Contribution

Functionalized Bacterial Cellulose with Nisin

Bacterial cellulose is an adaptable biopolymer that is compostable, biocompatible, and food safe which makes it an attractive sustainable material. For this reason, our team decided to use BC as the foundational material for Cellucoat.

However, the bacterial cellulose packaging materials on the market currently are solely used for packaging. Cellucoat aims to utilize the properties of bacterial cellulose to be functionalized and provide our product with a unique competitive edge against current BC packaging. We have chosen Nisin as an antimicrobial agent to be integrated into our bacterial cellulose to develop a food packaging material with preservative properties.

Not only is our bacterial cellulose itself biodegradable, biocompatible but nisin is also a food safe protein. Nisin has previously been used as a preservative in canned foods. Nisin itself is temperature stable and therefore it is able to survive the high pressure and temperature steam sterilization technique to remove any bacteria from the BC material.

This preservative and sustainable packaging serves as an alternative to clam shell and other plastic packaging to prolong the shelf life of produce. We have run tests to determine the efficacy of bacterial cellulose with integrated nisin to determine how effective it is in killing bacteria.

Future teams and companies who look to functionalize cellulose with other additives or intend to use our cellulose integrated with nisin would be able to do so with this product contribution. To read more about functionalization of our bacterial cellulose, click here.

Functionalized Bacterial Cellulose with PHB

The iGEM competition is no stranger to bacterial cellulose, with hundreds of teams having used BC as a component of their project. However, BC as a material has a core problem: poor mechanical strength and water absorption. Bacterial cellulose, when less than 2 mm thick, is 500x weaker than plastic of the same thickness, and has a strength akin to that of paper. So our team has to consider that the resource-intensive process of BC is not warranted if it produces a material that absorbs as much water and has similar mechanical strengths as paper. After all, packaging has to undergo many mechanical stresses throughout the transportation journey and the added issue of the material absorbing moisture from the produce causing the packaging to lose shape is not ideal.

To improve the mechanical strength and decrease the hydrophilicity of BC, our team decided to look into additives to integrate into the material, similar to plasticizers. Our aim was to find a substance that could be added into the BC without compromising the compostability and would be homogeneously distributed throughout the material.

Our search focused on polyhydroxybutyrate (PHB) to combine with BC to create a more durable, thermostable, and water-resistant material. This is because PHB and BC properties each compensate for areas where the other material falls short to create a combined, or nanocomposite material, that has the properties ideal for producing packaging.

PHB’s limitations of low thermal stability, difficult processability, and hydrophobicity (1) are mitigated by combining with BC as a nanocomposite material. Extracting PHB is an expensive process because it is hydrophobic, so processing PHB in a co-culture and the material is secreted and integrated in-situ within the fibers, then the processing issue is eliminated. The hydrophobicity of PHB helps prevent the rehydration of BC once dry, making it reasonable to use as a packaging for fruits that require gas exchange. PHB and BC are combined as a nanocomposite material, PHB acquires greater thermal stability as compared to PHB alone, the decomposition temperature of a PHB and BC nanocomposite by 48.5 °C (1). Combining PHB with cellulose to enhance its properties also ensures it retains 100% biodegradability (1), thus preventing the need for additional treatments that prevent degradation.

Future teams that look towards using BC as a material that requires mechanical strength that is comparable to that of plastics can use Cellucoat’s co-culture model to create a PHB and BC nanocomposite material.To read more about functionalization of our bacterial cellulose, click here.

NisQ Part Collection

We contributed 1 new basic part and 3 composite parts for the iGEM registry in regards to the design and expression of NisQ. While previous iGEM teams have worked with nisin before, NisQ (BBa_K4437004) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000). We created NisQ tagged with a 6XHis tag on the N-terminus end for protein purification from E. coli. Double enterokinase cut sites are included to remove the 6X His tag to isolate NisQ alone. Our fusion protein with both N-utilizing substance A (NusA,BBa_K4015004) and a 6X His tag on the N-terminus of NisQ was created to help increase the production and solubility of NisQ in E. coli. Using the Xpress expression vector (BBa_K3945014), we also created a novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. Using both GST and NusA increased the expected band size from 7kDa (NisQ alone) to 91kDa, which was easier to visualize on SDS-PAGE gels, allowing us to successfully produce NisQ protein.

By creating unique biobricks for expression of NisQ in E. coli, we can provide teams with a choice of other variants of nisin beyond NisA and NisZ that have greater antioxidant properties. To read more about our parts, click here.

PHB Part Collection

For the purposes of Cellucoat, increasing phasin production and therefore increasing PHB production and secretion into the cellulose fibers helps to improve the properties of the final material. However, future teams may want to fine-tune PHB expression and secretion for their purposes. Hence, our team tested the effect of varying ribosome binding site (RBS) strengths in the phasin-HlyA insert. There were four RBS’s of various relative strengths selected from the Community RBS family, which are from relative strongest to weakest strength: BBa_B0035, BBa_B0034, and BBa_B0030, with our team’s part names being BBa_K4437504, BBa_K4437503, and BBa_K4437502, respectively. Another RBS that was non-functional was also used as a point of comparison called DeadRBS (2) which has been submitted as BBa_K4437500, derived from Kosuri’s et al (2013) research methods and the part name for the Phasin-HlyA insert is BBa_K4437502. The varying RBS gave the opportunity to fine-tune levels of phasin expression, and therefore PHB production and secretion.

By testing the effect of varying RBS strengths on phasin expression levels, and the result of this differing phasin expression on PHB secretion and production, we can improve the existing registry part by providing a choice for teams to fine tune PHB secretion and production by up or down regulating phasin expression.To read more about the improvement of PHB, click here.

BioSculpting: The Production of Bacterial Cellulose Cardboard

One of the key developments of the BioSculpting subproject was the creation of bacterial cellulose cardboard, a stronger, more flexible version of BC. The technique involved creating three sheets of BC, two to serve as the cardboard liners and one to serve as the corrugated middle section. These sheets were then layered on top of eachother and adhered together as they dried.

To create the shapes necessary, we developed a variety of molds, aiming to replicate corrugation standards used in industry. These molds allow teams to easily replicate the BC cardboard produced for our packaging, while also providing thorough specifications from which to create their own molds. By designing our molds to produce nets of our desired 3D shapes, we also allow for flexibility in the shape that the mold produces.

The molds are designed to be 3D-printable, allowing for teams to quickly procure and prototype new molds as necessary. This also helps reduce the barrier of entry for teams seeking to use BC cardboard in the future. The files for the molds can be found here.To read more about BioSculpting, click here.

Standardization of Kirby-Bauer Measurements: KB-Perry

Kirby-Bauer tests are popular tests for testing the effectiveness of a variety of antimicrobial peptides. However, measurement for the zone of inhibition can be crude at times, with the primary method involving taking a ruler and trying to measure as accurately as possible. This method is, naturally, inaccurate, and errors can be quite common. To mitigate these issues, we developed KB-Perry to help standardize these measurements using image analysis techniques.

We began by creating a platform to gather standardized images from. Using the measurements of the petri-dishes used in the lab, we created a modular, modifiable platform for our phone to rest on. This ensured consistency between our images, mitigating error from different heights and tilts of the camera and allowed for us to continue towards the image analysis project. Files for this platform can be found here.

Using Fiji ImageJ software, we were able to capture zone of inhibition measurements in pixels, which, when converted to mm, allowed for a significantly more accurate measurement than a ruler alone could provide. This proved to be a great boon for our Kirby-Bauer measurements, and all subsequent measurements were taken using this system. To read more about our measurement tool, click here.

Predicting the Antimicrobial Properties of Peptides Using Machine Learning: INN

This year, our team explored the use of involutional neural networks to predict the antimicrobial properties of peptides. Involution is a type of neural network that processes its input data in a spatial-specific manner, unlike the convolutional method that many networks for predicting AMPs currently do. We replicated Veltri et al.’s CNN-LSTM, and the resulting INN-LSTM is as accurate as a conventional CNN-LSTM for the prediction of AMPs, and can be used to predict the antimicrobial properties of any given FASTA sequence. We used our network to verify the antimicrobial properties of our nisin insert, and can use it in the future to make predictions about other inserts in order to modularize Cellucoat, in combination with Golden Gate. Other teams can easily use the tool to make predictions about the antimicrobial properties about fragments of interest by providing its FASTA sequence. As the training and testing code is available in the repository, teams can also experiment with unique training and testing datasets, or modify the way that the network processes its data to suit their needs. To read more about our INN click here.

SAMARA: The Software And Model Aggregating Research Assistant

Conducting background research is a crucial part of the iGEM competition, helping teams see what’s been done in literature and by previous iGEM teams to carve out a niche for their projects. However, while tools exist to help assist teams in parsing through dozens of papers, it is very difficult and time-consuming to search through the work of past iGEM teams. SAMARA was designed to help solve this problem. SAMARA is a complete web-scraper-to-deployment pipeline, extracting information, parsing it, categorizing it, and summarizing it for deployment.

Originally designed as an upgrade to 2018 iGEM Calgary’s SARA, it quickly became a complete rework and redesign, developed from the ground up with modularity and customization at the forefront. Because of this design philosophy, SAMARA became a project of two parts.

First is a Scrapy web scraper, capable of automatically following links and extracting data based on the URL. This information can then be filtered, parsed, and processed according to the needs of the user, with ample documentation available to allow for fully customizable, purpose-built scraping without reinventing the wheel. Using the sshleifer distilbart cnn 12-6 model, we have incorporated summarization techniques to our processing pipeline to further refine the information extracted.

The second is a Django web-based deployment, allowing for seamless integration with the web scraper and letting users access the information from anywhere. This also offers the possibility of local deployments, letting teams utilize the tool on a more user-friendly interface without needing to push for a full-fledged deployment system. To read more about SAMARA, click here.

References

1. Ibrahim MI, Alsafadi D, Alamry KA, Hussein MA. Properties and applications of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biocomposites. Journal of Polymers and the Environment. 2020;29(4):1010–30.
2. Kosuri S, Goodman DB, Cambray G, Mutalik VK, Gao Y, Arkin AP, et al. Composability of Regulatory Sequences Controlling Transcription and translation in Escherichia coli. Proceedings of the National Academy of Sciences. 2013;110(34):14024–9.