified periplasmic protein and purified sample (M:Protein Marker; lane 1-2 were unpurified periplasmic protein,and lane 3-6 were purified
sample); (B) Western Blot of purified sample (M:Protein Marker; 1-4 were purified sample).

Purified anti-VEGF nanobody was tested in an MTT cell-proliferation assay. Data analysis revealed
that the monovalent anti-VEGF nanobody we designed, expressed, and purified—containing the P17
short peptide—effectively binds to VEGF and inhibits its cell proliferation effects.

![MTT96孔板图片+数据图](https://static.igem.wiki/teams/5918/antivegf/mtt-d1-5.webp)
Figure 26. The antiproliferative activity of anti-VEGF monovalent nanobody on
HUVEC cells.

We also used ProteinSol to simulate the solubility of nanobodies with and without P17; the
P17-containing variant showed higher solubility during expression and after secretion.

![溶解度对比](https://static.igem.wiki/teams/5918/antivegf/solution.webp)
Figure 27. Comparison of solubility of antibodies with and without P17 peptide
segments during antibody expression/secretion stage.

##### Learn

Although we have essentially achieved validation of antibody expression and function, we still
believe
there is potential for improvement in its application as a therapeutic drug. We must consider the
targeting
issue of antibodies. Diffusing antibodies carry a certain degree of systemic toxicity, which may
affect
normal angiogenesis in other parts of the body. Therefore, we have decided to further optimize the
anti-VEGF antibody to enable it to act specifically on the site of hemorrhoids.

## Cycle 4

##### Design

Considering potential safety concerns associated with the systemic diffusion of anti-VEGF
antibodies,
we designed and incorporated a masking peptide at the C-terminus of the antibody to ensure its
specific
targeting to the hemorrhoidal site. This peptide is linked to the antibody via a linker containing
an
MMP3 cleavage site. This linker can be cleaved by matrix metalloproteinase 3 (MMP3) expressed at the
hemorrhoidal site, causing the masking peptide to separate from the antibody and exposing the
antibody binding domain.

##### Build

We designed the gene sequences for the masking peptide and linker, fused them to the C-terminus of
the
nanobody sequence, and inserted them between the antibody sequence and the histidine tag.

![待放回路图](https://static.igem.wiki/teams/5918/antivegf/final.webp)
Figure 28. Anti-VEGF monovalent nanobody incorporating P17 peptide and masking
peptide.

##### Test

To validate the feasibility of the masking peptide, we analyzed all docking complexes with
the Anti-VEGF antibody using Rosetta InterfaceAnalyzer, and identified several sequences
with relatively weak interface energies. The representative complex was further examined
using PRODIGY, yielding a binding free energy of -9.3 kcal·mol⁻¹ and a dissociation constant
of Kd ≈ 2.6 × 10⁻⁷ M (260 nM), indicative of moderate affinity.
The binding interface was dominated by hydrophobic (apolar-apolar, 16) and polar-apolar
(10) contacts, while charged-charged (5) and polar-polar (1) interactions contributed less.
The nonpolar interface area (41.0%) exceeded the charged area (23.1%),
suggesting that binding is mainly driven by a hydrophobic core rather than electrostatic interactions.

![待放回路图](https://static.igem.wiki/teams/5918/antivegf/mask1.webp)>
Figure 29. Distribution of interface atom packing and binding free energy.

The reasonable range for packstact is 0.6-0.8,
indicating the degree of binding. The green band represents cyclic peptides that may dissociate.

To assess the dissociation process, steered molecular dynamics (SMD) simulations were performed
with GROMACS to generate the unbinding pathway,
followed by umbrella sampling and WHAM analysis to compute the potential of mean force (PMF).
The resulting binding free energy (plateau-minimum) was -9.8 kcal·mol⁻¹, corresponding to
Kd ≈ 1.22 × 10⁻⁷ M at 310 K. These results indicate that the masking peptide binds with
moderate affinity and reversible stability, consistent with its expected functional behavior.

![待放回路图](https://static.igem.wiki/teams/5918/antivegf/masks.webp)>
Figure 30. Potential of mean force.

##### Learn

We have designed an Anti-VEGF Probody for targeted hemorrhoids therapy,
which is activated by haemorrhoid-overexpressed MMP-3 to enable localized VEGF inhibition,
potentially reducing systemic toxicity while improving solubility and stability.

However, this study remains preliminary. Functional validation is limited to HUVEC assays,
and the full masked construct has not been experimentally built or tested.
Its activation mechanism relies solely on computational docking,
with no *in vivo* data on efficacy, stability, or immunogenicity.
Another key obstacle is the low oral bioavailability resulting from
the inability to penetrate intestinal epithelial cells.

To advance this candidate, future work must focus on constructing and validating the full protein,
testing it in animal models, and—critically—engineering it with a cell-penetrating peptide (CPP)
to enhance systemic absorption.
Overcoming these limitations is essential to advance this targeted nanobody
toward preclinical development.

# ROS Sensing

## Cycle 1

##### Design

In the area of hemorrhoid inflammation flare-up, the local region maintains a significantly higher
concentration of reactive oxygen species (ROS) than normal tissue. This characteristic
microenvironmental change becomes the key basis for our design of a targeted anti-inflammatory
strategy. Accordingly, we selected ROS as the specific signal trigger for the anti-inflammatory
functional module. Through genetic engineering modification, the engineered bacteria are endowed
with ROS sensing ability—when the bacteria colonize the hemorrhoid lesions and perceive the high ROS
signal, they precisely start-up the expression procedures of anti-inflammatory factors. Ultimately,
through the targeted release of anti-inflammatory factors, targeted sexual inhibition of the local
inflammation is achieved.

We selected SoxR/PSoxS oxidative stress response promoter as the regulatory element. SoxR is a
Transcription factor containing a [2Fe-2S] cluster, while PSoxS is a promoter sequence. In a high ROS
environment, SoxR binds between the -10 and -35 regions, and its Oxidized form distorts the promoter
conformation, promoting the binding of RNA polymerase and other transcription factors, thereby
activating transcription.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/soxr.webp)
Figure 31. Schematic diagram of the mechanism of action of the SoxR/PSoxS oxidative
stress response promoter.

##### Build

To validate the function of the SoxR/PSoxS oxidative stress response promoter, we cloned *amCyan* as a
reporter gene into the pUC57 vector, thereby constructing the corresponding reporter plasmid. The
recombinant plasmid was then transformed into *Escherichia coli* BL21(DE3) for subsequent
functional analysis.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/soxr-history.webp)
Figure 32. Flowchart for constructing *amCyan* into the pUC57 plasmid.

Agarose gel electrophoresis of colony PCR products showed that we successfully constructed the
expression vector.

![](https://static.igem.wiki/teams/5918/jiayin/soxr-pcr-2.webp)

Figure 33. Colony PCR and sequencing results.

(A) The result of electrophoresis of the colony PCR product initially attests to the
successful reorganization of Plasmid (M: DL1000 DNA Marker; lane 1-8: eight individual colonies
obtained after In-Fusion cloning).
(B) *srp-TagRFP* The sequencing results of the constructed Plasmid confirmed
the successful assembly.

Before oxidant induction, we used 15% SDS-PAGE electrophoresis to verify that soxR could be normally
expressed in *E. coli* BL21 (DE3).

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/soxr-pro-2.webp)
Figure 34. 12% SDS-PAGE results for verification of soxR expression.

control:Total
protein sample extracted from *E. coli* BL21(DE3) without plasmid introduction; lane 1: Total protein
samples extracted from expanded cultures of one different single colony that were successfully
transformed. A comparison of the two indicates that SoxR is successfully expressed.

##### Test

After verifying the expression of soxR, to verify the functions of the soxR and PSoxS, we
set up a series of tert - butyl hydrogen peroxide (tBHP) concentration gradients for induction (0
μM, 20 μM, 40 μM, 60 μM, 80μM, 100 μM, 120 μM, 140μM, 160 μM, 180μM, 200μM).

We found that when the concentration of tBHP was between 1-100 ppm, the expression of fluorescent
protein increased rapidly. We added 200 μL of induction medium inoculated with engineered Bacteria
into a 96 wells plate and used a microplate reader to detect the emission light intensity at 486
nm. Three parallel replicates were set for each induction concentration.

![](https://static.igem.wiki/teams/5918/jiayin/soxr-deta-2.webp)

Figure 35. Fluorescence intensity measured by microplate reader.

(A) Changes in fluorescence intensity over time in different concentrations of
tBHP-induced groups and the control group;
(B) Maximum fluorescence intensity in different concentrations of tBHP-induced
groups and the control group.

##### Learn

We observed that the activity of the PSoxS is strongly dependent on ROS concentration, and
its background expression remains low in the absence of ROS.

The current system relies solely on the positive activation of the PSoxS by SoxR and does not
incorporate Feedback loop such as the negative autoregulation by PSoxS itself. This may lead to
excessive expression of downstream therapeutic factors (such as Di-melittin) in a sustained High ROS
environment, increasing the cell metabolic burden or triggering Local toxicity.

Although the system responds well to low concentrations of ROS (20-100 μM tBHP), at higher
concentrations (>150 μM), the fluorescence intensity tends to saturate or even decline, possibly due
to oxidative damage caused by high ROS levels to the bacterial cells, affecting their survival and
expression capacity. This limits the applicability of the system in high-intensity inflammatory
environments.

# OMV delivery system

## Cycle 1

##### Design

*Escherichia coli* outer membrane vesicles (OMV) can be modified to construct the
intended anti-angiogenic hyperplasia module delivery system. To achieve precise
targeted delivery of OMV to the Hemorrhoids site, we selected the OmpA-CAR fusion
protein as the Targeting element.

To verify the function of OMV, we designed three independent circuits (and combined
them into two experimental groups): the three circuits are *ompA-CAR*,
*ompA-amCyan*, and *srp-TagRFP*; the two Combination schemes are:
Combination 1 (*ompA-amCyan* and *srp-TagRFP*), Combination 2 (*ompA-CAR*
and *srp-TagRFP*).

Among them, group 1 can assess the assembly efficiency of engineered OMV using
fluorescence co-localization techniques, while group 2 evaluates whether CAR can
exert the wound homing effect through cell-based assays, thereby clarifying its
targeted delivery efficacy.

##### Build

We integrated *ompA-CAR* and *srp-TagRFP* into the pET-28b(+) plasmid
respectively through one-step In-Fusion cloning, integrated *ompA-amCyan*
into the pET-28b(+) plasmid through two-step In-Fusion cloning, and transformed
the constructed plasmids into *Escherichia coli* Nissle 1917 (EcN) for functional
verification.

![](https://static.igem.wiki/teams/5918/jiayin/omv-zhili-2.webp)

Figure 36. Flowchart of Three-Plasmid Construction.

(A) *ompA-CAR* integrated into pET-28b(+);
(B) *srp-TagRFP* integrated into pET-28b(+);
(C) *ompA-amCyan* integrated into pET-28b(+).

We submitted the assembled plasmid for sequencing and intended to validate protein
expression upon successful sequencing. However, the results revealed that sequence
alignment failed for all three vectors, necessitating reconstruction before further
experiments could proceed. This indicates that the prior construction attempts
were not successful.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/omv-seq-2.webp)
Figure 37. The returned sequencing results showed significant problems.

##### Learn

Under these circumstances, the recombination process failed to generate the intended
plasmid, likely due to reverse insertion of the target fragment, which resulted in
unreadable sequencing data. Subsequent steps will involve redesigning the primers
and repeating the construction.

##### Redesign

Due to sequencing alignment failure, we redesigned the homologous arms flanking
each sequence to ensure proper directional insertion.

##### Rebuild

We proceeded with plasmid construction using new primers, submitted the assembled
plasmid for sequencing, and obtained successful results.

We introduced two plasmid combinations (*ompA-amCyan* with *srp-TagRFP*
and *ompA-CAR* with *srp-TagRFP*) into the EcN strain via double transformation.
PCR analysis of the transformed colonies revealed double-positive bands in both groups,
confirming the successful co-transformation of both plasmid sets into the EcN strain.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/omv-car-3.webp)
Figure 38. Colony PCR results of *ompA-CAR* and *srp-TagRFP*.
![待放回路图](https://static.igem.wiki/teams/5918/jiayin/omv-cfp-3.webp)
Figure 39. Colony PCR results of  *ompA-amCyan*  and
*srp-TagRFP*.

##### Test

To obtain high-purity OMV, we initially selected the density gradient centrifugation method
(DGS) for extraction. The key advantage of this approach is its ability to achieve
precise separation based on differences in density among various substances,
theoretically allowing efficient removal of impurities such as bacterial debris and
protein aggregates, thereby maximizing OMV purity. However, upon completion of the
experiment, no distinct signs of OMV accumulation were observed in the expected density
gradient regions.

##### Learn

Based on the experimental results, the density gradient Centrifugation method did not
effectively isolate and extract the target OMV. Therefore, we opted to use differential
centrifugation (DS) to repeat the extraction procedure.

## Cycle 2

##### Design

We redesigned the extraction protocol for OMV by employing DS for purification, as detailed
below:

1. Inoculate glycerol stock into 200 mL LB medium (supplemented with antibiotic) and culture
   at 37 °C until OD600 reaches 1.0.
2. Centrifuge the culture at 12,000 ×g for 20 min at 4°C. Discard the bacterial pellet and
   collect the supernatant. Filter the supernatant through a 0.45 μm sterile filter to
   remove residual bacteria and cell debris, obtaining a sterile filtrate.
3. Transfer the sterile filtrate into a 100 kDa ultrafiltration tube and centrifuge
   at 4,000 rpm. Retain OMVs in the upper chamber of the ultrafiltration device.
   Repeat centrifugation, wash with PBS, and concentrate the sample to one-tenth of
   the original culture volume.
4. Ultracentrifuge the concentrated sample using a Beckman Optima XPN ultracentrifuge
   (SW 41Ti rotor) at 150,000 ×g and 4°C for 4 h. After centrifugation, discard the
   supernatant, wash the pellet with 2 mL sterile PBS three times, resuspend, and
   store at -80°C (avoiding repeated freeze-thaw cycles).

##### Build

We performed the OMV extraction experiment following the protocol and obtained OMV precipitate.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/omv-chen-2.webp)
Figure 40. DC Results.

##### Test

We performed protein concentration determination on the extraction products of DGC and DC using
a micro-spectrophotometer, respectively. The results showed that the protein concentration of
the DGC product was close to the baseline level, while the protein concentration of the DC
product was significantly higher. This finding provides preliminary evidence that the DC
method successfully extracted OMV.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/pro.webp)
Figure 41. Comparison of protein concentrations between DGC and DC.

To further verify the extraction effect of OMVs, we selected the Outer
Membrane
Vesicle marker protein outer membrane protein A (OmpA, approximately 38.2 kDa) as a key validation
marker to conduct a 12% SDS-PAGE experiment. The characteristic band of the outer membrane
protein A can be clearly observed in the electrophoresis results, which provides
direct evidence for the successful extraction of OMVs; furthermore,
through intuitive comparison of the bands from extracts obtained by different centrifugation
protocols, the feasibility and success of the Differential centrifugation protocol are once
again confirmed.

![待放回路图](https://static.igem.wiki/teams/5918/jiayin/93830bb95bb332bb723a408ecad7b08a.avif)
Figure 42. 12% SDS-PAGE results

M: Protein marker; DC: Differential
centrifugation extract; DGC: Density gradient centrifugation extract.

##### Learn

In this round of cycles, we found that although DGC is pure, it almost loses all OMVs,
and DC can be used as the final experimental method. In the future, we will measure
the Zeta potential of the extract to clarify the stability of the extract dispersion
system and preliminarily determine the structure of OMVs. Subsequently, we will
conduct fluorescence confocal experiment to confirm the co-localization of AmCyan and
TagRFP, verifying the successful loading of Engineered outer membrane vesicle. Finally,
we will perform cell scratch assay to determine the role of CAR in promoting cell
migration and verify the targeting effect of Engineered outer membrane vesicle.

# PelB-Di-melittin

## Cycle 1

##### Design

Based on Di-melittin from NKU-China,2024(BBa\_K5332002), we fused an N-terminal PelB signal peptide
for secretion and a C-terminal 6×His tag for purification.

![PelB-Di-melittin ](https://static.igem.wiki/teams/5918/mel/pelb-di-melittin.webp)
Figure 43. Schematic representation of the engineered PelB-Di-melittin
construct.

The design includes an N-terminal PelB signal peptide for periplasmic secretion,
the Di-melittin coding sequence in a hairpin structure, a flexible linker (GGSSSGG), and a
C-terminal 6×His tag for purification.

##### Build

The sequence was cloned into the pET-28b(+) backbone using homologous recombination and
transformed into *E. coli* BL21 (DE3).

##### Test

Colony PCR and sequencing confirmed the presence of the correct construct.

![Plasmid Construction](https://static.igem.wiki/teams/5918/mel/zh1.webp)
Figure 44. pET-28b (+)-PelB-Di-melittin plasmid construction.

(A) Plasmid map of the recombinant expression vector
pET-28b (+)-PelB-Di-melittin. The plasmid contains a T7 promoter for transcription,
the PelB-Di-melittin construct. Kanamycin resistance is included for bacterial selection;
(B) Colony PCR results of recombinant clones. All seven tested colonies exhibited a clear
band at 739 bp, consistent with the expected size. A 1000 bp marker was used as reference;
(C) Sequencing verification of the recombinant plasmid. The sequencing results confirmed
that the PelB-Di-melittin insert was correctly integrated, with no mutations or frame
shifts, and fully consistent with the design.

##### Learn

The vector was successfully constructed, enabling us to proceed with expression analysis.

## Cycle 2

##### Design

Since PelB should guide the peptide into the periplasm, we aimed to extract the protein from the
periplasmic fraction.

##### Test

Cultures were induced with IPTG, and proteins were isolated using osmotic shock methods. SDS-PAGE of the periplasmic extract showed no visible bands at the expected size (9.6 kDa).

![SDS](https://static.igem.wiki/teams/5918/mel/dbj3.webp)
Figure 45. The shown gel was obtained from electrophoresis on a 15% SDS-PAGE.

Lane 1: periplasmic extract with no target band; Lane 2-5: periplasmic extract after purification, showing no visible target band.

##### Learn

The peptide might not have been cleaved/secreting correctly, or degradation may have
occurred in the periplasm.

## Cycle 3

##### Design

To check whether expression occurred intracellularly, we decided to analyze the
whole-cell lysate.

##### Test

Cells were directly lysed, and proteins were purified using a Ni-NTA column. SDS-PAGE revealed a clear band at 12 kDa, consistent with uncleaved PelB-Di-melittin(12.1 kDa).

![SDS](https://static.igem.wiki/teams/5918/mel/dbj1.webp)
Figure 46. The shown gel was obtained from electrophoresis on a 15%
SDS-PAGE.

Lane 1-2: periplasmic extract after purification, showing no visible target
band; Lane 3: purified product from whole-cell lysates, where a distinct band of 12
kDa was observed; Lane 4: total protein extract, also displaying a clear 12 kDa band.
The observed size corresponds to uncleaved PelB-Di-melittin (12.1 kDa), rather than
the expected 9.6 kDa after signal peptide cleavage. The 10 kDa marker band, highlighted
in red, appeared faint during gel imaging and was specially indicated for clarity.

##### Learn

Di-melittin was expressed but remained uncleaved, suggesting that the PelB tag was not
processed as expected. This guided our future plans to test alternative signal
peptides or improve periplasmic extraction conditions.

# Suicide and Medicine-Food Collaboration

## Cycle 1

##### Design

We selected *E. coli* Nissle 1917 (EcN) as the chassis strain for our engineered bacteria.
*EcN* is a clinically approved probiotic with excellent safety, strong intestinal colonization
ability,
and suitability for gut-related therapeutic applications.

To prevent uncontrolled colonization or leakage of engineered bacteria after therapeutic effects,
our goal was to construct a suicide module responsive to food-derived small molecules. Based on
literature research, we identified hippuric acid—a downstream metabolite of anthocyanins enriched
in black rice—as a candidate signaling molecule. We designed a genetic circuit in which a hippuric-
acid-responsive riboswitch regulates expression of the toxin gene *ccdB*. When hippuric
acid is present, the riboswitch remains "off" allowing survival during treatment; once
hippuric acid is depleted due to diet cessation, the riboswitch is activated, driving
*ccdB*
expression and inducing cell death.

![回路图](https://static.igem.wiki/teams/5918/suicide/schematic-diagram-of-suicide-system.webp)
Figure 47. Schematic diagram of suicide system.

##### Build

Since no hippuric-acid riboswitch could be identified or verified, we used the thiamine
pyrophosphate (TPP) riboswitch as a functional analogue for preliminary validation.

We assembled the toxin gene *ccdB* downstream of the T7 promoter into pET-28b(+). After
sequencing confirmation, the recombinant plasmid was transformed into *E. coli* BL21(DE3)
for functional testing.

![](https://static.igem.wiki/teams/5918/suicide/ccdb1.webp)

Figure 48. pET-28b(+)-*ccdB* plasmid construction.

(A) The constructed plasmid map;
(B) Gel electrophoretic map after transforming the constructed plasmid into
*E. coli* BL21 (DE3);
(C) Alignment map of sequencing results vs. expected plasmid sequence.

In parallel, the *thiM* riboswitch was placed upstream of the reporter *amCyan*. The TPP
riboswitch-AmCyan module was assembled under constitutive PJ23100 in pUC57. After
sequence verification, the plasmid was transformed into *E. coli* BL21(DE3) for testing.

![](https://static.igem.wiki/teams/5918/suicide/ricfp1.webp)

Figure 49. pUC57-TPP *riboswitch-amCyan* plasmid construction.

(A) The constructed plasmid map;
(B) Gel electrophoretic map after transforming the constructed plasmid into
*E. coli* BL21 (DE3);
(C) Alignment map of sequencing results vs. expected plasmid sequence.

##### Test

Toxin protein validation. Engineered bacteria carrying the T7-*ccdB* construct were
cultured in LB with IPTG at 0, 0.1, 0.5, and 1 mM; an empty-plasmid control was included. OD600
monitoring showed significant growth inhibition at 0.5 mM IPTG. Plating every 30 min under 0.5 mM
IPTG revealed time-dependent decreases in colony number and size.

![](https://static.igem.wiki/teams/5918/suicide/ccdb-toxin-protein1.webp)

Figure 50. Growth dynamics and viability of bacteria.

(A) Bacterial OD600 over time;
(B) Viable cell counts over time.

![](https://static.igem.wiki/teams/5918/suicide/plate.webp)

Figure 51. Plate after IPTG induction.

(A) Plate after 0 hour of IPTG induction.
(B) Plate after 2.5 hours of IPTG induction.
(C) Plate after 3 hours of IPTG induction.

*thiM* riboswitch validation. Engineered bacteria carrying the *thiM* riboswitch-*ccdB*
construct were cultured in LB with TPP at 0, 10 μM, 100 μM, 1 mM, and 5 mM. OD600 showed inhibition
at
1 mM and 5 mM, indicating dose-dependent regulation by the *thiM* riboswitch.

![预实验](https://static.igem.wiki/teams/5918/suicide/ricfp-od600-pre.webp)
Figure 52. Relative fluorescence intensity over time under different TPP
induction.

##### Learn

Preliminary testing showed no response at low TPP, whereas induced *ccdB* expression
effectively
inhibited growth. Further work is needed to quantify the dynamic range and basal leakage of the
*thiM* riboswitch.

## Cycle 2

##### Design

We introduced the *thiM*-*ccdB* module to achieve orthogonal validation of
the suicide system: with TPP present the riboswitch stays "off" and cells survive; without
TPP it turns "on", drives *ccdB* expression, and induces cell death—a complete
proof-of-concept.

##### Build

The widely characterized *thiM* riboswitch was placed upstream of *ccdB*. The entire *thiM*-*ccdB* module was assembled under PJ23100 in pUC57. After
sequence confirmation, the construct was transformed into *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/5918/suicide/ricfp1.webp)

Figure 53. pUC57-TPP *riboswitch-ccdB* plasmid construction.

(A) The constructed plasmid map;
(B) Gel electrophoretic map after transforming the constructed plasmid into
*E. coli* BL21(DE3);
(C) Alignment map of sequencing results vs. expected plasmid sequence.

##### Test

Strains carrying the *thiM*-AmCyan construct were cultured in M9 with a TPP gradient
(0-15 mM, 1 mM gradient). OD600 and fluorescence were recorded in parallel. Fluorescence remained
unchanged at low TPP but dropped markedly at high TPP, confirming dose-dependent repression by the
riboswitch.A four-parameter logistic (4PL) fit yielded IC50=7 mM.

![拟合](https://static.igem.wiki/teams/5918/suicide/ricfp-4pl.webp)
Figure 54. Dose-response curve of the *thiM* riboswitch showing fluorescence
intensity
under different TPP concentrations, with IC50=6.81±1.77 mM.

To verify the suicide module, strains carrying the *thiM*-*ccdB* construct were
cultured in M9 with 0 mM vs. 7 mM (IC50) TPP. OD600 dynamics showed significant growth inhibition at
7 mM, confirming that the *thiM* specifically activates *ccdB* and triggers
suicide.

![折线](https://static.igem.wiki/teams/5918/suicide/riccdb-od6001.webp)
Figure 55. OD600 measurement under 7 mM TPP induction.

##### Learn

Induced CcdB expression effectively inhibited growth, and the suicide system exhibited clear
dose dependence on TPP. For topical hemorrhoid applications, this module supports safe control;
for oral use, the rapid response suggests optimizing concentration-time delivery.

## Cycle 3

##### Design

Hippuric acid is a broad-spectrum metabolite widely present in legumes and cereals; its level
fluctuates with diet and lacks tissue specificity. We therefore proposed personalized adaptation:
patients choose specific foods to activate matching riboswitches, precisely timing bacterial
suicide and reducing off-target risks.

Experiments further indicated that once signal supply stops, the suicide system activates within
2-3 h, and CcdB clears most bacteria within ~1 h. To build a clinically relevant time window, we
integrated a genetic delay (expiry-date) circuit, extending activation to >40 h, thereby reducing
dosing frequency.

##### Build

To predict the interval from signal withdrawal to toxin-induced death, we modeled the expiry-date circuit
and ran simulations with signaling molecules of different half-lives, obtaining corresponding CcdB
concentration trajectories. We also simulated different initial doses to derive time-course curves
under varied intake levels.

![待放回路图](https://static.igem.wiki/teams/5918/suicide/expiry-date.webp)
Figure 56. Conceptual diagram of the delay circuit.

Based on the patient's selected diet, the corresponding molecules are identified. These
molecules function as signals to activate the riboswitch, thereby regulating the expression of
the CcdB toxin. In the presence of the signaling molecules, the riboswitch remain in the "off"
state,
allowing the engineered bacteria to survive during treatment. Through the ODE model, we know that
after the intake of signal molecules is stopped, the suicide system will be activated after a
delay of at least 40 hours.

##### Test

With the initial concentration fixed at X\_init = 20 μM, varying decay constants (per half-life) were
used to numerically integrate CcdB expression over time; both shorter and longer half-lives
significantly
delayed toxin accumulation.

At a fixed half-life, varying initial doses (5, 10, 20, 50 μM) all reduced the accumulation rate of
CcdB due to the delay circuit, indicating robustness across intake levels.

![待放回路图](https://static.igem.wiki/teams/5918/suicide/simulation-results.webp)
Figure 57. Final results of the mathematical model.

Dynamic curves of CcdB concentration based on the constructed circuit, with the dashed line
indicating
the concentration at which the engineered bacteria are completely eliminated: the top figure, CcdB
dynamics
induced by TPP as the signaling molecule; the middle figure, simulated CcdB dynamics for signaling molecules
with different half-lives under the same dosage; the bottom figure, simulated CcdB dynamics for a specific
signaling molecule under varying dosages.

With TPP as the signaling molecule, the engineered bacteria are fully cleared after 41.87 hours.
With a signaling molecule half-life of 1000 min-1, the engineered bacteria are fully cleared after
41.41
hours. With a signaling molecule concentration of 10 µM, the engineered bacteria are fully cleared
after 41.54 hours. The suicide circuit can be triggered with a minimum delay of 40 hours across
varying
types and concentrations of signaling molecules.

##### Learn

Using the time to 10% of maximum CcdB as the "toxicity-threshold trigger time," response surfaces
showed that both half-life and dose exert limited influence on CcdB accumulation when the delay
circuit is present—supporting reliable timing control across broad conditions.

## Cycle 4

##### Design

For hippuric acid—the small molecule candidate we selected—there are currently no publicly reported
riboswitch aptamer sequences corresponding to it, nor is there sufficient research on its molecular
interaction mechanisms. Consequently, it is difficult to rely on traditional molecular simulations
or
pure mathematical modeling to make predictions. Given this limitation, we turned to deep learning
models
for exploration, leveraging the "black-box" capability of neural networks to compensate for the gaps
n mechanism research. This design concept is exploratory and innovative: it not only adapts to the
current
reality of insufficient experimental validation but also opens up a new aptamer prediction pathway
based
n generative models. Therefore, we propose the Mol2Aptamer model, which generates candidate RNA
aptamer
sequences by inputting the SMILES representation of small molecules.

##### Build

First, we constructed a dataset for small molecules and their corresponding riboswitch
aptamers. We integrated and filtered three public, experimentally validated nucleic acid
aptamer databases: **AptaDB**,
**Aptagen**, and
**the Global Nucleic Acid Aptamer
Database**.
Ultimately, we obtained 292 small molecules and their corresponding 794 aptamer sequences.
All these data are derived from actual experimental results, thus exhibiting high biological
reliability. It should be noted that the number of experimentally validated small molecule-riboswitch aptamer pairs that can be found in the literature is inherently limited, making it
difficult to further expand the dataset size at this stage. This objective limitation reflects
the initial exploratory state of this research field and is precisely the motivation for our attempt
to use deep learning models to expand predictive capabilities.

On the other hand, considering that aptamer sequences in different databases include both DNA
and RNA formats, we uniformly converted all sequences into RNA representation. This processing
may give rise to certain controversies, but we believe its rationality is primarily reflected
in two aspects: First, the molecular recognition function of aptamers mainly depends on their
secondary structure and spatial conformation. Since DNA and RNA share high similarity in base-pairing rules and structure formation mechanisms, sequence-level conversion will not alter
their intrinsic "structure-function mapping". Second, from a modeling perspective, we regard
the small molecule-aptamer prediction task as a sequence generation problem similar to translation.
Unifying base types helps reduce the complexity of model learning and avoids noise caused by
different coding systems. In summary, this preprocessing is not only biologically self-consistent
but also improves the stability and generalization of model training from a methodological
perspective.

Subsequently, we trained a dedicated BPE (Byte Pair Encoding) tokenizer based on this dataset to better tokenize and model SMILES strings and RNA sequences. We then constructed a deep learning framework with an encoder-decoder architecture: the encoder is responsible for extracting the structural features of small molecules, while the decoder gradually generates RNA aptamer sequences based on these features. The model architecture incorporates an attention mechanism, enabling the generation
process to selectively capture key information in the molecular structure. Through this approach, we established a neural network model capable of end-to-end learning of the small molecule-RNA mapping
relationship, laying the foundation for subsequent experiments and inference.

##### Test

We evaluated the model using an independent validation set, focusing on assessing the diversity
and rationality of the generated sequences. Additionally, we conducted preliminary structural and
affinity validation of the candidate sequences using secondary structure prediction software and
molecular
docking tools. Meanwhile, we performed cross-validation on some existing small molecule-RNA data to
test the
model's generalization ability. Furthermore, we systematically integrated different generation
strategies,
including greedy decoding, Top-k sampling, Top-p (nucleus) sampling, and temperature adjustment, to
balance
exploratory generation and sequence rationality. Through these tests, we can preliminarily determine
whether
the RNA aptamers generated by the model have potential biological value and provide reliable
candidate sequences
for subsequent wet-lab screening.

![待放回路图](https://static.igem.wiki/teams/5918/suicide/aptamer.webp)
Figure 58. Training curves of the Mol2Aptamer across 500 epochs.

The top-left subplot shows the training loss (blue) and validation loss (orange), both
decreasing and
gradually converging, with the minimum observed at epoch 486 (red dashed line). The top-right
subplot
shows token-level accuracy (green), which steadily increases and reaches its highest value
(0.6659) at
epoch 486. The bottom-left subplot shows sequence-level accuracy (purple), which remains lower
than
token accuracy but still improves during training, reaching 0.2242 at epoch 486. The
bottom-right subplot
shows perplexity (brown), which consistently decreases.

During training, the model exhibited stable convergence: both training and validation loss decreased
and
eventually converged, indicating effective feature learning and good generalization. Token accuracy
steadily increased, showing that the model progressively improved its ability to predict individual
tokens
correctly, while sequence accuracy, though lower, also increased, reflecting the greater difficulty
of
predicting entire sequences correctly. Meanwhile, perplexity consistently declined, further
confirming
convergence. Taken together, these results demonstrate that epoch 486 corresponds to the model's
optimal
performance, where loss is minimized and accuracy is maximized, highlighting a well-trained and
balanced
model.

##### Learn

We fed back the results of the test phase into model iteration. Through statistical analysis of the
generated sequences and extraction of structural features, we can observe the small molecule-RNA
interaction
rules implicitly captured by the model, thereby providing insights for understanding the aptamer
recognition
mechanism. Future work can be optimized in two aspects: data and model. On the data side, we will
gradually
expand the training corpus to improve molecular coverage. On the modeling side, we will explore
introducing
graph neural networks (GNNs) to enhance molecular structure representation, or adopt contrastive
learning strategies
to strengthen the association representation between small molecules and RNA sequences. Ultimately,
these
improvements not only enhance the model's ability to generate aptamers but also lay the foundation
for
constructing a closed-loop optimization system combined with wet-lab SELEX (Systematic Evolution of
Ligands
by Exponential Enrichment). As experimental validation results continue to be fed back, the model
will undergo
continuous iteration, gradually forming an automated Design-Build-Test-Learn (DBTL) cycle that can
drive the
design of small molecule-RNA aptamers.

# Future Work

In our current project, we have achieved significant results, including the successful validation of
key biological parts through both wet lab and dry lab approaches. However, HemorrEaser still
requires considerable development before it can be considered a fully realized therapeutic. In
future work, we plan to characterize the feasibility of delivering bioPROTAC via outer membrane
vesicles (OMVs) and investigate whether the OmpA-CAR fusion can maintain the wound-healing
properties of CAR while achieving effective targeting. After completing the characterization of all
individual components, we will proceed to assemble the complete genetic circuit and introduce it
into the chassis organism. It is important to note that since nanobodies require a specific chemical
microenvironment for proper expression in prokaryotic systems, we will also constitutively express
the disulfide bond isomerase DsbC in the engineered *E. coli* Nissle 1917 (ΔnlpI) strain to optimize
expression efficiency.

Looking ahead, we are committed to systematically addressing these challenges through rigorous
characterization and optimization. By integrating these efforts, we aim to advance HemorrEaser from
a proof-of-concept study toward a viable therapeutic candidate, ultimately contributing to the
development of innovative treatment strategies for hemorrhoidal disease.

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2016;
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![contentdot](https://static.igem.wiki/teams/5918/main-pic/contentdot.webp)

# Experiments

![headflower](https://static.igem.wiki/teams/5918/main-pic/headflower.webp)

[Part1](#Part_1)
[Part2](#Part_2)
[Part3](#Part_3)
[References](#Part_4)

# 区块一 一级标题

## 二级标题

### Ⅰ.三级标题（罗马数字）

**加粗**
普通正文文字内容
*斜体*
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另起一行须另起一对p标签。

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# 区块二 一级标题

![待放回路图](图片链接)
Figure 1. Figer和数字有空格，数字和点无空格，点之后有一空格

## 二级标题

* 无序列表
* 缩进无序列表（添加class="suojin"）
* 缩进无序列表（添加class="suojin"）
* 无序列表

1. 有序列表
2. 直接复制li填写，自动标号。
3. 缩进有序列表（添加class="suojin"）
4. 缩进有序列表（添加class="suojin"）
5. 一对ol标签内连续编号，无论缩进与否。

## 二级标题

表注如table1

[ ]

表格实际标题

| Parameter | Description | Value | Unit | Source |
| --- | --- | --- | --- | --- |
| kmHrpS | Degradation rate of HrpS mRNA | 4.62×10-2 | min-1 | [3] |
| KHrpS | Translation rate of HrpS | 2.98 | nM·min-1 | [2] |
| kHrpR | Degradation rate of HrpR | 5.775×10-4 | min-1 | [5] |
| βHrpL | Maximum transcription rate of the *P*HrpL** | 5 | nM·min-1 | [-] |
| K2 | Dissociation constant of HrpRS for the promoter *P*HrpL.** | 3.78×102 | nM | [[Calculated]](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D) |
| n2 | Hill coefficient of HrpRS to *P *HrpL** | 2 | -- | [6] |
| kmHolin | Degradation rate of Holin mRNA | 4.62×10-2 | min-1 | [3] |

![dots](https://static.igem.wiki/teams/5918/main-pic/tabledot.webp)

# 区块三 一级标题

Lorem ipsum dolor sit amet consectetur, adipisicing elit. Necessitatibus ad nobis, suscipit perspiciatis dolorum eius sapiente porro animi eum officia assumenda, quibusdam ducimus similique obcaecati fugit incidunt ipsa earum! Unde?

## 一定要改id和对应的labelclass!

![](https://static.igem.wiki/teams/5335/model/molecular-model/figure-3-rmsd2.png)

Figure 2. Figer和数字有空格，数字和点无空格，点之后有一空格
![](https://static.igem.wiki/teams/5335/model/molecular-model/figure-4-ram-temp.jpg)

Figure 3. Figer和数字有空格，数字和点无空格，点之后有一空格

图片的进一步解释，在span标签内用p标签包裹。示例：
**A.**273K: 92.5% (322/348) of all residues were in favored (98%) regions. 99.4% (346/348) of all residues were in allowed (>99.8%) regions.
There were 2 outliers (phi, psi):54 Pro (-38.5, -24.5), 259 Ser (-175.0, 0.3).
**B.**288K: 94.5% (329/348) of all residues were in favored (98%) regions. 99.4% (346/348) of all residues were in allowed (>99.8%) regions.
There were 2 outliers (phi, psi):194 Ser (46.1, -6.2), 269 Pro (-51.2, -1.0).
**C.**301K(RT): 93.4% (325/348) of all residues were in favored (98%) regions. 98.3% (342/348) of all residues were in allowed (>99.8%) regions.
There were 6 outliers (phi, psi):20 Asp (-36.7, -23.7), 84 Arg (60.3, -26.9), 194 Ser (47.6, -11.7), 246 Val (-68.5, 39.9), 269 Pro (-52.1, 7.0), 309 Lys (33.9, 84.8)

# References

[https://2009.igem.org/Team:PKU\_Beijing/Modeling](https://2009.igem.org/Team%3APKU_Beijing/Modeling)

Johnson GE, Lalanne JB, Peters ML, Li GW. Functionally uncoupled transcription-translation in Bacillus subtilis. Nature. 2020 Sep;585(7823):124-128.

Hambraeus G, von Wachenfeldt C, Hederstedt L. Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs. Mol Genet Genomics. 2003 Aug;269(5):706-14.

Park HH, Lim WK, Shin HJ. In vitro binding of purified NahR regulatory protein with promoter Psal. Biochim Biophys Acta. 2005 Sep 15;1725(2):247-55.

引用跳转链接可添加href（如上文[1]）。

新增条目时记得修改数字。

![contentdot](https://static.igem.wiki/teams/5918/main-pic/contentdot.webp)

# Results

![headflower](https://static.igem.wiki/teams/5918/main-pic/headflower.webp)

[Global Regulation Module](#Part_1)
[Validation for BioPROTAC](#Part_2)
[Validation for Anti-VEGF Nanobody](#Part_3)
[Anti-inflammatory Module](#Part_4)
[OMV Delivery Module](#Part_5)
[Suicide and Medicine-Food Collaboration Module](#Part_6)
[References](#Part_7)

# Global Regulation Module

To prevent the potential risk of overproliferation when engineered bacteria are applied
*in vivo*, we designed a LuxR/TetR dual-factor cascade system. This system utilizes
a quorum-sensing mechanism to monitor population density and incorporates a negative
feedback loop to precisely regulate downstream gene expression, thereby achieving global
control.

![](https://static.igem.wiki/teams/5918/srcgraph/qsvec.webp)

Figure 1. pET-28b(+)-*luxI*-*luxR*-*tetR*-*amCyan* plasmid construction.

(A)The constructed plasmid map;
(B)Gel electrophoretic map for colony PCR;
(C)Alignment map of sequencing results vs. expected plasmid sequence.

As shown in **Figure 1.A**, the *amCyan* gene was placed downstream of the Ptet.
After the construct was successfully introduced into *E. coli* BL21(DE3), colony PCR
was performed, and as shown in **Figure 1.B**, the bands matched the expected length. Sequencing
analysis further confirmed that the recombinant plasmid was consistent with the design
as shown in **Figure 1.C**.

## Expression Visualization Validation

To visually assess the expression level of the downstream gene, the recombinant
plasmid was expressed in *E. coli* BL21(DE3). Once visible fluorescence was observed
under ambient light, 10 mL of the bacterial culture was preserved in 50% glycerol.
Subsequently, 100 μL aliquots were inoculated hourly into 10 mL of M9 medium supplemented with
50 μg/mL kanamycin. After 48 hours of incubation, samples from the experimental and
empty-plasmid control groups were subjected to **visual observation and imaging**.

![宏观](https://static.igem.wiki/teams/5918/quorum-sensing/qs-fluorescence.webp)
Figure 2. Fluorescence intensity over time.

As shown in **Figure 2**, the AmCyan protein expression level in the engineered bacteria demonstrated a
gradual increasing trend over time.

## Expression Quantification Experiment

To further quantify the regulatory effect of the quorum-sensing circuit, the recombinant plasmid
described
above was expressed in *E. coli* BL21(DE3). When the culture reached an OD600 of
0.4-0.6,
a glycerol stock was prepared, and 1 μL of the glycerol stock was inoculated into 200 μL of LB
medium for
relative fluorescence intensity measurement. A preliminary experiment was designed to determine the
total
detection duration and time intervals. Based on the results, measurements were taken every 20
minutes
starting from inoculation, for a total duration of 5.5 hours. Data analysis and ODE fitting were
subsequently
performed.

![拟合1](https://static.igem.wiki/teams/5918/quorum-sensing/growth-curve-new.webp)
Figure 3. Measured OD600 values and the corresponding logistic fit of
the one-step growth curve (R2 = 0.9943).

As shown in **Figure 3**, the growth kinetics of the engineered bacteria, measured by OD600,
exhibited a progressive increase that followed a logistic model (R2 = 0.9943) over the
course
of the experiment.

![拟合2](https://static.igem.wiki/teams/5918/quorum-sensing/output.webp)
Figure 4. Measured relative fluorescence units (RFU) and the corresponding fitted
curve of the global regulation circuit dynamics (R2 = 0.9078).

As shown in **Figure 4**, the dynamic expression profile of the global regulation circuit was
quantitatively
monitored by measuring relative fluorescence units (RFU) over a 500-minutes cultivation period. The
kinetic
trajectory was fitted using a two-parameter model, which accurately captured the characteristic
progression
of circuit activation. The fitted curve demonstrates the density-dependent activation dynamics of the
quorum-sensing regulatory system, showing an initial rapid increase to a peak RFU value, followed by
a
decline and eventual stabilization at a steady state.

# Validation for BioPROTAC

To validate the functional capacity of bioPROTAC, the following experimental
investigations were conducted:

1. Yeast two-hybrid (Y2H) and bacterial **protein co-purification assays** were employed to
   confirm that the fusion construct does not compromise VHH212 nanobody engagement with HIF-1α;
2. ***In vitro* ubiquitination assays** were performed to demonstrate that the VHL moiety
   within the fusion protein successfully mediates polyubiquitination of HIF-1α.

## Validation of Interaction

​​In the yeast two-hybrid system​​, all groups grew on SC-2 medium, confirming successful transformation.
The experimental group co-transformed with pGADT7-vhh-vhl and pGBKT7-*hif-1α* plasmids, along with the positive control,
exhibited robust growth on SC-4 medium, whereas the negative control showed slow or no growth[2]. These results
verify that the interaction between the VHH nanobody and Hif-1α remains intact despite the fusion protein
construction (**Figure 5.A**).

​​Similarly, in the bacterial protein co-purification assay​​, *E. coli* BL21(DE3) was co-transformed with pET-28b(+)-*hif-1α*-His and pUC57-*vhh-vhl*-Myc.
After sonication, Hif-1α-His was purified using Ni-NTA affinity chromatography. If VHH-VHL interacts with HIF-1α, both proteins should be detectable by SDS-PAGE.
Indeed, compared with the control, whole-cell lysates showed bands corresponding to Hif-1α-His (94.0 kDa) and VHH-VHL-Myc (34.6 kDa).
Furthermore, the co-purification of VHH-VHL-Myc with Hif-1α-His was confirmed in the elution fractions, while neither band was detected in wash fractions containing non-specific proteins.
This further demonstrates that the VHL ubiquitination domain does not interfere with the VHH module's ability to recognize and bind its target protein (**Figure 5.B**).

![](https://static.igem.wiki/teams/5918/bioprotacwet/result-inter.webp)

Figure 5. Validation of VHH-VHL interaction with HIF-1α.

(A)Yeast two-hybrid assay. Experimental group: pGADT7-*vhh-vhl* +
pGBKT7-*HIF-1α*.
Controls: Positive-pGADT7-T + pGBKT7-53; Negative-pGADT7-T + pGBKT7-lam. Successful
transformation
confirmed by SC-2 growth. Representative growth on SC-4 shown;
(B)Protein co-purification assay by 12% SDS-PAGE. Ct: Whole-cell lysate
from *E. coli* BL21(DE3)
transformed with empty pET-28b(+) and pUC57; Total: Co-transformant lysate
(pET-28b(+)-*HIF-1α*-His +
pUC57-*vhh-vhl*-Myc); Lanes 1-3: Ni-NTA affinity purification
eluates (protein concentrations: 0.116, 0.431, 0.936 mg/ml); Wash: Background
proteins from affinity purification. Molecular
weights: HIF-1α-His (94 kDa), VHH-VHL-Myc (34.6 kDa).

## Validation of Ubiquitination

​​For the *in vitro* ubiquitination assay​​, purified bioPROTAC protein (VHH-VHL) was incubated with truncated HIF-1α substrate (aa 1-365),
E1 activating enzyme UBA1, E2 conjugating enzyme UBC8, ubiquitin-His, and ATP. Reactions were terminated at different time points (2 h, 4 h, 6 h)
and analyzed by Western blot using an anti-ubiquitin antibody, with two replicates per group. It should be noted that, due to experimental
equipment limitations, two nitrocellulose membranes were used for the assay.

Compared with the negative control lacking UBA1 and UBC8, the experimental group showed time-dependent enhancement of HIF-1α ubiquitination.
In contrast to the blank control without VHH-VHL, the group supplemented with VHH-VHL exhibited significantly stronger ubiquitination signals(**Figure 6.**).

These results demonstrate that the bioPROTAC can efficiently mediate ubiquitination of HIF-1α *in vitro*,
and strongly suggest its potential to promote ubiquitination of non-hydroxylated HIF-1α under hypoxic
conditions *in vivo*.[3]

![](https://static.igem.wiki/teams/5918/bioprotacwet/ub3.avif)

Figure 6. *In vitro* validation of VHH-VHL-mediated HIF-1α ubiquitination​.

​​Western blot analysis of *in vitro* ubiquitination reactions at 2 h, 4 h and 6 h. Reactions without UBC8 and UBA1 served as negative controls; reactions without VHH-VHL served as blank controls; all control reactions were terminated at 6 h; HIF-1α(truncated)-Ubn, above 44.3 kDa; UBC8-Ub, 33.54 kDa.

## Discussion

Through computational rational design and complementary *in vivo/in vitro* validation, the
engineered
bioPROTAC molecule VHH-VHL has demonstrated capacity to both retain the high-affinity binding of its
VHH212 module to the target protein HIF-1α and functionally recruit the ubiquitination machinery to
facilitate targeted polyubiquitination. These findings indicate that the designed bioPROTAC
effectively
binds to and mediates ubiquitin-dependent degradation of HIF-1α under hypoxic conditions *in
vivo*,
thereby
attenuating hypoxia-driven angiogenesis at hemorrhoidal sites and therapeutically inhibiting disease
progression.

# Validation for Anti-VEGF Nanobody

## Antibody Expression Validation

To achieve the expression of P17-Anti-VEGF nanobody, we fused a 6×His-tag to the C-terminal
of our PelB-P17-Anti-VEGF nanobody gene and cloned it into the pET-28b(+) plasmid for
purification, and controlled the expression by the lac operon (**Figure 7**). Then the plasmid
was transformed into *E. coli* SHuffle for expression. Transformants were selected on LB agar plates
containing kanamycin, as only bacteria harboring the recombinant plasmid possess the kanamycin
resistance gene. Positive colonies were picked for culture expansion.

![map](https://static.igem.wiki/teams/5918/antivegf/monovalent-pelb-p17.webp)
Figure 7. Plasmid map used to express P17-Anti-VEGF nanobody.

The transformed bacteria were cultured at 37 °C until the OD600 reached 0.5,
at which point protein expression was induced with 0.5 mM IPTG for 16 hours.
The bacterial cells were harvested by centrifugation,
and the periplasmic proteins were extracted using the osmotic shock method.
Successful expression of the P17-Anti-VEGF nanobody fusion protein was confirmed by
SDS-PAGE and Western blot analysis of the obtained protein samples.(**Figure 8.**)

![map](https://static.igem.wiki/teams/5918/srcgraph/abpr.webp)
Figure 8. SDS-PAGE and Western blot analysis of purified samples.

(A) 15% SDS-PAGE of unpurified periplasmic protein and purified sample: M:Protein Marker; lane 1-2 were unpurified periplasmic protein , and lane 3-6 were purified sample;
(B)Western Blot of purified sample; M:Protein Marker; lane 1-4 were purified sample.

b

## Antibody Function Validation

To further validate the anti-VEGF activity,
cell proliferation was assessed in VEGFR2-positive HUVECs and VEGFR2-negative HEK293 cells
using the MTT assay at 24 hours post-treatment. The results showed (**Figure 9**) that
treatment with the nanobody for 24 hours significantly inhibited the proliferation of HUVECs
in a dose-dependent manner, with inhibition rates ranging from 40% to 50% (P < 0.05) compared to the
control. Conversely, no significant inhibitory effect was observed in HEK293 cells at any
concentration tested, with inhibition rates below 5% (P > 0.05).

![map](https://static.igem.wiki/teams/5918/antivegf/mtt-d1-5.webp)
Figure 9. The antiproliferative activity of nanobody on HUVEC cells.

# Anti-inflammatory Module

To alleviate hemorrhoid symptoms, treat local inflammation, and reduce swelling, pain, and effusion of hemorrhoidal tissue,
we designed the anti-inflammatory module.

## SoxR/PSoxS oxidative stress response promoter

At the inflammation site of hemorrhoids, the concentration of reactive oxygen species (ROS) is very
high, and we selected it as the signal trigger of the anti-inflammatory module. We chose SoxR/PSoxS
oxidative stress response promoter as the regulatory element. SoxR is a transcription factor
containing [2Fe-2S] cluster, while PSoxS is a promoter sequence. In a high ROS environment, SoxR
binds between the -10 and -35 regions, and its oxidized form distorts the promoter conformation,
promotes the binding of RNA polymerase (RNAP) and other transcription factor, and activates
transcription.

To verify the function of the SoxR/PSoxS oxidative stress response promoter, we cloned *amCyan* gene
as the
reporter gene into the pUC57 vector and constructed the corresponding reporter plasmid (**Figure 10.**).

![](https://static.igem.wiki/teams/5918/srcgraph/rosvec.webp)

Figure 10. pUC57-*soxR*-*soxS*-*amCyan* plasmid construction.

(A)Plasmid map used to verify the SoxR/PSoxS oxidative stress response promoter;
(B)The result of electrophoresis of the colony PCR product initially attests to the
successful reorganization of plasmid. M: DL1000 DNA Marker. Lane 1-8: eight individual colonies
obtained after in-fusion cloning;
(C)The sequencing results of the constructed plasmid confirmed the successful
assembly.

We transformed the recombinant plasmid into *E. coli* BL21 (DE3) for subsequent functional
verification.
Before oxidant induction, we used 12% SDS-PAGE electrophoresis to verify that SoxR could be normally
expressed in *E. coli* BL21 (DE3) (**Figure 11**).

![1](https://static.igem.wiki/teams/5918/jiayin/soxr-pro-2.webp)
Figure 11. 12% SDS-PAGE results for verification of SoxR expression.

control:Total protein sample extracted from *E. coli* BL21 (DE3) carrying the empty plasmid.
Lane 1: Total protein samples extracted from expanded cultures of one different single colony that were successfully transformed. A comparison of the two indicates that SoxR is successfully expressed.

After verifying the expression of SoxR, to verify the functions of the SoxR and PSoxS,
we set up a series of tert-butyl hydrogen peroxide (tBHP) concentration gradients for induction (0
μM, 20 μM, 40 μM, 60 μM, 80μM, 100 μM, 120 μM, 140μM, 160 μM, 180μM, 200μM). We added 200 μL of
induction medium inoculated with engineered bacteria into a 96-well plate and used a microplate
reader to detect the emission light intensity at 486 nm. Three parallel replicates were set for
each induction concentration (**Figure 12**).

![](https://static.igem.wiki/teams/5918/jiayin/soxr-deta-2.webp)

Figure 12. Fluorescence intensity measured by microplate reader.

(A)Changes in fluorescence intensity over time in different concentrations
of tBHP-induced groups and the control group;
(B)Maximum fluorescence intensity in different concentrations of tBHP-induced
groups and the control group.

We found that the activity of the PSoxS is extremely dependent on the ROS concentration
, and the background expression of PSoxS is low in the absence of ROS.

## PelB-Di-melittin

##### Ⅰ.Search for anti-inflammatory factors

To identify a suitable anti-inflammatory effector, we reviewed extensive literature and data,
and ultimately selected Di-melittin, designed by NKU-China, 2024. To facilitate
expression and purification, we added an N-terminal pelB signal peptide and a
C-terminal 6×His-tag.

Melittin itself is known for its strong anti-inflammatory activity through multiple mechanisms
, making it highly attractive for our project. However, the clinical application of melittin
is severely limited by its strong cytotoxicity. The Di-melittin variant designed by
iGEM24\_NKU-China significantly reduces this cytotoxicity by connecting two melittin monomers
with a linker to form a unique hairpin structure, thereby making its application feasible.

##### Ⅱ.PelB-Di-melittin

After contacting NKU-China, we learned that their experiments involved eukaryotic
expression of Di-melittin in yeast cells for subsequent validation. For biosafety
considerations, however, we selected *Escherichia coli* Nissle 1917 (EcN) as our
chassis strain. After careful discussion, we concluded that since Di-melittin consists of
only 75 amino acids and has a relatively simple structure, it can be feasibly expressed in
*E. coli*. Moreover, the modified Di-melittin shows greatly reduced cytotoxicity to host
bacteria, making prokaryotic expression in *E. coli* achievable. Since NKU-China,2024
has already conducted comprehensive functional characterization and thoroughly validated
the anti-inflammatory properties of Di-melittin, building upon their work, our work primarily
focused on verifying its feasibility for prokaryotic expression. Therefore, we performed
Di-melittin expression verification in *E. coli*BL21(DE3).

To this end, we engineered Di-melittin with an N-terminal PelB signal peptide for
secretion into the periplasmic space and a C-terminal 6×His-tag for downstream
purification. The 6×His-tag was connected to Di-melittin through a flexible linker (GGSSSGG)
to minimize structural interference (**Figure 13**).

![PelB-Di-melittin ](https://static.igem.wiki/teams/5918/mel/pelb-di-melittin1.webp)
Figure 13. Schematic representation of the engineered PelB-Di-melittin construct.

The design includes an N-terminal PelB signal peptide for periplasmic secretion, the Di-melittin
coding sequence in a hairpin structure, a flexible linker (GGSSSGG), and a C-terminal 6×His-tag for
purification.

##### Ⅲ. Plasmid Construction

To achieve prokaryotic expression of Di-melittin, we constructed a recombinant plasmid based on
pET-28b(+) through homologous recombination and transformed it into *E. coli*
BL21(DE3) (**Figure 14.A**).The transformed colonies were cultured on LB plates containing kanamycin at 37 °C for 16 h.
Colony PCR (**Figure 14.B**) and sequencing (**Figure 14.C**) confirmed the correct insertion
of the target gene.

![Plasmid Construction](https://static.igem.wiki/teams/5918/mel/zh1.webp)
Figure 14. pET-28b(+)-*pelB*-*di*-melittin plasmid construction.

(A)Plasmid map of the recombinant expression vector
pET-28b(+)-*pelB-di-melittin*;
(B)Colony PCR results of recombinant clones. All seven tested colonies exhibited a clear
band at 739 bp, consistent with the expected size. A 1000 bp marker was used as reference;
(C)Sequencing verification of the recombinant plasmid. The sequencing results confirmed
that the PelB-Di-melittin insert was correctly integrated, with no mutations or frame
shifts, and fully consistent with the design.

##### Ⅳ. Protein Expression Analysis

Positive clones were induced at OD600≈0.6 with 1 mM IPTG and cultured at 16 °C for 18 h.
Cells were harvested, lysed, and total protein was purified with Ni-NTA affinity
chromatography.
SDS-PAGE analysis showed a clear band of approximately 12 kDa in whole-cell lysates, while
no band was detected from periplasmic extractions (**Figure 15**). Based on design,
the expected size of Di-melittin after PelB cleavage was
~9.6 kDa. However, the observed 12 kDa band corresponded to the uncleaved PelB-Di-melittin,
consistent with its theoretical molecular weight. Sequencing analysis verified the integrity of the
PelB sequence, suggesting that the observed molecular weight discrepancy did not result from errors
in the tag sequence.

![SDS](https://static.igem.wiki/teams/5918/mel/dbj4.webp)
Figure 15. The shown gel was obtained from electrophoresis on a 15% SDS-PAGE.

Lane 1-2: periplasmic extract after purification, showing no visible target
band. Lane 3: purified product from whole-cell lysates, where a distinct band of 12
kDa was observed. Lane 4: total protein extract, also displaying a clear 12 kDa band.
The observed size corresponds to uncleaved PelB-Di-melittin (12.1 kDa), rather than
the expected 9.6 kDa after signal peptide cleavage. The 10 kDa marker band, highlighted
in red, appeared faint during gel imaging and was specially indicated for clarity.

Owing to time constraints, we were not able to perform further experiments to reach a
definitive conclusion. We propose several possible explanations:

1. Interference from protein folding. The target protein may adopt secondary structures or fold
   during intracellular synthesis or membrane translocation, thereby hindering the access of signal
   peptidase to the cleavage site.
2. Degradation of periplasmic peptides. Anti-inflammatory peptides are relatively small and
   may be unstable during periplasmic extraction or subsequent handling. Endogenous periplasmic
   proteases could degrade the peptide, and although PMSF was applied as an inhibitor, the
   concentration may have been insufficient to prevent degradation.
3. Inclusion body formation. The protein may aggregate in the cytoplasm as inclusion bodies,
   producing strong bands in whole-cell lysates but yielding little or no soluble protein in
   the periplasmic fraction.

While the functional activity of Di-melittin had been comprehensively verified by iGEM24\_NKU-China,
our results further demonstrated that the peptide can be efficiently expressed in a prokaryotic
system,
paving the way for scalable production and *in vivo* applications.

## Discussion

This experiment engineered an *E. coli* Nissle 1917 strain with the SoxR/PSoxS oxidative
stress-responsive promoter to express the anti-inflammatory peptide Di-melittin specifically
in high-ROS hemorrhoidal environments, thereby alleviating swelling, pain, and exudation.

# OMV Delivery Module

To achieve precise targeted delivery of *Escherichia coli* outer membrane vesicles
(OMVs) to the hemorrhoid site, we selected the OmpA-CAR fusion protein as the
targeting element. The α-helix of the OmpA signal peptide efficiently inserts into
membrane structures, while CAR specifically recognizes angiogenic endothelial cells
abundant at the hemorrhoid site. Thus, the OmpA-CAR fusion protein effectively mediates targeted
OMV transport to the hemorrhoid site.

We constructed expression vector containing *ompA-CAR*, *ompA-amCyan*,
and *srp-TagRFP* on
pET-28b(+), respectively. We co-transformed two plasmid pairs, *ompA-amCyan* with *srp-TagRFP* and *ompA-CAR* with *srp-TagRFP*, into *E. coli* Nissle 1917 (ΔnlpI), a mutant known for its increased OMV yield. PCR appraisal of the transformed colonies showed that both groups
exhibited
double positive bands, indicating that both sets of plasmids had been successfully double
transformation
into the EcN strain (**Figure 16,17**).

![map](https://static.igem.wiki/teams/5918/jiayin/omv-p-car-4.webp)
Figure 16. Colony PCR results of *ompA-CAR* and *srp-TagRFP*.
![map](https://static.igem.wiki/teams/5918/srcgraph/omvbacp.webp)
Figure 17. Colony PCR results of  *ompA-amCyan*  and
*srp-TagRFP*.

Subsequently, we employed the differential centrifugation method (DC) to isolate the OMV
precipitate
to verify the extraction efficiency, we further utilized the OMV marker protein OmpA
(approximately
38.2 kDa) as the core validation indicator and performed a 12% SDS-PAGE experiment.[1] The
electrophoresis
results showed a clear characteristic OmpA protein band in the extract, directly confirming the
success
of the OMV extraction procedure (**Figure 18**).

![](https://static.igem.wiki/teams/5918/srcgraph/omvex-final.webp)

Figure 18. OMV Extraction Results.

(A)DC Results;
(B)12% SDS-PAGE results(M: Protein marker. DC: Differential centrifugation extract;
DGC:
Density gradient centrifugation extract.).

## Discussion

We designed and constructed the OmpA-CAR fusion peptide, whose N-terminus retains the positively
charged-hydrophobic amphiphilic region characteristic of the OmpA signal peptide, enabling stable
insertion and anchoring into the OMV lipid bilayer membrane; the C-terminus is linked to the CAR
sequence with specific recognition functionality, exposing it on the outer surface of the vesicles.
This structure simultaneously achieves dual functions of membrane anchoring and targeted recognition,
guiding drug-loaded OMV to precisely accumulate at the hemorrhoid lesion site, thereby laying the
molecular and carrier foundation for the subsequent directional delivery of the antiangiogenic module.

Limited by the current experimental progress, the loading efficiency and targeting effect of OMV have
not yet been clarified. In subsequent studies, we will first measure the Zeta potential of the extract
to clarify the stability of its dispersion system and preliminarily confirm the structural characteristics
of OMV; then, through fluorescence confocal microscopy experiment, observe the co-localization of AmCyan and TagRFP
to verify whether the loading of Engineered outer membrane vesicle is successful; finally, conduct cell
scratch assay to test the promoting effect of CAR on cell migration, thereby verifying the targeting
efficacy of engineered OMV.

# Suicide and Medicine-Food Collaboration Module

To ensure the safety of engineered bacteria, we designed a suicide module that kills bacterial
strains
both in the external environment and in the digestive tract once the food-derived small molecules
are
no longer supplied, thereby avoiding potential leakage of engineered bacteria.

## Functional validation of TPP riboswitch *thiM*

To validate the activity of the *thiM* riboswitch and its response range to TPP, we placed the
AmCyan protein downstream of the riboswitch and introduced the construct into *E. coli* BL21(DE3). As shown in **Figure 19.A**, the *amCyan* reporter was placed downstream of the riboswitch. As
shown in **Figure 19.B**, after the construct was successfully introduced into *E. coli* BL21(DE3), colony PCR was
performed and the bands matched the expected size. As shown in **Figure 19.C**, sequencing analysis further confirmed that
the recombinant plasmid was consistent with the design.

![](https://static.igem.wiki/teams/5918/srcgraph/rbsvec-2.webp)

Figure 19. pUC57-TPP *riboswitch-amCyan* plasmid construction.

(A)The constructed plasmid map;
(B)Gel electrophoretic map after transforming the constructed plasmid into
*E. coli* BL21(DE3);
(C)Alignment map of sequencing results and expected plasmid sequence.

When cultures reached OD600 = 0.4-0.6, the bacteria were transferred to M9 minimal medium. We set up a
preliminary experiment (TPP concentrations of 0, 10 μM, 100 μM, 1 mM, and 5 mM) to determine the
approximate range in which TPP exerts its inhibitory effect (**Figure 20.A**). As shown in **Figure 20.A**, functional testing demonstrated that
the TPP
riboswitch gradually repressed downstream gene expression as TPP concentration increased,
with obvious
inhibition observed at concentrations above 1 mM. These results confirmed the successful
construction of the
expression vector and defined the approximate inhibitory range of the riboswitch ligand TPP.

![](https://static.igem.wiki/teams/5918/srcgraph/tpp-results.webp)

Figure 20. Validation of the *thiM* riboswitch.

(A)Relative fluorescence intensity over time under different TPP induction;
(B)Dose-response curve of the *thiM* riboswitch showing fluorescence intensity
under different TPP concentrations, with IC50 = 6.81±1.77 mM.

The samples were divided into groups and supplemented with 0-30.0 μL of TPP stock solution in 2.0 μL
increments,
corresponding to final concentrations of 0-15 mM in 1 mM increments. Fluorescence intensity (Ex: 453
nm, Em:
486 nm) and OD600 were measured every 30 minutes for 5 hours at 37℃ with shaking at 200
rpm.
Relative
fluorescence intensity was calculated as fluorescence divided by OD600.

As shown in **Figure 20.B**, the *thiM* riboswitch gradually repressed downstream gene expression as
the TPP
concentration
increased, with the inhibitory effect reaching a plateau above 13 mM. Data fitting using the
four-parameter
logistic (4PL) model yielded an IC50 = 6.81 ± 1.77 mM. This experiment validated that the TPP
riboswitch can
nearly completely suppress gene expression under high TPP concentrations.

## Functional validation of CcdB Toxin Protein

In our experiment, we constructed a plasmid vector with CcdB toxin protein connected after the
lactose
promoter and introduced it into *E. coli* BL21(DE3). IPTG was used to induce expression and
OD600
was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to
determine
viable cell counts.

![](https://static.igem.wiki/teams/5918/srcgraph/ccdb.webp)

Figure 21. pET-28b(+)-*ccdB* plasmid construction.

(A)The constructed plasmid map;
(B)Gel electrophoretic map after transforming the constructed plasmid into
*E. coli* BL21(DE3);
(C)Alignment map of sequencing results vs. expected plasmid sequence.

As illustrated in **Figure 21.A**, the *ccdB* gene was inserted downstream of the T7 promoter. Subsequent transformation of this construct into *E. coli* BL21(DE3) enabled verification by colony PCR, which yielded bands corresponding to the anticipated molecular size (**Figure 21.B**). Additionally, sequencing analysis further confirmed that the recombinant plasmid conformed precisely to the designed specifications (**Figure 21.C**).

![](https://static.igem.wiki/teams/5918/srcgraph/ccd-results.webp)

Figure 22. Growth dynamics and viability of bacteria.

(A)Bacterial OD600 over time;
(B)Viable cell counts over time.

As illustrated in **Figure 22.A**, OD600 analysis revealed that the CcdB protein induced a potent bactericidal effect, leading to rapid bacterial cell death within one hour post-induction. **Figure 22.B** further demonstrated a drastic decline in viable cell counts and a significant reduction in colony diameters over time. Consistent with these observations, **Figure 23.A-C** confirmed a sharp decrease in both viable cell numbers and colony sizes with prolonged incubation. These findings collectively establish that CcdB exhibits a pronounced lethal activity against bacterial cells, in contrast to weakly toxic proteins that solely inhibit growth.

![](https://static.igem.wiki/teams/5918/suicide/plate.webp)

Figure 23. Plate after IPTG induction.

(A)Plate after 0 hour of IPTG induction;
(B)Plate after 2.5 hours of IPTG induction;
(C)Plate after 3 hours of IPTG induction.

## Functional validation of *thiM* riboswitch regulatory CcdB toxin protein

To validate the functionality of the *thiM* riboswitch and the toxin protein CcdB, a genetic circuit was constructed utilizing the pUC57 plasmid as the backbone. The *ccdB* gene was inserted downstream of the *thiM* riboswitch, with the entire circuit regulated by the PJ23100 promoter, and subsequently transformed into *E. coli* BL21(DE3). As illustrated in **Figure 24.A**, the design positioned *ccdB* under the control of the riboswitch. **Figure 24.B** demonstrates that colony PCR performed after successful transformation yielded bands corresponding to the expected size. Additionally, sequencing analysis, as depicted in **Figure 24.C**, confirmed that the recombinant plasmid conformed to the intended design.

![](https://static.igem.wiki/teams/5918/srcgraph/tpp-ccdb.webp)

Figure 24. pUC57-TPP *riboswitch-ccdB* plasmid construction.

(A)The constructed plasmid map;
(B)Gel electrophoretic map after transforming the constructed plasmid into
*E. coli* BL21(DE3);
(C)Alignment map of sequencing results vs. expected plasmid sequence.

Cultures were grown to an OD600 of 0.4-0.6, transferred to M9 medium, and induced with 7 mM TPP (corresponding to the half-maximal inhibitory concentration, IC50). Toxicity was assessed in 96-well plates by monitoring OD600 using a Synergy™ H1 microplate reader, with comparisons made between experimental and control groups. As shown in **Figure 25**, at the IC50 concentration of the riboswitch ligand TPP, the suicide system elicited a pronounced response, effectively suppressing bacterial growth and confirming the potent bactericidal activity of the CcdB protein under riboswitch regulation.

![OD](https://static.igem.wiki/teams/5918/suicide/riccdb-od6001.webp)
Figure 25. OD600 measurement under 7 mM TPP induction.

# References

Jingyuan SONG, Xiulei QI, Huaizhong GUO, et al. Lipidomics
analysis of glycine-induced bacterial outer membrane vesicles (J/OL).
Chinese Journal of Chromatography, 2025, 43 (5): 547-555.

Wang, S(2024). Y2HGold Yeast Two-Hybrid Screening and Validation
Experiment Protocol. Bio-protocol Preprint.

Zhao, Q. and Xie, Q. (2013).In vitro Protein
Ubiquitination Assays.Bio-protocol3(19): e928.

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