Design

Our project seeks to develop an engineered microbial system aimed at overcoming beta-lactam antibiotic resistance in bacteria. Specifically, we are designing a conjugative bacterial system that enables horizontal gene transfer of a plasmid carrying a beta-lactamase inhibitory protein (BLIP) from one bacterial strain to another. The BLIP is intended to inhibit the activity of beta-lactamase enzymes, which degrade beta-lactam antibiotics, thereby resensitizing resistant bacteria to beta-lactam based treatment.

A. Early Idea: CRISPR-Cas9 in Bacteriophages System

Our initial project idea was inspired by a research article that explored combining engineered bacteriophages with CRISPR-Cas9 technology.[1] The concept involved three major design elements: phage engineering, CRISPR-mediated gene targeting, and conditional survival circuits. Phages are viruses that naturally infect and kill bacteria, and by tailoring them to specifically recognize antimicrobial-resistant pathogens such as MRSA or CRKP, they could selectively target harmful bacteria while leaving non-resistant strains unharmed. For CRKP in particular, phages carrying CRISPR-Cas9 systems could be programmed to cut resistance genes, weakening or killing the bacteria and preventing further spread of resistance elements. To ensure the biosafety, we also planned on designing a conditional survival circuit, allowing engineered phages to replicate only in the presence of AMR bacteria by linking replication to the detection of specific bacterial signals.

While this approach was scientifically promising, we recognized practical challenges. Researchers at Academia Sinica in Taiwan have significant expertise and prior experience with CRISPR-Cas9 and phage-based systems. We reached out in hopes of learning from their work and gaining background knowledge or technical guidance, but unfortunately did not receive a response. This highlighted both the complexity of the design and the limitations of our current resources.

Acknowledging these constraints, we pivoted toward a more accessible and feasible synthetic biology strategy using bacterial transformation and recombinant plasmids. This new direction still allowed us to address antimicrobial resistance in a meaningful way, while ensuring that our experiments could be safely and effectively conducted within the biosafety level and resources available to our team.

B. Major Turning point: The PlasMission Spy Design

Our initial idea was to design a CRISPR-Cas9 system to target carbapenem-resistant Klebsiella pneumoniae (CRKP). While this approach was promising, we soon realized it was too challenging to implement with the limited resources available to us, and it would also exceed the iGEM safety guidelines[2] for high school teams, which only allow BSL-1 level projects.

In response, we refocused our efforts on a more accessible synthetic biology strategy: using bacterial transformation and recombinant plasmids to address resistance to beta-lactam antibiotics. This adjustment kept our project within the required safety framework while still tackling a critical global health issue.

As we researched potential genetic targets, we identified beta-lactamase as a key enzyme responsible for resistance. [3] During this process, we came across BLIP (beta-lactamase inhibitory protein), [3] which provided us with a natural inhibitor that could be engineered for our system.

Building on this discovery, we designed a conjugation-based delivery system to transfer the BLIP gene into recipient bacteria, offering a novel and feasible way to combat beta-lactam antibiotic resistance at the BSL-1 level. We named this system PlasMission Spy, reflecting its role in “spying on” and counteracting resistance mechanisms by delivering the BLIP containing plasmid directly into the resistant bacteria.

Beta-lactamase inhibitory protein (BLIP), originally found in Streptomyces clavuligerus, a small two-domain protein that potently inhibits ambler class A β-lactamases, [4] such as TEM-1. It acts as a competitive inhibitor, blocking the enzyme’s active site, preventing β-lactam antibiotic from being hydrolyzed by beta-lactamase. There are two types of BLIP that are being used in this project– bliA (BLIP-I) and bliB (BLIP-II).

Although they have very similar mechanisms through creating hydrogen, salt bridges, and aromatic bonds with the beta lactamase enzyme, [5] they have completely different sequences and are not mutations of BLIP. [6]

Both the BLIP-I and BLIP-II sequences were obtained by reverse translating the protein sequence of BLIP from the National Institute of Health (NIH). [7][8] We chose this approach because, after comparing the coding sequence of BLIPs with their protein sequences, we observed inconsistencies between the two. Although we explored other possible sources, most either traced back to the same NIH dataset or lacked sufficient reliability for cross-reference. Ultimately, we decided to reverse translate the protein sequence from NIH, as protein sequence reflects direct functional relevance. In contrast, coding gene sequence may vary, sometimes containing annotation errors, frame shifts, ambiguous bases, or codon usage differences issues depending on the host organisms.

Figure 1. DNA Sequence Translated Into Amino Acid Sequence of BLIP-I from NIH

Figure 2. Amino Acid Sequence Translated Into DNA Sequence of BLIP-I from NIH

The biggest difference between BLIP-I and BLIP-II apart from sequences is its affinity against beta-lactamase. Both proteins inhibit beta-lactamase effectively with high potency. Typically, a larger binding interface would provide more opportunities for specific interactions that contribute to high affinity and selectivity for the target, which can increase potency. Even though BLIP-II uses a smaller binding interface than BLIP-I, it binds much tighter because each of its contacts is stronger and more important. BLIP-II focuses on a few key “hotspot” residues on TEM-1 that provide most of the binding energy, while BLIP-I spreads its interactions more broadly but less efficiently. As a result, BLIP-II forms a more stable complex that comes apart more slowly, giving it much higher binding strength overall. [9]

Conjugation is a natural mechanism by which bacteria exchange genetic material, and it is also one major pathway of antibiotic resistance spread. For conjugation to occur, the plasmid must carry an origin of transfer (oriT) and the appropriate tra functions. In our PlasMission Spy Design, we plan to use this conjugation model to transfer Beta-Lactamase Inhibitory Protein (BLIP) into the bacteria with antibiotic resistance.

Components of the PlasMission Spy Design:
- Cargo plasmid:Carries the oriT sequence along with our target gene, BLIP
- Helper system: Provided by the donor strain containing tra1/tra2 functions
- Expected outcome: The system mobilizes our BLIP construct efficiently into recipient strains, E. coli DH5ɑ for downstream testing.

Donor Bacteria Organism: E. coli S17-1
Genotype: recA pro hsdR RP4-2-Tc::Mu-Km::Tn7
Description: Recommended mobilization host for pARO180, pARO190, pARO181, and pARO191 (ATCC 77123-77126, respectively) because it contains chromosomally integrated tra genes. A kanamycin-sensitive strain devoid of the E. coli K-12-specific restriction system allowing efficient uptake of foreign DNA. The properties of streptomycin, trimethoprim, and spectinomycin resistance were verified. RP4-Tc::Mu-Km::Tn7 is integrated into the chromosome.
Source: https://www.atcc.org/products/47055

We chose to use S17-1 as our donor because S17-1 contains tra1/tra2 genes in its chromosomes, and it contains pilus proteins, creating the prime setups for a complete conjugation system[12]. From past iGEM projects such as 2019 newOrleans, [13] S17-1 is the optimal choice for systems requiring conjugation methods.

Donor Plasmid components:
- oriT BBa_K125320
- Backbone pSB1K3 - Kanamycin required for antibiotic resistance marker
- Promoter BBa_J23103
- ScarRBS BBa_B0030 and BBa_B0034
- BLIP-I (bliA) Expression / BLIP-II (bliB) Expression
- Fluorescent Marker T2T3_ymCherry - for fluorescent protein conjugation validation marker

Figure 3. BLIP-I Version I plasmid

In this design, we decided to use one set of promoter and terminator with two different ribosome-binding sites in between to ensure a fixed ratio of protein and reduce variability between them. Avoiding two sets of promoter and terminator is also for the purpose of reducing metabolic burden on the bacteria and stress in transcription processes. But most importantly by avoiding redundant regulatory elements, there would be a lower chance of mutation or recombination, especially for such a new coding that is prone to accumulating mutations.

In order to activate the RP4 mechanism, the origin of transfer (oriT) genes were required in either chromosomal or plasmid sequence. In which case we use BBa_K125320 [14] from the iGEM registry.

For expression, we chose to use a constitutive promoter BBa_J23103[15]. Compared to the Anderson promoter family it has a relatively moderate strength, providing ideal protein expression while maintaining an acceptable metabolic burden on the host.This balance made BBa_J23103 the most appropriate choice for our design.

Pertaining to backbone, we chose to use pSB1K3[16]. As one of the iGEM registry’s standard set of vectors it ensures assembly it ensures compatibility with biobrick assemblies. Additionally, pSB1K3 contains Kanamycin resistance, which allows selectivity after transformation, and is later on used for validation in this version of the plan.

We selected RBS BBa_B0034 and BBa_B0030 [17][18]because they are iGEM standard parts that ensure reliable initiation of translation. Its strong and consistent activity makes it a benchmark for protein expression, while maintaining compatibility with BioBrick assembly and comparability to other iGEM projects.

Figure 4. BLIP-II Version I plasmid

Recipient Bacteria
Description
HIT Competent Cells *™-DH5a are unique chemically competent cells, which utilise a novel transformation protocol - High Ice Transformation. A simple ice incubation from 1-10 minutes is all that is required to transform the cells to high efficiency. Source: http://www.real-biotech.com/index.asp?modules=product&files=p2&ID1=130&ID2=84

We chose to use non-pathogenic DH5ɑ because it is a K12 and one of the most commonly used for cloning strain.

Recipient Bacteria components:
- Backbone pSB1C3 - the Chloramphenicol us required for antibiotic resistance marker
- Green Fluorescent protein BBa_E0040 - for fluorescent protein conjugation validation marker

Figure 5. Recipient Plasmid Version 1

Validation method
Originally, the idea of testing BLIP on antibiotic resistance proteins was not considered because we initially thought that using bacteria with penicillin resistance was not on the whitelist of the categories we selected to test on, therefore we decided to use three alternative ways to validate whether conjugation was successful.

- Method 1 - Fluorescent Marker
In the plasmid designs for both recipient and donor, there were fluorescent proteins embedded into each. Green for recipient and red for donor. Ideally if the conjugation was successful, the transconjugant should emit both green and red light, green under 395 nanometer light and red under 540 nanometer light. We expected the two proteins to shine separately under the two distinctive light and we would be able to merge the pictures together later.

- Method 2 - Antibiotic Resistance Marker

Figure 6. Antibiotic Resistance Marker Visual

We planned to validate the conjugation success through manipulation of different antibiotics on different plates. If the conjugation was successful, this was the ideal survival status of the donor, the recipient, and the transconjugant.

- Method 3 - PCR Amplification of the BLIP sequences For this we designed primers aiming for the whole of BLIP-I (558 bp) and the majority of BLIP-II (939 bp). By amplifying the target gene from the transconjugant after plating on agar plate with both Kanamycin and Chloramphenicol, we further validate the existence and success of conjugation of BLIP from donor to recipient.

After additional research, we further optimize our plan with a better and more effective validation method with alternated plasmids in the recipient bacteria.

In the second version of our plan, no changes were made to the donor and recipient bacteria. However, upon receiving the iGEM distribution kit, we adjusted our design to make the most of the available resources. Specifically, we modified the plasmid backbone in the donor to pSB1C3 and used BBa_J435320 as the plasmid for the recipient.

These changes were made for two main reasons:
To take advantage of the plasmids provided in the distribution kit.
To modify the recipient into an ampicillin-resistant strain, allowing us to test the effectiveness of BLIP after conjugation while still adhering to the iGEM safety whitelist.

We decided to replace the recipient plasmid with BBa_J435320, as it encodes both the Bacillus anthracis β-lactamase (bla) gene and the LacZ sequence. The bla gene is particularly important because it belongs to Ambler class A β-lactamases, making it highly relevant for validating the effectiveness of BLIP, our β-lactamase inhibitory protein. In addition, the LacZ gene enables blue-white screening after conjugation, allowing us to easily identify transconjugants.

Donor Plasmid (Plasmid 1) components:
- oriT BBa_K125320
- Backbone pSB1C3
- Promoter BBa_J23103
- ScarRBS B0034
- BLIP-I (bliA)Expression/BLIP-II (bliB) Expression
- ScarRBS B0030
- T2T3_ymCherryM10L BBa_J43413
- Terminator BBa_B0012

Figure 7. BLIP-I Version II Donor Plasmid

Figure 8. BLIP-II Version II Donor Plasmid

Recepient Plasmid (BBa_J435320, Plasmid 2):

Figure 9. Version II Recipient Plasmid

Validation Method
- Method 1 - Antibiotic Resistant Marker
We decided to use a similar method of design for antibiotic resistant marker validation here with the alternated antibiotic. In order to compare the effectiveness of BLIP, the use of double antibiotics was implemented. Supposedly, if the BLIP proved its effectiveness, then under conditions of both antibiotics, the ampicillin resistance should be inhibited therefore no bacteria would survive in that case.

- Method 2 - Blue-White Screening
After the conjugation, the samples will be plated on LB agar plates with X-Gal and appropriate antibiotics, as shown in Table 6. If the conjugation is successful, the transconjugant should contain both plasmid 1 and 2, which allows it to survive chloramphenicol selection (ChR from plasmid 1) and show blue colonies in blue-white screening (LacZ from plasmid 2). If BLIP successfully inhibits beta-lactamase’s activity, the transconjugant won’t survive the ampicillin selection.

- Method 3 - PCR Amplification of the BLIP sequences
After the conjugation, the transconjugant (blue colonies on the ChR plate) will be selected and subcultured. Then, the plasmid will be harvested by miniPrep for further PCR amplification using the corresponding primers. The sizes of the amplicons will be further confirmed to ensure that plasmid 1 is successfully transferred from the donor bacteria to recipient bacteria.

- Method 4 - Restriction Digestion
After the conjugation, the transconjugant (blue colonies on the ChR plate) will be selected and subcultured. Then, the plasmid will be harvested by miniPrep. As a quick tool to check, the extracted plasmid will be digested with both EcoRI and PstI enzymes to cut out the gene inserts. The sizes of the gene inserts will then subsequently be confirmed with a gel electrophoresis.

As a new team, we encountered several challenges throughout the project, including time constraints, limited experience, delayed arrival of materials, and restricted lab access. Despite these hurdles, we stayed motivated and continued working toward demonstrating the effectiveness of BLIP. In this revised design, instead of using conjugation to introduce BLIP into the target bacteria, we adopted a more direct approach — constructing a recombinant plasmid containing BLIP, mCherry, and ampicillin resistance, which will then be transformed into E. coli DH5ɑ for testing.

Figure 10. Plasmid Design for the Additional Plan.

To verify the effectiveness of BLIP, we plan to perform a comparative antibiotic susceptibility test between transformants expressing BLIP and the control strains without it. The presence of ampicillin resistance in the recombinant plasmid allows us to evaluate whether BLIP successfully inhibits β-lactamase activity and restores sensitivity to ampicillin. Additionally, the inclusion of mCherry serves as a visual reporter to confirm successful transformation and expression on a non-selective plate. By monitoring growth patterns and fluorescence, we can assess both the expression of the construct and the functional impact of BLIP.

1. Asmamaw, M.; Zawdie, B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. Biologics : Targets & Therapy 2021, 15 (1), 353–361. https://doi.org/10.2147/BTT.S326422
2. Reygaert, W. C. An Overview of the Antimicrobial Resistance Mechanisms of Bacteria. AIMS Microbiology 2018, 4 (3), 482–501. https://doi.org/10.3934/microbiol.2018.3.482
3. Bush, K.; Bradford, P. A. β-Lactams and β-Lactamase Inhibitors: An Overview. Cold Spring Harbor Perspectives in Medicine 2016, 6 (8), a025247. https://doi.org/10.1101/cshperspect.a025247
4. Hanes, M. S.; Jude, K. M.; Berger, J. M.; Bonomo, R. A.; Handel, T. M. Structural and Biochemical Characterization of the Interaction between KPC-2 β-Lactamase and β-Lactamase Inhibitor Protein. Biochemistry 2009, 48 (39), 9185–9193. https://doi.org/10.1021/bi9007963
5. Gyun Kang, S.; Ung Park, H.; Sook Lee, H.; Tae Kim, H.; Joon Lee, K. New β-Lactamase Inhibitory Protein (BLIP-I) from Streptomyces Exfoliatus SMF19 and Its Roles on the Morphological Differentiation. Journal of Biological Chemistry 2000, 275 (22), 16851–16856. https://doi.org/10.1074/jbc.m000227200
6. Streptomyces exfoliatus beta-lactamase inhibitory protein BLIP-I precu - Nucleotide - NCBI. Nih.gov. https://www.ncbi.nlm.nih.gov/nuccore/AF201389?report=genbank (accessed 2025-10-04)
7. UniProt. https://www.uniprot.org/uniprotkb/Q9KJ90/entry?utm_source.com (accessed 2025-10-04)
8. Raval, N.; Maheshwari, R.; Kalyane, D.; Youngren-Ortiz, S. R.; Chougule, M. B.; Tekade, R. K. Importance of Physicochemical Characterization of Nanoparticles in Pharmaceutical Product Development. Basic Fundamentals of Drug Delivery 2019, 369–400. https://doi.org/10.1016/b978-0-12-817909-3.00010-8
9. Fryszczyn, B. G.; Adamski, C. J.; Brown, N. G.; Rice, K.; Huang, W.; Palzkill, T. Role of β-Lactamase Residues in a Common Interface for Binding the Structurally Unrelated Inhibitory Proteins BLIP and BLIP-II. Protein Science 2014, 23 (9), 1235–1246. https://doi.org/10.1002/pro.2505
10. Brown, N. G.; Chow, D.-C.; Ruprecht, K. E.; Palzkill, T. Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class a β-Lactamases. Journal of Biological Chemistry 2013, 288 (24), 17156–17166. https://doi.org/10.1074/jbc.m113.463521
11. Adamski, C. J.; Palzkill, T. BLIP-II Employs Differential Hotspot Residues to Bind Structurally Similar Staphylococcus Aureus PBP2a and Class a β-Lactamases. Biochemistry 2017, 56 (8), 1075–1084. https://doi.org/10.1021/acs.biochem.6b00978
12. Ferrières, L.; Hémery, G.; Nham, T.; Guérout, A.-M.; Mazel, D.; Beloin, C.; Ghigo, J.-M. Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia Coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery. Journal of Bacteriology 2010, 192 (24), 6418–6427. https://doi.org/10.1128/jb.00621-10
13. Team:Orleans/Results - 2019.igem.org. https://2019.igem.org/Team:Orleans/Results (accessed 2025-10-04)
14. Part:BBa K125320 - parts.igem.org. https://parts.igem.org/Part:BBa_K125320 (accessed 2025-10-04)
15. Part:BBa J23103 - parts.igem.org. https://parts.igem.org/Part:BBa_J23103 (accessed 2025-10-04)
16. Part:pSB1K3 - parts.igem.org. https://parts.igem.org/Part:pSB1K3
17. Part:BBa B0034 - parts.igem.org. https://parts.igem.org/Part:BBa_B0034
Part:BBa B0030 - parts.igem.org. https://parts.igem.org/Part:BBa_B0030