D e s i g n

AIDA-I Autotransporter System

Our project aims to optimize the expression of tyrosinase at the membrane level. Our main objective is to enable our bacteria to produce and present melanin on their surface. To achieve this, we are focusing on optimizing the expression of tyrosinase, the key enzyme that catalyzes the production of melanin from tyrosine.

Our strategy revolves around the autotransporter AIDA-I (Adhesin Involved in Diffuse Adherence), a type V secretion system. AIDA-I is expressed in certain pathogenic strains of Escherichia coli. It allowed the bacteria to attach to the host's epithelial cells, leading to virulence. Its modular structure makes it an ideal vector for our application. [1]

The main domains of AIDA-I are:

• Passenger domain: The adhesion domain, the functional part
• Autochaperone: C-terminal of the passenger domain, aids in the folding of the protein and increases the efficiency of transport through the outer membrane
• Linker: Region between the C-terminal domain of passenger domain and the beginning of the β barrel domain. It allows the passenger domain to pass through the β barrel domain by providing flexibility to the protein
• Β-barrel domain/translocator domain: Composed of 12 β strands in the outer membrane. Forms the canal and ensures anchoring to the outer membrane, forming a pore [2]

Figure 1: Structure of the AIDA-I autotransporter system

Plasmid Design

The strategy is to replace the adhesion domain of AIDA-I with tyrosinase. This will not affect the functioning of AIDA-I neither the bacteria viability, as this surface display approach allows the enzyme to be located outside the cell where its action is required without affecting bacterial viability through the production of large amounts of melanin.

For this purpose we based our design on "Molecular optimization of autotransporter-based tyrosinase surface display" [3]and we chose the pAIDA plasmid from Addgene. This allowed us to avoid overly complex and time-consuming cloning.

The plasmid pAIDA already contains all the elements for expression, translocation and anchoring on the outer membrane such as:

• pLacUV5: A strong promoter inducible with IPTG
• Signal peptide: To guide the protein to the periplasm
• Linker: To help the protein fold correctly

We chose this design mainly because of the two tags, in order to control the expression and integrity of our fusion protein. The His tag in N-terminal would be for the detection of the complete tyrosinase with no degradation while the Myc tag in C-terminal is for the detection of any protein whether they are degraded or not. Thus, these tags provide us with multiple choices of tests.

Figure 2: Design of pAIDA-Tyr1 plasmid

Toxin/Antitoxin System CcdB/CcdA

For safety reasons and to prevent any unwanted transfer of our pAIDA-Tyr1 plasmid to other bacteria present in the environment we decided to add a biological containment system, the toxin/anti-toxin system.

We added the gene encoding the CcdB toxin to our pAIDA-Tyr1 plasmid. CcdB, the toxin targets the DNA gyrase, a topoisomerase. CcdB binds to the gyrase while it is bound to the cleaved DNA, as a result the replication is blocked.

The antitoxin CcdA neutralizes CcdB by forming a stoichiometric complex (CcdA)2-(CcdB)2, thus the toxin is unable to interact with the gyrase. The CcdA antitoxin neutralizes CcdB by forming a stoichiometric complex (CcdA)2-(CcdB)2, thereby preventing the toxin from interacting with gyrase. [4]

The toxin will be added to our plasmid pAIDA-Try1, its expression is controlled by the pLAC promoter and the B1006 terminator. While the antitoxin CcdA would be introduced into the genome of our E. coli W3110 ΔompT by using λ Red recombination technique.

Thus if bacterial conjugation occurs, the plasmid should be lethal for any bacterium lacking the gene CcdA unlike our strain and the CcdB toxin would be expressed and kill the cell. Consequently, we avoid any unwanted melanin production.

Figure 3: Toxin/antitoxin system CcdB/CcdA for biological containment

Auxotrophic Strain Design

To control the proliferation of our bacteria, we designed an auxotrophic strain, i.e., one that is dependent on a nutrient that it cannot synthesize itself. We made our bacteria dependent on DL-glutamate, an essential component for the formation of peptidoglycan, which is essential for the cell wall.

By invalidating the murI gene that codes for glutamate racemase, we have created a strain that is unable to produce this vital metabolite on its own.

In order to test the auxotrophy of our strains with DL-glutamate, we had to design a complementation plasmid with a copy of the functional murI gene. Without MurI, our auxotrophic bacteria become dependent on our hydrogel which will provide them with DL-Glutamate.

Golden Gate Assembly

To carry out the complementation plasmid, we used the Golden Gate method as well as the bricks provided and the plasmid in the 2025 kit. This synthetic biology technique is based on the use of type IIS restriction enzymes, making it possible to cut and assemble several DNA fragments in a single tube, in a single cycle, and especially without leaving any "scars" sequences. This cloning technique as well as the kit provided by iGEM allowed us to make the process simpler and faster.

For robust and constitutive expression of the murI gene, we opted for a combination of well-characterized biological parts:

• Promoter: BBa_J23119 (a strong constitutive promoter from the iGEM collection)
• RBS: BBa_J428038 (an effective ribosomal binding site for optimizing translation)
• Gene: murI (the gene encoding glutamate racemase)
• Terminator: BBa_J428092 (ensuring correct termination of transcription)

This genetic circuit was assembled in the BBa_J428341 vector using the Golden Gate method, a fast and efficient technique provided by the iGEM 2025 kit. This methodological choice allowed us to construct the plasmid in a standardized and reliable manner.

Figure 4: Design of the complementation plasmid pMurI assembled by Golden Gate method