Overview

Throughout this project, the Immunibee team remained committed to the principles of modularity and reproducibility. Consequently, we adhered to the Modular Cloning standard (MoClo). MoClo enables the straightforward assembly of various DNA building blocks, ultimately yielding a fully functional gene. This capability is made plausible through the use of Type II restriction enzymes, which cleave the DNA of each component in a manner that generates single-stranded DNA overhangs, complementary to the subsequent component within the composite part. ^{generating...}

Plasmids

The plasmids utilized in this project are:

• pAGM9121 ^{generating...}
• pICH47732 ^{generating...}
• pSEVA3b67Rb ^{generating...}

In constructing our parts, we utilized standard level 0 (pAGM9121) and level 1 (pICH47732) plasmids. Both of these plasmids are categorized as high-copy plasmids. All of our newly developed parts were integrated into the level 0 plasmid: these components contained specific MoClo flanks, which facilitated their effortless assembly and the formation of our composite parts within the level 1 plasmid.

Figure 1: pAGM9121 Plasmid

Figure 2: pICH47732 Plasmid

The level 0 plasmid, pAGM9121, is resistant to Spectinomycin. In contrast, the level 1 plasmid, pICH47732, is resistant to Ampicillin. ^{generating...} This distinction enabled us to consistently identify and select bacteria containing the desired plasmids. We must note that we conducted all of our construct assembly within E. coli.

Figure 3: pSEVA3b67Rb Plasmid

Since MoClo plasmids are not designed for use in B. Subtilis, once we wanted to export our final constructs into B.subtilis we used pSEVA3b67Rb. ^{generating...} This high-copy plasmid is a shuttle vector, making it compatible for utilization in E. coli and B. Subtilis. The plasmid contains resistance to the antibiotic Chloramphenicol.

Moreover, this plasmid contains the gene mRFP1, a red fluorescent protein from Discosoma sp. To integrate our constraints into this new plasmid we introduce an XbaI restriction site at the 5 prime end and a SpeI restriction site at the 3 prime end. This allowed us to insert our constructs precisely into the corresponding restriction sites within the pSEVA3b67Rb plasmid using the Golden Gate method.

Best Basic Part: CotB -> BBa_K4733008

For the 2023 iGEM competition, the 2023 UBC-Okanagan nominates CotB (BBa_K4733008) for the Best Basic Part award. Spore coat protein B (CotB) allows the display of heterologous and foreign proteins on the extracellular surface of live bacterial cells. This display system has various applications in a wide array of life sciences industries, such as pharmaceuticals, medicine, and biotechnology. Some relevant examples include the production of antibodies, enzymes, and vaccine delivery systems. ^{generating... generating... generating...} We retieved the nucleotide sequence for CotB from Uniprot which was sumbimitted by Prescan et al.. ^{generating...}

How does it works?

CotB is a protein found in the spore coat of Bacillus Subtilis cells. CotB contains an anchoring motif that allows it to attach to the cell’s outer membrane. CotB is 59 kDa in size and has a highly hydrophilic C-terminus formed by amino acid repeats containing, glutamine, lysine, and serine residues. ^{generating...}

It has been known for some time that when a protein of interest is fused to the C-terminus or N-terminus of B. Subtilis anchor protein (i.e. CotB, CotB, CotC, CotG, CotZ, etc), the fusion protein will be displayed on the surface of B. Subtilis spores. ^{generating...} However, Han et al. showed that a recombinant protein fused to CotB can also be exported to the outer membrane of vegetative B. Subtilis cells. ^{generating...} These researchers used this innovative display system to express on the surface of B. Subtilis cells the foreign fusion protein: immunodominant ovalbumin T-cell epitope fused with the cholera toxin B subunit. They fused this recombinant protein to the C-terminus of CotB and confirmed it was successfully exported to the extracellular membrane through flow cytometry, immunofluorescence microscopy, and western blotting.

Advantages and Disadvantages

The main advantage of CotB is its flexibility and ability to display foreign proteins on the outer membrane of B. subtilis. Additionally, CotB belongs to B. subtilis a not pathogenic species meaning it will not be harmful to hosts in case of use as a drug or vaccine delivery system. So much so that Bacillus subtilis 168 was modified to produce Natto (fermented soybeans). ^{generating...}

That being said, the heterologous proteins expressed on the surface of B. Subtilis have been shown to degrade significantly over time. Furthermore, Han et al. stated that the amount of heterologous protein expressed by this display system is not very high, and as such more work needs to be done to increase the yield. ^{generating...}

Usage and Community

How did we use it? How did we use it? The 2023 UBC-Okanagan team used this part for a vaccine delivery system. The system involves fusing CotB to a capsid protein, VP3, belonging to the Deformed Wing Virus. A linker was used to maintain the integrity of both of these proteins. To the lvl 0 CotB part, the team added the MoClo flanks 8 and 9 to the N and C terminus of the protein (refer to figure 4) so that during the assembly of parts, CotB would be on the C-terminus of the fusion protein. The idea behind this system is to have VP3 function as an antigen displayed on the outer surface of B. Subtilis, which will be ingested by a Bee: thus eliciting a favorable immune response in the bee, developing an immunity.

Figure 4: CotB construct

CotB’s sequence was modified by the 2023 UBC-Okanagan team so that it does not contain any illegal restriction type II sites (BbsI and BsaI) since we followed Modular Cloning (MoClo) standards to assemble our constructs. More information on how we built our constructs can be found in Engineering

How is it useful to the community? As stated earlier, CotB allows the display of recombinant proteins on the surface of live bacterial cells, which is extremely beneficial for the development of drug and enzyme production systems, other vaccine delivery systems, and even control of environmental pollution through its use as a bioremediation tool for adsorbing heavy metal ions. ^{generating...}

This part (BBa_K4733008) can help other iGEM teams take their innovative ideas to the next level by providing them a base for which their prototypes can be exported out and displayed in the outer surface layer of cells, which will enable them to better reach their desired targets.

Modeling

Using protein sequence and Alphafold we modeled the shape and structure of CotB and vizualized it using ChimeraX (Figure 5). Moreover, using Orientations of proteins in membranes database and software (OPM) by The University of Michigan we generated arough estimate of how CotB would look like on the surface of a gram positive bacteria.

Figure 5: CotB modeled

Figure 6: CotB modeled on the surface of a Gram-Positive bacteria

Similarly, using alphafold and other computational tools we modeled how our protein of interest (VP3) would look attached to CotB, as well as how the recombinant protein CotB-Linkers-VP3 would be expressed on the surface of a membrane. For more information please refer to (Modeling).

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Introducing DNAadapter: Streamlining MoClo Standardization and DNA Synthesis

DNAadapter is a tool we developed born out of the necessity to standardize parts for modular cloning (MoClo) and to simplify DNA design for synthesis. Created with the wetlab community in mind, DNAadapter is designed to reduce errors and streamline the construction and modularization of standard biological parts, making it the go-to tool for iGEMers and researchers in the field.

The Challenge

In our design stage we struggle dealing with MoClo standardization and DNA synthesis as it can not only time-consuming but also error-prone if multiple people are designing and typing by hand sequences. Last year and this year, we experienced firsthand how cumbersome and tedious it can be to navigate this process for each individual part.

The Solution: DNAadapter

DNAadapter was conceived to alleviate these challenges and provide a user-friendly interface that simplifies the MoClo standardization process. With DNAadapter, you can say goodbye to manual errors and time wasted on repetitive tasks.

Key Features

1. Standardization: DNAadapter allows you to standardize your biological parts with ease. The intuitive interface ensures that your parts are consistent and conform to the MoClo standard effortlessly. Just paste or type your DNA sequence and select from a range of standardized flanking sites depending of which type of part is it (e.g. promoter, CDS, terminator) or choose your own endings.

2. Error Reduction: By automating many of the processes involved in DNA synthesis and MoClo, DNAadapter significantly reduces the risk of human error, leading to more accurate results. Pre selected flanks are already uploaded and checked with the MoClo standard!

3. Streamlined Building: Our software streamlines the building and modularization of parts, eliminating the need for redundant manual labor already taking into account spacers and avoiding illegal sites, undesired ATG sequences, and giving you more time to focus on your research.

4. User-Friendly Interface: We understand the importance of a good user interface in a field were bionformatic tools are usually made with function as its priority which require great understanding of the molecular concepts. DNAadapter is designed with the end user in mind, providing a seamless experience for researchers of all skill levels providing guides and further information.

5. iGEM Integration: DNAadapter is tailored to the needs of iGEMers, providing a platform that aligns with the unique requirements of iGEM projects.

Join the DNAadapter Communit!

At Okanagan iGEM, we believe in collaboration and the power of the scientific community. Join us in our mission to standardize MoClo and the DNA synthesis workflow. We are committed to making iGEMers and academics research journey easier, faster, and more precise.

Feel free to get in contact with our developer team for further integration and improvement of our tool.