Proof of Concept

Wet Lab

GOAL FOR OUR PROOF OF CONCEPT

Our goal is to demonstrate that by conjugating the β-lactamase inhibitory protein (BLIP) DNA from our donor (E. coli S17-1) to our recipient (E. coli DH5α), the recipient can successfully take in the DNA and inhibit β-lactamase in antimicrobial-resistant bacteria, thereby restoring the effectiveness of antibiotics.

Key Concepts and Milestones of Our Project:
1. Beta-Lactamase Inhibitory Protein (BLIP)
2. Plasmid Construction and Transformation
3. Conjugation of S17-1 to DH5α
4. Final Result

1. Beta-Lactamase Inhibitory Protein (BLIP)

In search of a better solution to combat antimicrobial resistance, we aim to experiment with both kinds of BLIP: BLIP-I and BLIP-II. Although both BLIPs inhibit the same beta-lactamases through similar mechanisms, BLIP-II achieves greater binding affinity (binding with beta-lactamase) despite using fewer contact residues and having a smaller interface than BLIP-I [2], which is a peculiar reversal of the usual trend [1]. It is also a more potent inhibitor of β-lactamases than BLIP-I. Testing both BLIP-I and BLIP-II will strengthen our Proof of Concept, showing that our BLIP conjugation system can support different variants, and helping us evaluate which version has the strongest effect on inhibiting beta-lactamase. The plated results on antimicrobials were examined and analyzed.

2. Plasmid Construction and Transformation

A. Construction of Donor Bacteria

Upon receiving the BLIP sequences from GenScript and Twists Bioscience, we amplified the gene sequences with PCR. The pSB1C3 plasmids were transformed into E. coli DH5α for plasmid propagation and subsequently extracted using a miniprep procedure. To avoid the presence of inhibitors such as residual PCR reagents that could interfere with restriction digestion, we purified the PCR products using the GeneDireX PCR Clean-Up and Gel Extraction Kit.
During the early stages of our experiments, we experienced significant DNA loss during gel extraction, which led to faint or nearly undetectable bands in subsequent gel electrophoresis. Consequently, we often could not obtain sufficient DNA for downstream restriction digestion and ligation.

To improve purification recovery, we performed several troubleshooting cycles. We eventually identified the acidic ddH₂O used for elution as a likely cause of the low yield (see the wet-lab section of the Engineering page for details). Replacing it with Tris buffer, which maintains a stable and slightly alkaline pH, effectively minimized DNA binding to the silica column and preserved DNA integrity [4]. This adjustment consistently improved both the yield and quality of our purified DNA for cloning and digestion workflows.

After numerous attempts at restriction digestion and multiple procedural refinements, we successfully inserted BLIP-I and BLIP-II into the pSB1C3 backbone using EcoRI and PstI, followed by ligation with T4 DNA ligase. Each stage of the process—restriction digestion, purification, ligation, and transformation into E. coli DH5α for verification, presented its own challenges.

During the early stages, transformations repeatedly failed. However, through persistence and optimization of our protocols, we eventually observed a single pink colony on the chloramphenicol (Ch) plate (Figure 5). This indicated successful construction and transformation of the BLIP-I recombinant plasmid, as the transformed E. coli DH5α expressed both the chloramphenicol resistance gene from the pSB1C3 backbone and mCherry fluorescence encoded by the BLIP-I insert.

In contrast, the BLIP-II construct did not initially show colony growth on the Ch LB plate (Figure 6) but exhibited growth in Ch-supplemented LB broth (Figure 7). We subsequently subcultured the broth and spread it onto a Ch-LB plate, where obvious bacterial growth is observed (Figure 8), confirming successful transformation of the BLIP-II recombinant plasmid.

Using pSB1C3 provides a high-copy plasmid with chloramphenicol resistance, ensuring stable replication and strong gene expression. The pSB1C3 backbone carries a pMB1-derived origin of replication, which enables replication to high copy numbers, typically around 100–300 copies per cell in standard E. coli strains such as DH5α [5]. Inserting BLIP into the pSB1C3 backbone before introducing the plasmid into donor E. coli S17-1 allows the construction of a transfer-ready vector for subsequent conjugation experiments. This design enables the targeted horizontal transfer of BLIP to recipient bacteria, forming the foundation for the PlasMission Spy system.

Following successful plasmid construction and verification in E. coli DH5α, the recombinant pSB1C3-BLIP-I-mCherry and pSB1C3-BLIP-II-mCherry plasmids were purified by miniprep and subsequently transformed into donor E. coli S17-1. Transformation plates containing chloramphenicol (Ch) showed the appearance of distinct pink colonies (Figure 9-10), indicating successful uptake and expression of the BLIP-mCherry constructs. The presence of both chloramphenicol resistance and visible mCherry fluorescence confirmed that the transformation was successful and that S17-1 now carried the transfer-ready BLIP constructs for subsequent conjugation experiments.

B. Construction of Recipient Bacteria
We selected E. coli DH5α_BBa J435320 as our recipient strain to represent an antimicrobial-resistant bacterial model in our BLIP conjugation system. DH5α [6] is a widely available and well-characterized laboratory strain, making it suitable for its stable propagation of plasmids and high transformation efficiency.

We obtained our BBa J435320 plasmid through the distribution kit and inserted them into the DH5α through transformation. The transformants are then plated on Amp-LB plates to select the recipient strains ready for conjugation. There was no significant colony formed on the Amp-LB plate (Figure 11), but observable growth was found in the Amp-LB broth (Figure 12). We then subculture the bacteria on Amp-LB plate to ensure the selection again. (Figure 13).

3. Conjugation Experiments

Conjugation represents the final stage of our project, serving as the ultimate test of the PlasMission Spy system. Due to limited time before the wiki freeze, we conducted our conjugation experiments on October 6, just two days before submission. To maximize our chances of success, we performed both solid and liquid conjugation methods in parallel.

A. Solid Conjugation
Solid-surface conjugation was chosen because it promotes stable and prolonged contact between donor and recipient cells, which is essential for efficient plasmid transfer. [7] On solid agar, bacteria remain immobilized and in close proximity, enabling sustained mating-pair formation. This spatial stability facilitates higher transfer efficiency compared to liquid cultures, where cells are constantly moving and have limited opportunities for contact. [8]

Our donor strain, E. coli S17-1, utilizes the RP4 conjugation system, which forms rigid pili that enhance cell-to-cell adhesion during plasmid transfer. [9] These structural features make solid conjugation particularly effective for our system and suitable for visual confirmation of successful transfer events.

B. Liquid Conjugation
In parallel, we also performed conjugation in liquid LB medium, allowing us to compare transfer efficiency across conditions. Although conjugation in liquid culture is typically less efficient due to reduced physical contact, it offers easier scaling, uniform mixing, and faster cell growth. Conducting both approaches provided us with complementary data on how environmental conditions influence the transfer dynamics of our BLIP-containing plasmids.

C. Conjugation Results
Side note: Although we could not get IPTG to our lab in time, we still attempted blue-white screening using X-gal only.

Solid Conjugation Results
For solid conjugation, we co-cultured the donor strain (E. coli S17-1 carrying pSB1C3-BLIP) and the recipient strain (E. coli DH5α carrying BBa_J435320) on LB agar plates and LB agar supplemented with X-gal. After incubation at 37 °C for six hours, the plates exhibited visible pink and blue coloration, corresponding to the characteristic pigmentation of the donor (mCherry expression) and the recipient (lacZ expression), respectively. The presence of both colors indicated that both strains were actively growing and in close contact, a necessary condition for conjugation to occur on solid media.

Following incubation, we gently scraped the mixed bacterial growth from the plate surfaces and resuspended the cells in 1 mL of sterile PBS. From this suspension, 100 µL aliquots were plated onto chloramphenicol (Ch), ampicillin (Amp), and dual-antibiotic (Ch + Amp) LB plates, grown overnight for subsequent selection and validation.
Detailed observations and interpretations of the final results are presented in the Validation and Final Results section.

Liquid Conjugation Results
For liquid conjugation, we co-cultured the donor (E. coli S17-1 carrying pSB1C3-BLIP) and recipient (E. coli DH5α carrying BBa_J435320) strains in LB broth at 37 °C for four hours. The culture became noticeably turbid, indicating robust bacterial growth.

Due to limited time and materials near the end of our experimental schedule, we focused our efforts on the most critical validation step and plated the culture onto dual-antibiotic (Ch + Amp) LB plates. Although we were unable to prepare additional X-gal plates for this trial, this prioritization allowed us to directly test both conjugation success and BLIP’s functional inhibition of β-lactamase activity under selective conditions. The results of this validation are discussed in detail in the Validation and Final Results section.

4. Validation and Final Results

To validate successful conjugation and assess the functional impact of BLIP, we combined antibiotic selection with blue-white screening [10]. Although we could not obtain IPTG in time, we still proceeded with blue-white screening using X-gal alone. Because the lac operon (especially in parts like BBa_J435320) exhibits basal (leaky) expression even without induction [11], it is possible to see some background LacZ activity. In our context, this leakage could lead to some blue coloration in uninduced conditions. This leakiness is well known in classic lac systems due to incomplete repression; the lac promoter still allows a low basal transcription rate in the absence of an inducer.

Our donor strain (E. coli S17-1) carries the pSB1C3-BLIP plasmid, which provides chloramphenicol resistance (ChR), while the recipient strain (E. coli DH5α) carries a plasmid containing a functional lacZ gene and ampicillin resistance.

In our design, if conjugation occurs, the transconjugants should receive the donor’s ChR marker and retain the recipient’s lacZ, forming blue colonies on chloramphenicol (Ch) plates containing X-gal. The blue colonies on Ch plates — represent the presence of both donor and recipient genetic markers, confirming successful plasmid transfer.

However, BLIP inhibits β-lactamase activity, which means that ampicillin resistance may be suppressed in successful transconjugants. As a result, no colonies are expected on dual-antibiotic (Ch + Amp) plates. This outcome does not indicate failure; rather, it demonstrates the functional effectiveness of BLIP in inhibiting β-lactamase, validating the biological purpose of our system.

Predicted Outcomes
- Ideal: Blue colonies on Ch plates and no colonies on Ch + Amp plates — conjugation successful and BLIP functional.
- Failed conjugation: Blue colonies on Amp plates only — no plasmid transfer.
- Inactive BLIP: Colonies on Ch + Amp plates — transfer succeeded, inhibition failed.

Together, these findings confirm both the successful horizontal transfer of BLIP and its intended inhibitory function within recipient bacteria.

Final Results
On October 7, one day before the wiki freeze, we successfully obtained conjugation results that matched our predicted ideal outcome:
- BLIP-I: Pink and blue colonies on Ch plates and a single pink colony on Ch + Amp plate, indicating successful transfer and partial β-lactamase inhibition. (Figure 14)
- BLIP-II: Pink and blue colonies on Ch plates and no colonies on Ch + Amp plate, demonstrating complete inhibition of β-lactamase activity. (Figure 15)

Left: Pink and blue colonies on the Ch-LB plate indicate donor and transconjugant cells.
Middle: Blue colonies on the Amp-LB plate show recipient and transconjugant strains carrying lacZ.
Right: Only one pink colony on the dual Ch-Amp-LB plate demonstrates effective BLIP-mediated inhibition of β-lactamase activity.

Left: Pink and blue colonies on the Ch-LB plate indicate donor and transconjugant cells.
Middle: Blue colonies on the Amp-LB plate show recipient and transconjugant strains carrying lacZ.
Right: No colonies on the dual Ch-Amp-LB plate demonstrate complete BLIP-mediated inhibition of β-lactamase activity.

These results confirm that our BLIP plasmids were conjugatively transferred from E. coli S17-1 to E. coli DH5α and that the BLIP proteins functionally inhibited β-lactamase, restoring ampicillin sensitivity in the recipient cells. This fulfills the core proof of concept for our project.

Limitations and Future Validation
Due to the time constraint of the wiki freeze, we were unable to conduct final verification steps such as miniprep, PCR, or restriction digestion of the transconjugant plasmids. Nevertheless, the clear phenotypic outcomes—antibiotic selection patterns and blue-white differentiation, provide strong experimental evidence that the PlasMission Spy system works as intended.

Future validation will include molecular confirmation of plasmid sequences, quantification of conjugation efficiency, and functional assays under controlled induction to further characterize system performance.

Dry Lab

GOAL FOR OUR PROOF OF CONCEPT

Our goal is to demonstrate, using computational modeling, that the β-lactamase inhibitory protein (BLIP) DNA can be successfully transferred from donor (E. coli S17-1) to recipient (E. coli DH5α) via bacterial conjugation [15], and that this transfer could enable the recipient to inhibit β-lactamase in antimicrobial-resistant bacteria, thereby restoring antibiotic effectiveness. In addition, we aim to compare the relative effectiveness of BLIP-I and BLIP-II in plasmid transfer efficiency and potential β-lactamase inhibition, providing quantitative insights into which system may be more effective.

As a purely computational study, experimental conjugation was not performed. To guide our modeling, we referred to approaches suggested by the NYCU-Formosa dry lab team, focusing specifically on plasmid transfer (conjugation) as the primary process.

CONJUGATION MODEL

We chose to simulate the conjugation process to track donor, recipient, and transconjugant populations over time. By calculating conjugation rates (γ₁ for BLIP-I and γ₂ for BLIP-II) and modeling population dynamics, we can predict the number of transconjugants expected under different donor-recipient conditions. This framework allows us to compare the efficiency of BLIP-I and BLIP-II plasmid transfer. Figures 18 and 19 visualize the modeled process and corresponding symbol annotations.

Designing Equations
Difference equations were applied to model conjugation dynamics, adapting parameters from previous work (SDU-Denmark 2019) [16] to fit the BLIP systems. The equations track changes in recipient and transconjugant populations over discrete time intervals:

Our simulations used donor and recipient populations of 1×10⁶ and 1×10⁷ cells/mL, reflecting the standard 1:10 ratio in conjugation experiments. In the donor-growth scenario, all populations increased exponentially over time. In the constant-donor scenario, donors remained constant, recipients declined, and transconjugants increased logistically, demonstrating the expected inverse relationship. Comparisons between BLIP-I and BLIP-II showed differences in conjugation efficiency, with BLIP-I producing a slightly higher number of transconjugants under identical conditions. Figures 20-22 depict these trends for both systems under both scenarios.

Discussion
The model predicts that conjugation efficiently transfers BLIP plasmids to recipients, supporting the concept that BLIP-expressing transconjugants could restore antibiotic sensitivity. Importantly, the comparison between BLIP-I and BLIP-II provides insight into which system may achieve higher plasmid transfer efficiency, guiding future experimental designs. While wet lab validation was not performed, these computational simulations establish the feasibility of BLIP transfer via conjugation and provide a quantitative framework for evaluating system effectiveness.

Using the equations developed for our model, we simulated two scenarios to evaluate BLIP plasmid transfer by conjugation. In the first scenario, donor and recipient populations were allowed to grow, and the model predicted exponential increases in cell numbers over time due to continuous cell division. In the second scenario, donor and recipient growth were excluded. Under these conditions, donor cells remained constant, acting as a stable plasmid reservoir; recipient populations gradually declined; and transconjugants followed a logistic growth curve [18]. The model isolates the effect of the conjugation process, showing an inverse relationship between transconjugants and recipients in which ongoing gene transfer leads to a proportional decrease in recipients as transconjugants increase.

GOAL FOR OUR PROOF OF CONCEPT

Our goal is to design a kinetic model that can effectively simulate the inhibitory interactions between BLIP (β-lactamase inhibitor protein) variants and β-lactamase, as well as their impact on ampicillin degradation. By building a simplified computational system in COPASI, we aim to verify that the model can reproduce expected biological behaviors — specifically, that BLIP-II shows stronger inhibition of β-lactamase than BLIP-I. This proof of concept serves as an initial validation of our model framework, showing that the equations, parameters, and rate laws used can reasonably capture enzyme–inhibitor dynamics, even before being expanded to include more complex biological processes such as plasmid replication and stochastic gene expression.

BLIP EXPRESSION MODEL

The model was parameterized using experimentally derived or literature-supported constants, including mRNA half-lives [19], translation elongation rates, transcription frequencies, and enzyme kinetic constants. Specifically, transcription and degradation parameters were drawn from E. coli datasets, while binding affinities (kₒₙ and kₒff) [21] and catalytic rates [20] were adapted from previous studies quantifying BLIP–BLA interactions. These values allowed realistic values being input yet computationally tractable to evaluate inhibition dynamics.

Design
The simulation results show that BLIP-II exhibits stronger inhibition toward β-lactamase than BLIP-I, as comparing figure 23 and 24, aligning with experimental findings that BLIP-II has a well and tight binding with the enzyme and forms a more stable complex [19]. In the modeled system, BLIP-II rapidly reduces β-lactamase activity, whereas BLIP-I inhibition proceeds more gradually, allowing faster antibiotic degradation. This contrast presented by the graphs supports the model’s predictive capacity to reflect subtle variations in inhibitory strength.

Discussion
The results of the BLIP expression models show that our kinetic model can successfully simulate how BLIP-I and BLIP-II interact with β-lactamase and affect antibiotic activity. The COPASI simulations indicate that BLIP-II is a more effective inhibitor than BLIP-I, as it binds more tightly to β-lactamase and slows down the degradation of ampicillin for a longer time. Research papers also support the idea that BLIP-II has an efficient inhibitor towards BLA. [22] However, the model also contains several limitations. It does not include plasmid replication, random variations in gene expression, or changes in the cell environment, and it simplifies enzyme behavior to make the system easier to analyze. Because of this, the model does not fully represent what happens in real-life biological situations. Even so, the simulation provides a strong starting point for understanding how different BLIP variants influence β-lactamase activity. Future work should include experimental testing and more detailed modeling to confirm these trends and bring the results closer to real biological conditions.

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