iGEM 2022 — Team calgary

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## Notebook

[Wetlab](#header1)
[Drylab](#header2)
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## Wet Lab

**Week 1**

## Whole team

Wetlab members had a tour of the lab and learned basic synthetic biology lab techniques such as minipreps, aseptic technique, transformations, overnights, viewing gels, and how to troubleshoot experiments. Throughout this week, the team brainstormed what project they wanted to go forward into the iGEM season with, and Cellucoat was the one that the team voted for.

**Week 2**

## Co-Culture

We presented a wet-lab subproject presentation to the team in which literature reviews presented G.hansenii to be a better bacterial cellulose producer in terms of the fruit waste media we hope to develop. However, we wanted to continue to use K.xylinus as it is still a moderately great bacterial cellulose producer at a pH of 5 and was readily available for us as our RA had glycerol stocks present in his lab.
We began to evaluate the major differences between G.hansenii and K.xylinus. The assessment of these key differences played a critical role in making alterations when developing the co-culture protocol which was based on a paper that successfully developed a co-culture between G.hansenii and recombinant E.coli. Furthermore, the initial co-culture protocol was made along with several other protocols including HS media preparation protocol, HS-agar plate protocol, streak/spread plate protocols and serial dilution protocols.
Our team began to set up preliminary experiments to test whether G. xylinus and E. coli would be compatible to grow together. We made LB plates of G. xylinus, collected from Dr. Hu’s lab at the Schulich School of Engineering. One streak plate was made using contaminated media, four spread plates were made using the contaminated stock HS media. We also prepared several HS-agar plates to grow our bacteria in the following week. These plates were labeled and spread plates were made using contaminated HS media. Two standing culture tubes were also made using freshly made HS media.

## Fruit Waste Media

We explored potential fruits to use, and decided to narrow the focus of the proof of concept for this subproject to specifically oranges. This would effectively demonstrate the breakdown of orange pulp and peels. As such, we decided to incorporate cellulase and pectinase in the pre-treatment process to maximize the yield of extracted sugars from the fruit waste. This contributes to the overall goal of this subproject to reduce the cost of the culture conditions. We researched and adapted procedures to experimentally extract sugars from oranges.
In addition, we discussed potential quantification methods to measure the amount of glucose (and other sugars) in the orange extracts to accurately match the glucose concentrations outlined in the conventional HS media.
Finally, we discussed avenues for designing a fermentation strategy to break down orange peels.

## Nisin

We characterized the mechanism of action of nisin towards gram positive bacteria and fungi. We explored the mechanism of action of nisin against gram negative bacteria and the potential implications of recombinantly synthesizing nisin in a gram negative bacterium (E.coli). However, the team has decided to continue with developing a method to recombiantly express nisin in E.coli as there are slight structural differences between Lipid II of the peptidoglycan which nisin binds to between gram positive and negative bacteria, indicating that the efficacy of nisin to limit peptidoglycan synthesis and therefore harm the peptide production chassis is decreased as compared to the chance that nisin was recombinantly expressed in a gram positive bacteria. The team also explored the structural properties of nisin and its derivatives. The findings were promising as it indicated that autoclaving at 121 degrees celsius at a pH of 3 is a viable method of sterilizing the BC to prepare it for commercial use. This is because as the pH decreases, nisin displays increased thermostability, allowing it to maintain functionality at higher temperatures. Hence, the team had to pivot from sterilizing the BC with a base wash to autoclaving to preserve the function of the BC integrated antimicrobial.
We developed preliminary procedures for testing the antimicrobial activity of nisin and designed various nisin constructs with different plasmids on Benchling. Using HP feedback, we have also confirmed that we not only should use a signal peptide, we must use a signal peptide. The signal peptide supported by the literature that the production and purification nisin experiment is based on uses a larger signal peptide, NusA, which is what was anticipated to be used for transporting the nisin precursor into the periplasm. However, Dr. Burkinshaw recommended that we integrate a well characterized signal peptide known to be used within E.coli, such as NusA or MalE, to the N-Terminus of our construct to produce a peptide that is both the nisin precursor and the signal peptide. Dr. Burkinshaw indicated that this would be the most efficient choice to move forward with because once the signal peptide delivers the precursor nisin to the periplasm, the E.coli cell has proteases that cleave the signal peptide off of the nisin peptide, creating the mature nisin product.

## Detection

We narrowed down different detection systems that could work for either use ((i) detection of antimicrobial peptide activity or (ii) detection of pathogens. We also investigated sensor systems that involved GFP and some that did not (ex: colourimetric, pH). As a team we decided that the detection system will be revisited once we receive more feedback from HP.

**Week 3**

## Co-Culture

For this entire week, the coculture subteam worked on a set of goals to accomplish this week to determine through research and develop a set of protocols to improve the production/efficiency of K.Xylinus. We first did some research into the best way to undergo a coculture experimental workflow, and a three-step plan was recommended, where the first step includes having a monoculture of K.Xylinus and E.coli to determine individually for each microbe what are the optimal growth conditions. The second step is to take the media from the monocultures and switch them so the impacts of the extracellular secretions of E.coli and K.Xylinus on each other, and how it may positively or negatively impact the yield of BC and the eventual desired antimicrobial peptide. Lastly, the third step is to place K.Xylinus and E.coli together in a coculture. These three steps are the backbone of what the wetlab coculture workflow is based upon, with a step between each 1-2-3 experiments to determine the impact of each condition on the BC yield and cell growth.
Protocols for the 1-2-3 experiments, cell counting, and determining BC yield were developed and placed into a master document outlining the wet lab coculture workflow. Once the wetlab coculture workflow was completed, the second half of the co culture subteam goal was to develop an experimental plan on how to simulate the integration of nisin, the antimicrobial peptide, with GFP in our BC. GFP was used because its distribution within the BC product can be observed using fluorescent microscopy without hindering the structural integrity of the BC product. These sets of protocols were known as the GFP co culture workflow, where sets of experiments on how to transform E.coli to express GFP which would be used for two procedures to determine the best way to integrate GFP into the BC. The first method would be to integrate the GFP-expressing E.coli into the co culture for the lysis procedure where E.coli would be integrated into the membrane of the BC and would be lysed during sterilization thus releasing its contents into the BC membrane. The alternative procedure is to dehydrate the BC product and rehydrate it with GFP-enriched solution, allowing the GFP to saturate the BC.
Lab work included beginning the intermittent feeding experiments and developing a method to streak and get single K.Xylinus colonies to use for the monoculture and co-culture.

## Fruit Waste Media

This week, we finalized our procedures and developed a detailed workflow for the enzymatic hydrolysis and processing of various orange components. We included pectinase into our procedure, and decided on the testing method for the fruit waste media; a BC seed will be incubated with the FWM from various components of the oranges, and with the HS media for the control. We removed antibiotics from our procedure and worked on matching optimal enzyme temperatures to streamline the hydrolysis process. We explored different methods for recombinantly producing cellulase, but ran into obstacles with identifying a specific type of cellulase to focus on. We also briefly discussed the logistics procuring the oranges we planned to use in our experiments.

## Nisin

We decided that instead of solely producing nisin alongside nusA in E. coli, we would create a library of various AFPs and signal peptides that we could test. To create a module design, we reconstructed our nisin sequence to be compatible with the RFP flipper device using Golden Gate assembly.
After researching immobilization methods of peptides onto bacterial cellulose, we realized that our nisin peptide may be too small to design with a cellulose binding domain. We looked into alternative methods of immobilization, specifically chemical methods, with the goal of retaining bacterial cellulose’s mechanical properties (water retention and flexibility) that are required for Cellucoat’s application as a fruit coating. Our goal is to test a dehydration/rehydration method using GFP to visually assess the success of immobilizing a protein without chemical modification, and compare the results to a literature-established chemical immobilization method.
We wrote a protocol for growing fungi and are in touch with Dr. Addy and Fran who will support us with growing fungi. In addition, we spoke with a grocery store and will be visiting next week to get fruit samples.
We wrote a protocol on characterizing the antimicrobial activity of nisin through an agar disc assay, which can be applied to both bacteria and fungi. Furthermore, we wrote protocols on determining the stability of nisin, particularly through the temperature and pH.

## Detection

During the meeting with Dr. Stietz, it was indicated that she had not had much success with using a split GFP detection system. She believed we may face the same difficulties because tagging massive cellulose with GFP will be difficult and the GFP may get lost as it is a small molecule. Furthermore, we learned that for application on the fruit, lysing may not be the most successful method without an additional step, due to the presence of endotoxins and other debris. Dr. Stientz also suggested that since BC production has already been established, that we should focus on methods of quantification. In relation to the detector system, she suggested that the sensor system should be targeted towards detecting pathogens as a way of tracking the functionality of the antimicrobial peptide.

**Week 4**

## Co-Culture

The coculture team conducted research on additional properties of BC, and if a symbiotic relationship between K.Xylinus and E.coli were possible. What the research yielded was that there is an additional molecule, acetan, that enriches K.Xylinus BC and gives it additional antioxidant properties and gives the BC additional crystallinity. This also goes to show that K.Xylinus is the best BC-producing bacteria for industrial purposes because of the ability of K.Xylinus to produce acetan as a product of its metabolism, which also has the effect of increasing both cell count and BC yield as well as decreasing the adhesion of cells onto the BC. A synbio symbiotic relationship between K.Xylinus and E.coli is possible according to literature, but not feasible with the time and resources at hand.
The BC from both the 24 and 48 hr demonstrated that the intermittent feeding did result in homogenous layers being formed. These BC were put into the autoclave to be sterilized and were brought back for experimentation. Then, we used two different types of soaps (dawn dish soap and tween soap), NaOH base wash, and plain water to determine the best method to purify the BC to get rid of the unpleasant smell and color. Next week we will develop a protocol to determine the level of purification of each method, as well as determining the best method to dry the BC.
Experimentation surrounding the 3-step co-culture protocol has been a bit slower than expected because the first step, developing a K.Xylinus and GFP expressing E.coli monocultures, have not been yielding successful cultures. Hence, we took two approaches to solve the problem. The first was to increase the air-to-media surface area available to increase growth of both cultures by growing the standing cultures in a petri-dish instead of a tube. The next was to change the components in the HS-media used and to add more tryptone, an amino acid source, to encourage E.coli growth.

## Fruit Waste Media

This week, an order list for missing materials for this workflow was prepared. The procedures were revised, and plans to begin experimental work next week were made. Order list was sent to Tian, going to check with Deirdre. Oranges and juicer were acquired and prepped for experiments starting the next week.

## Nisin

This week we began testing the RFP flipper device from the 2019 iGEM team’s glycerol stock. Both the “improved flipper” and “old flipper” bacterial stocks showed growth on chloramphenicol resistance plates. We made overnight cultures for both, and isolated the plasmid DNA via miniprep.
We continued our research on finding an effective method for immobilizing nisin onto our BC membrane. Our HP meeting with Dr. Hu from the University of Calgary informed us of our decision to go ahead with a rehydration/dehydration method, however, we were made aware of the potential for nisin to get “lost” within the BC, and may need to explore other methods.
To understand the microbes and fungi common on fruit from grocery stores, PDA media and plates were prepared, and the fruits donated from a local grocery store (in various stages of rot) were swabbed and plated. Although dry cotton swabs were used, plans were made to improve the sampling procedure in the coming week.

## Detection

No updates.

**Week 5**

## Co-Culture

At the beginning of the week, we took a look at the cultures that have been growing over the weekend. From the step 2 E.coli monoculture grown in conditioned media, it was evident that there was biofilm growth, and under the UV light there was a slight green indicating that there was GFP expressing E.coli embedded within the biofilm. We then added ampicillin to determine what was the identity of the biofilm, as it could be an E.coli biofilm, E.coli and K.Xylinus biofilm, K.Xylinus biofilm, or something else entirely due to contamination. Adding the antibiotic caused a brown clump of cells to form within the biofilm and no green glow to emit from the biofilm when placed under UV. Hence, the step 2 conditioned media experiment was restarted and new conditioned media was made through centrifugation and filtration.
We also created a new HS media using the addition of tryptone, an amino acid source, in hopes of promoting E.coli growth. K.Xylinus grew, but E.coli did not. E.coli could, however, grow in unmodified HS media. Lastly, the drying protocol was attempted on the NaOH washed BC, which resulted in a less than paper thin BC product that was stuck on the tin foil and ripped.

## Fruit Waste Media

This week marked the kick-off of experiments for the fruit waste media. We peeled oranges: the peels were processed in the food processor, and juiced the oranges. The peels and pulp were laid out to air dry as we waited for our oven to arrive. The juice was initially gravity filtered with a coffee filter, and residual juice and foam was collected (unfiltered). Next, the juice was passed through a vacuum filter. A portion of the filtered juice was passed through the filter again. Enzymatic hydrolysis was performed on all three of these samples. The peels were ground and filtered, and enzymatic hydrolysis was performed. Finally, the pulp was dried in the oven, then ground, filtered, and enzymatically hydrolysed.

## Nisin

We started the week off by checking on the plates swabbed from fruit for fungal growth, there was quite a bit. Later, we visited Fran who we showed our plates to as well as received pure cultures of fungi. We made yeast dextrose peptone broth for the fungi to grow on and created fresh plates to isolate cultures of fungi from the fruit into pure cultures.
Several attempts were made to digest our improved and old RFP flipper samples containing the RFP flipper device in a PSB1C3 backbone. Our goal was to confirm whether the minipreps prepared last week from the 2019 team’s glycerol stock solutions were indeed the RFP flipper device. We ran our digest using the BsaI restriction enzyme cut sites to determine whether we could successfully use the samples for eventual golden gate assembly with our nisin G-block. We had difficulty visualising the correct DNA band sizes on our gels, and had to make multiple procedural changes including using a different RedSafe dye and using a different thermocycler. Eventually we were able to visualise bands that corresponded to the RFP flipper device size, but the bands were very faint even though we had used 1uL of DNA for both the improved flipper (5526.8 ng/uL) and the old flipper (3443.3 ng/uL) in our digests. As well, we noticed that there was a large amount of DNA at approximately 10,000 bp in every successful well of the gel, indicating that our samples may not be as pure as the Nanodrop had indicated.

## PHB

In order to understand the industrial viability and need for our solution, we met with Christopher Clark. We learned that any container produced with BC needs to be able to maintain its structure and withstand the force of other packages stacked on top of it. Based on our experience with the BC we had in the lab, we decided to explore some ways to reinforce the BC.

**Week 6**

## Co-Culture

A coculture with K.Xylinus and E.coli expressing GFP was successful, yielding a BC material that fluoresces under a UV light. When the material was autoclaved, the fluorescence got brighter as it got further from the center of the BC disk. This experiment provided the initial confirmation that allowed us to move forward with coculture experimentation, as now we know that that coculture works in our application.

## Fruit Waste Media

The order list for the FWM subproject was finally finalized and ordered this week. The order list items are needed to continue experiments. The order list needed to be updated to match the new and simpler DNS Assay procedure we will be using, that protocol was finalized this week as well. Other items that will arrive are enzymes needed to further treat the oranges.

## Nisin

The order list for the nisin subproject was finally finalised and ordered this week. No other major updates occurred for this week.

## PHB

We explored possible strengthening approaches, and decided to further research the addition of PHB, a bioplastic. PHB had previously been integrated into BC in a co-culture, and this compatibility with our existing work with co-culturing was encouraging. We reviewed literature to understand more about the PHB production process, and started to consider how we would go about including it in our BC. Moreover, Dr. Mayi informed us that the 2017 iGEM Calgary team had done extensive work with PHB for their project. We started looking into their work, and researched which other iGEM teams had produced PHB. This research provided preliminary protocols for future PHB expression, and also inspired us to potentially improve PHB-producing parts to express higher levels of PHB with the hope of further strengthening our BC.

**Week 7**

## Co-Culture

Based off of previous experiments to test if what the literature indicated would be applicable in the lab were successful and rendering results that aligned with existing literature, the coculture team went forward with qualitatively and quantitatively recording results for the Step 1, 2, and 3 to establishing the best conditions for growing E.coli and K.Xylinus and if a co culture between these two bacterial pieces would be possible. This week, steps 1 and 2 were performed and step 2 has now been completed.
The intermediate feeding experiment has also been started and completed this week, demonstrating results that are imperative to applying this project on an industrial scale. The results indicate that if we were to want a thicker single palette, then we would have to go with the 24 hour intermittent feeding, while if we wanted multiple discrete palettes then we would have to go with the 48 hour intermittent feeding schedule.
Lastly, because BC becomes slightly more brittle than anticipated when dry, the coculture team has begun investigating ways to improve the elasticity of the material through adding bioplastics such as PHB-co-HV from the 2017 iGEM Calgary team. The plan is to recombinantly express and secret this bioplastic in the coculture by improving upon the plasmid design from the 2017 iGEM Calgary team.

## Fruit Waste Media

This week, we carried on with the orange peel hydrolysis upon the arrival of pectinase. Moreover, we ran preliminary tests to evaluate the potential for both K. xylinus and E. coli to grow within our modified media, made with the extracts hydrolysed with cellulase only. The results indicated that both the E. coli successfully grew, and more importantly, the co-culture K. xylinus successfully grew and produced BC. This is promising for the potential of the FWM to be incorporated in our co-culture subproject. Invertase also arrived this week, meaning the full enzymatic treatment to the orange components can be done. The workflow for FWM experiments was fleshed out and 5 new oranges will be bought this weekend to begin processing next week for a new round of processing.

## Nisin

This week we began to conduct preliminary experiments for the effect of various antibiotics at different concentrations against E.coli. The goal of these experiments was to generate data in order to compare the effectiveness of nisin relative to antibiotic strength. We ran a Kirby-Bauer disc diffusion test for 5 different antibiotics that we found in our lab: ampicillin, kanamycin, chloramphenicol, tetracycline, and erythromycin. We scaled the concentrations of each antibiotic based on the typical volume used per 1 LB plate, and ran tests at 25%, 50%, and 75% of the maximum volume, per quarter of a plate. After discussion with our advisors, we realized that we needed to rerun the experiment with the same concentrations for each antibiotic, in order to compare the effectiveness between groups (rather than solely within groups). Our plan moving forward is to run Kirby-Bauer disc diffusion tests at a constant concentration for each antibiotic against E.coli, and once our nisin solution arrives, run the same test with the same concentration for nisin against E.coli. To test the effectiveness of nisin against different fungi, we will use the same antibiotics as well as thymol as a negative control.
We also ran PCR experiments on several stock solutions from the 2019 iGEM Calgary team that may be compatible with our RFP Flipper device. Of the parts we tested, our gel results showed bands for several plasmid backbones containing signal peptides, including pSB1A3-MalE, pSB1C3-TorA, and pSB1C3-PhoA. We may be able to use these parts to test our Golden Gate assembly for when we want to express nisin using the same methodology.

## PHB

We focused on designing our primers and part for PHB this week. First, using the Calgary 2017 team’s phasin and secretion system part (BBa\_K934001) as a basis, we added a FLAG tag upstream of the Hly-A coding region. Our research assistant, Seb, and our teaching assistant, Tian, emphasised the importance of being able to purify and later quantify the amounts of phasin produced. Next, with the help of Seb and Tian, we designed a set of primers designed to amplify this part and produce versions with varying RBS types. Rather than ordering four versions of each part, we designed the following types of primers: a forward primer, with a unique restriction enzyme site present in the Tokyo 2012 PHB plasmid and one of four RBS types; and a reverse primer, with another unique restriction enzyme site. We made versions with a non-functional RBS as a negative control (called DeadRBS), as well as with B0030, B0034, and B0035, which have varying strengths and are present in the iGEM registry. We made two copies of the forward and reverse primer sets: one with SpeI and PstI sites, and one with BstBI and BstAPI sites. As such, if one of the sets proved problematic when we later ligated our phasin part into the PHB plasmid, the other could be used as an alternative. Given the timeframe of our project, this would also allow us to continue without restarting entirely.

**Week 8**

## Co-Culture

New intermittent feeding was started this week, however it was not continued as data collected was sufficient enough. Monoculture was also started in order to develop a growth curve. These monocultures were contaminated with E.coli and therefore will need to be restarted next week.

## Fruit Waste Media

5 new oranges were bought this week to begin processing. All enzymes are now accounted for in the lab and so 2 sets of 24 hour incubations could be performed on all parts of the oranges including the 3 varieties of orange juice, the pulp, and the peel. These 2 sets consisted of cellulase and pectinase in round 1 and invertase in round 2. Controls were also made for no enzymatic activity and just cellulase and pectinase activity. While the CP treatments were incubated in the shaking incubator due to its limitations a static water bath was used for the invertase treatment. All these samples were afterwards freezed to later be assayed when all DNS reagents arrive and to begin media replacement experiments.

## Nisin

A mini-prep was run on the overnight culture made from the 2019 golden gate assembly signal peptides. We ran into some trouble with our concentration values as they were low, however, the A260/A280 ratio showed the plasmid was pure. The kirby bauer test was re-ran using new calculations with two different concentrations of 50mg/uL and 50ug/uL to see the effect of each concentration. Upon analysis of the results, it was determined the 50ug/uL worked the best as it was the concentration which gave us the best measurable results. The zones of inhibition were calculated for all of them and the results showed ampicillin to be the weakest antibiotic and tetracycline to be the strongest antibiotic.

## PHB

We continued to finalise our primer designs, and checked the important specifications (including melting temperature compatibility, GC content, and to ensure no primer dimers would form) with Tian and Seb. We also designed a method to insert a his-tag upstream of the PhaB gene. This would produce a his-tagged β-ketothiolase, which is a key enzyme in the PHB biosynthetic pathway. Measuring Β-ketothiolase would allow us to compare phasin levels (with FLAG tag) to PHB levels, and quantify the effect of increased phasin expression on PHB yield. In this insertion approach, the primer was designed with a his-tag sequence in the middle, flanked by two sequences that anneal upstream of PhaB. During PCR, the his-tag insert would anneal, and the his-tag would be included in the PCR product and amplified.

**Week 9**

## Co-Culture

Samples of BC were prepared for the drying and purifying experiments throughout the week, and before the weekend the samples were autoclaved and then placed into their respective buffer solutions to purify over the weekend on the rotator at mode 2. BC seeds for the co culture were also planted in preparation for monday.

## Fruit Waste Media

No further updates for this week.

## Nisin

We conducted a PCR on the 2019 signal peptide constructs and ran the pcr product on a gel. Multiple bands were observed, which was not expected. The plasmid maps may indicate that there was an issue with the primers used.
After a successful PCR amplification of our Gblock 1 (GB1) and Gblock 2 (GB2), we performed a digestion using EcoRI and XbaI and ligation of both inserts into the successfully miniprepped “pSB1A3-dummy” (henceforth “pSB1A3”) sample from the 2019 glycerol stock. We transformed GB1+pSB1A3 and GB2+pSB1A3 into Top 10 E. coli cells. We also transformed our pSB1C3 (Part:BBa\_J04450) plasmid into Top 10 E. coli cells. All samples yielded results with sufficient DNA concentrations and purity ratios. We then transformed both C3037 and Top 10 cells (from our 2021 stock) with our isolated plasmids to see if there was a difference between the chemically competent cell type and the growth of our colonies after plating. We performed another set of minipreps, and unfortunately our DNA concentrations were extremely low, so we re-transformed our previous ligation products for GB1-pSB1A3 and GB2-pSB1A3 into newly purchased Top 10 cells. In addition to these set of experiments, we also began running our third attempt (see last weeks summary) of inserting nisin into a plasmid, by amplifying both GB1 and GB2 with new primers containing EcoRI and PstI for subsequent digestion and ligation into linearized pSB1C3 provided by the iGEM registry.
To characterize nisin’s properties, we ran a Kirby-Bauer disc diffusion test with various concentrations (stock = 20,000 to 40,000 IU/mL, 1:10 dilution, and 1:20 dilution) against Bacillus subtilis and the bacteria swabbed off of an eggplant and nectarine from our local grocery store. After talking to Josh McGinnis, a bioprospector who studies novel fungi strains, we discovered that the bacteria on the eggplant and nectarine were likely Bacillus strains, which is why using B. subtilis was a comparable choice for our experiments. We used thymol - an antibacterial agent - as our comparison to nisin, and phenol - a standard control for antimicrobial tests - as our positive control.

## PHB

We finalised and ordered our his-tag insert, and organised our workflow for the remainder of the summer. We also planned and prepared the reagents for the transformations planned for next week.

**Week 10**

## Co-Culture

Coculture has come to its conclusion with the finishing of the step 1 and step 3 experiments. However, observations and experiments surrounding the properties of BC and how we can better apply it to a commercial setting will be observed using the techniques and strategies established via experimentation.

## Fruit Waste Media

This week we conducted the BC growth experiment. We tested different ratios of fruit waste media with HS to determine the rate of BC growth with different concentrations. We will be conducting experiments on the impact of different ratios of fruit waste in the media on E.coli growth in the coming weeks.

## Nisin

PCR was conducted for the 2019 signal peptide constructs however it showed multiple bands and was re-done. Another miniprep was done on the 20189 signal peptide constructs as supplies were low. The first miniprep failed, the second miniprep was successful. A digest was conducted (not run on a gel).
The re-transformed ligation products of GB1-pSB1A3, GB2-pSB1A3, as well as pSB1C3/RFP were miniprepped. We had two samples in the pSB1A3 backbone with GB2 which had sufficient nanodrop results. A diagnostic digest was done on pSB1A3+GB1 and pSB1A3+GB2 with EcoRI/XbaI and EcoRI/NotI. Based on the gel results we only saw two bands in lane 8 and 9 which contained Top 10 cells - GB2+pSB1A3 (1) digested with EcoRI/XBaI and EcoRI/NotI, respectively. There were also faint bands in lane 10 and 11 which contained Top 10 cells - GB2+pSB1A3 (3) digested with EcoRI/XBaI and EcoRI/NotI, respectively. NotI was used because we believed the backbone (“pSB1A3 dummy”) may have had extra base pairs. These two ligation samples were sent for sequencing. For the pSB1C3 backbone we had 2 good nanodrop results and these were digested and ligated with GB1 and GB2. A bacterial transformation was done with these ligated products and miniprep. The minipreps were not successful. Another attempt was made by using GB4 and new primers with EcoRI and PstI. After a PCR was done we put GB4 into a linearized and transformed it. However, the transformation failed.
Characterization of Nisin was on hold this week.

## PHB

The PHB g-block and primers arrived this week. We started our wetlab experiments for PHB by locating the PHB-producing part (BBa\_K934001) designed by the Tokyo 2012 team in our collection of iGEM distribution kits. Using the distribution kit from 2019, we rehydrated the BBa\_K934001 part and transformed it into TOP10 Oneshot chemically competent cells. The transformations were then miniprepped, which yielded low DNA concentrations. The transformations will be re-done next week. Furthermore, the positive control primers (B0034; these are the same RBS as in the original Calgary 2017 BBa\_K934001 part) were used and yielded bright bands of the anticipated size from the PCR products. Based on this success, the remaining amplification of the phasin G-block with the remaining primers will be attempted using the same touchdown PCR program in the coming week.

**Week 11**

## Co-Culture

Experimentation on dying BC in and ex vivo has begun using cabbage juice after it has been boiled. BC will be grown over the weekend using HS media with cabbage juice as a substitute for distilled water.

## Fruit Waste Media

Following last week’s growth experiments, the BC was purified and prepared for uniaxial testing.

## Nisin

We started the MIC protocol by making MH media plates and Bacillus Subtilis culture tubes. A PCR reaction was conducted on the 2019 signal peptide constructs. The PCR products were run on a gel; many lanes contained no DNA, and some lanes contained unexpected banding. Due to this, the PCR product was not purified. For our nisin gblocks, we successfully transformed E.coli Top 10 cells with GB1+pSB1A3 and GB2+pSB1A3. We then successfully miniprepped the samples and ran a diagnostic gel which indicated banding at the expected size for linearized GB1+pSB1A3 and GB2+pSB1A3. Once our samples are confirmed with sequencing we can move onto protein expression.

## PHB

We amplified the remaining primers with the phasin G-block via touchdown PCR, and confirmed that the PCR was successful with a diagnostic gel. As a result, we now have eight phasin parts, with each of the four RBS types (one full set of SpeI/PstI, and one full set of BstAPI/BstBI). We also re-transformed the Tokyo plasmid into TOP10 Oneshot chemically competent cells and miniprepped the samples. Three samples with good purity and high DNA concentration were kept. To confirm that the DNA was of the desired plasmid, we ran a PCR with the standard VF2 and VR primers. This PCR was unsuccessful, so we repeated the PCR with different tubes of the VF2/VR primers. We also included samples with the His-tag primers, as they were designed with specific complementarity to the Tokyo BBa\_K934001 plasmid. This was accomplished via touchdown PCR. The diagnostic gel with these samples showed streaking in the lanes with the VF2/VR-amplified samples, but bright, distinct banding in one of the lanes with the his-tag primer samples. The bright band was within the expected banding range for the BBa\_K934001 plasmid, so the gel was stored for future gel purification. Finally, we digested and ligated the SpeI/PstI phasin-B0035 and SpeI/PstI phasin-B0030 parts into the Tokyo plasmid, and transformed them into TOP10 Oneshot chemically competent cells. The spread plates produced from these samples were used to prepare overnight cultures, which were refrigerated for incubation next week.

## Compost Tests

As our PHB subproject progressed, one important consideration was whether or not it would compromise the biodegradability of BC. Although both BC and PHB are biodegradable according to literature, we were interested in conducting some actual tests to develop a timeline for how quickly a PHB-BC composite would degrade. We also had some questions about the eligibility of bioplastics in municipal composting programs, which would influence the use of our product. As such, we reached out to Natalia Gonzalez, an expert in the Calgary Waste Management (CWM) services who specialises in residential and commercial waste. We sent Natalia a summary of our questions, and scheduled a virtual meeting for next week.
In parallel, we also started our own compost system in the hopes of actually testing BC and composite PHB-BC biodegradability. Using a starter compost sample provided by Tian, our teaching assistant, we prepared a large plastic container with air holes and added dried grass, vegetable scraps, and old leaves to the starter. The compost was misted and left in a warm area outdoors.

**Week 12**

## Co-Culture

No updates.

## Fruit Waste Media

No updates.

## Nisin

This week our team was busy hosting our 4th annual JulyGEM conference so we spent limited time in the lab. The time we did spend was to set up a timelapse experiment to determine nisin’s efficacy against rotting on fruit. We did a spot test to compare nisin-soaked BC discs, BC discs alone, and nisin alone, on a grape and on a cherry. We plan to repeat the experiment and film over a longer span of time.

## PHB

We redid the PCR amplification of the BBa\_K934001 plasmid with the His-tag primers and ran a diagnostic gel. The gel produced clear, distinct bands in the desired range. This indicates the success of the first stage of the incorporation of the His-tag, by producing the two individual PCR products. These were combined in the next round of touchdown PCR. However, a diagnostic gel was run and the banding (at ~960bp) was smaller than the desired 1200 bp. This indicates that the second step of the PCR was unsuccessful. This process was repeated, but again, was unsuccessful. Additionally, the overnight cultures of the SpeI/PstI phasin-B0035-Tokyo and SpeI/PstI phasin-B0030-Tokyo plasmids were miniprepped and nanodropped. The unlinearized DNA was run on a diagnostic gel, and intense streaking and multiple bands were observed in each lane. According to Dr. Mayi, this occurred because unlinearized DNA can supercoil and does not result in clear banding. Based on this advice, the DNA samples were digested with SpeI to linearize them and run on a diagnostic gel. However, the presence of streaking and multiple bands in each lane indicated that the linearization was unsuccessful.

## Compost Tests

We met with Natalia, which gave us critical insights on municipal composting standards and practices. Importantly, we learned that despite education efforts, many compostable waste products are still sent to the landfill despite the city’s well established green-cart composting system. As our project has a focus on sustainability, we expressed interest in collaborating with the CWM to improve this issue through further educational efforts. The CWM has an app called the Calgary Garbage Day app, which guides users on how to compost their waste and runs tips on its homepage. These tips are developed annually. We offered to help in the tip development process, and decided to correspond further next week about the specifications for this project.
Natalia also shared that, while bioplastics are not currently accepted by the CWM, the service conducts annual tests to assess how well new polymers compost, which may determine their future eligibility to be accepted for composting. One of the key restrictions to accepting bioplastics, as Natalia explained, is that the dirt produced by the facility is returned to citizens, and the presence of “plastic”-appearing particles could have serious negative consequences on the program’s reputability. We kept this in mind for future developments of our prototype, including considerations about colour and appearance. Natalia also mentioned that the CWM offers tours, which we expressed interest in.
Meanwhile, we continued to feed our compost sample, and Natalia shared that the CWM gives compost samples away for free. We considered replacing our compost with the municipal compost sample for a more accurate measure of how our samples would decompose. We arranged a pickup with Natalia in the event we scheduled a tour.

**Week 13**

## Co-Culture

No updates.

## Fruit Waste Media

New oranges were bought and physically processed into the pulp, peel and juice. The juice was then centrifuged 7 times to produce “double-filtered” juice. All these samples are being prepared for enzymatic hydroylsis that will begin next week.

## Nisin

After receiving inconclusive sequencing results for our miniprepped GB1+pSB1A3 and GB2+pSB1A3 samples from last week, we realized that there were design issues in our primers. Our primers contained VF and VR sequences, meaning that they would bind to both the plasmid and the Gblock sequence. We noticed, however, that our GB1+pSB1A3 sample contained almost a perfect sequence match for nisin and the T7 terminator, indicating that the samples may contain the correct sequence, if unique, gblock-specific primers were used. We redesigned a new set of primers and hope to resequence next week. Importantly, we also ran a PCR amplification of our GB1+pSB1A3 sample using promoter and terminator specific primers (Note: these primers were meant for GB4, and consequently the Tm’s were abnormally low for GB1, so we did not feel they were usable for sequencing) and successfully yielded a band at our GB1 insert size. In parallel, we also transformed and miniprepped GB4+pSB1A3 but our diagnostic gel did not yield the band sizes we expected. For our golden gate work, we performed a PCR amplification with BsaI primers on GB1, GB2, and GB4. Our gel revealed only a successful band for GB2.

## PHB

This week, the insertion of the RBS-phasin insert into the Tokyo plasmid, which is a key component of the PHB subproject, was tested with B0030 and B0035. Although three samples showed potential success, this will need to be confirmed with sequencing. Otherwise, the experiment will need to be repeated, including for the remaining two RBSs that were not yet tested. These tests will confirm the experimental compatibility of the RBS-phasin part with the Tokyo part, and allow us to troubleshoot in parallel with the addition of the his-tag to the Tokyo part.
In the meantime, PCR experiments to insert the his-tag into the Tokyo plasmid were performed. Despite successful results from the diagnostic gel, weak banding was observed, so the experiment will be repeated next week and a gel purification will be performed with the existing banding.
The PHB and BC nanocomposite sample dried today, and similar to the negative control it was extremely brittle and flaked off very easily. It appears that this is caused by using the NaOH purification method, so in the future only sodium bicarbonate will be used for purification.

## Compost Tests

We continued to work on organising a tour of the Shepherd Compost Facility, but were delayed because the organiser was unavailable on vacation. Moreover, Natalia had shared that the CWM’s annual composting tests were currently accepting samples, as the test in January had to be redone. Rather than using our own compost, this test would be ideal as it would accurately show how well our BC and composite PHB-BC would degrade in an industrial compost setting. We started arranging when we could drop our samples off.

**Week 14**

## Co-Culture

No updates.

## Fruit Waste Media

New orange samples were treated with enzymes and DNS was performed on them. Data shows some promise that glucose numbers increased but more trials need to be done and a “spiking” method might be used.

## Nisin

This week our team was preparing for our Faculty Talk event and the IndigeSTEAM event, so we spent limited time in the lab. The time we did spend was to prepare the required media and reagents for protein expression of our GB1+pSB1A3 sample (even though we were still waiting on sequencing results). We planned to resequence our sample with our redesigned primers early next week. We also spent time preparing for a minimum inhibitory concentration (MIC) test using nisin against B. subtilis.

## PHB

PHB
In order to assess the experimental compatibility of the four RBSs (B0030, B0034, B0035 and DeadRBS) with the Tokyo plasmid, and to troubleshoot this process in parallel to the insertion of the his-tags into the Tokyo plasmid, the RBS-phasin inserts were digested and ligated into the Tokyo backbone. Ligation products were transformed. However, growth was only observed on the B0030 plate. Bands were not in the correct size. Another digestion, ligation and transformation is necessary.
Due to the low concentrations from gel purifying the His-ttag, the PCR reaction was redone and no gel purification was conducted. This approach yielded higher DNA concentrations. Unfortunately, we ran out of the SpeI enzyme, so an order was placed for more enzyme and the digestion was postponed.

## Compost Tests

We organised a more specific timeline for the deliverables of the composting tips. We aimed to complete a draft of 20 tips by August 22, receive feedback from the CWM by August 26, and send the finalised tips by August 31.

**Week 15**

## Co-Culture

No updates.

## Fruit Waste Media

No updates.

## Nisin

After receiving our sequencing results, we confirmed that we were able to successfully clone our GB1 sequence into a pSB1A3 backbone! This meant that we could continue moving forward with protein expression for our sample. We created auto inductions and began preparation for an SDS-PAGE gel. Additionally, we set up and ran another time lapse video where we place BC paper discs soaked in nisin (following our immobilization protocol) on the surface of grapes. Kirby-Bauer test results for nisin against B.subtilis were collected and statistical analyses were performed - thus, inspiring the formation of a new subproject, KB-Perry.

## PHB

We evaluated the progress of this subproject, and assessed the amount of work remaining. As the iGEM season was drawing to a close, which would greatly limit our lab access, we decided to pursue a partial success route with our PHB subproject. According to this approach, rather than integrating the RBS-phasin and the his-tag into the Tokyo PHB construct together, we would attempt both of these separately. As such, in order to quantify the different amounts of phasin secreted, we planned to use a Western blot and determine expression amounts based on band brightness. With this new goal in mind, we digested, ligated and transformed the His-tag into the Tokyo PHB part, and separately repeated this process with the B0034, B0035 and DeadRBS phasin parts into the PHB plasmid. Finally, we transformed a BL21 E. coli strain with the unmodified Tokyo plasmid in order to express PHB, and ideally test a proof of concept in a co-culture with K. xylinus to make composite PHB-BC. The transformations were miniprepped and stored for next week.

## Compost Tests

We prepared two samples of pure BC, which had undergone our standard growth and processing steps. We also prepared two samples of PHB-BC, using intermittent feeding with PHB dissolved in acetic acid. Two samples of each test were provided in case one sample was lost in the compost, according to Natalia’s advice. The samples were submitted for testing. We also finished arranging the tour of the compost facility.

**Week 16**

## Co-Culture

Step 1 co culture experiments were redone to get results that can actually be compared to the coculture results. The experiment was conducted in mini petri dishes rather than regular sized petri dishes to ensure all conditions other than the species present in the culture were controlled. Results were shared with the dry lab to conduct coculture modeling again.

## Fruit Waste Media

No updates.

## Nisin

We continued our work with protein expression, running an SDS-PAGE and a Western Blot. Unfortunately, we did not see any bands at the expected size on either of the gels. Nisin is a relatively small peptide - with only 7 kDa in size - so we assumed that we were running into difficulty due to this reason. We decided to reattempt digesting, ligating, transforming and miniprepping our GB2 sequence into a different backbone (Xpress expression vector). More Kirby-Bauer results were collected, along with a round of our minimum inhibitory concentration tests for nisin against B.subtilis.

## PHB

We digested the three miniprepped products from last week, including the SpeI/PstI RBS-phasin-Tokyo samples, the plain Tokyo samples, and the his-tagged Tokyo samples. For the RBS-phasin samples, the DNA was digested with PstI and EcoRI. The transformation of the unaltered Tokyo plasmid in BL21 was unsuccessful, as was the ligation of the RBS-phasin parts into the Tokyo plasmid. Although the size of the his-tag was too small to identify in the gel, despite using a 0.5% gel, the samples that had bands in the anticipated range were sent for sequencing. The sequencing results came back negative, and based on discussions with Dr. Mayi, the enzyme used to linearize the plasmid, ApaI, was likely not cutting efficiently and resulting in the failed digestions. Due to the lack of success with the SpeI/PstI RBS-phasin insertion, we digested, ligated and transformed the Tokyo plasmid with the BstAPI/BstBI versions of the RBS-phasin parts. Based on the diagnostic gel, this second attempt was successful, which means all of the RBS-phasin parts (including DeadRBS, B0030 and B0034) had been successfully inserted into the Tokyo plasmid. These samples were sent to sequencing for confirmation. We also repeated the transformation of BL21 cells with the unmodified Tokyo plasmid.

## Compost Tests

We toured the Shepherd Compost Facility, and learned a great deal about the steps involved in industrial composting, as well as gained some insight about the future of bioplastics in composting. While Craig More was optimistic about the redirection of biowaste in Calgary as a result of the green-cart program, he reiterated Natalia’s previous point that some contamination still occurs, and that the composting facility currently lacks the capacity to process all of the city’s waste. While the facility is planning to expand, this still represents an interesting problem that we hope to improve through the tips we are developing for the Calgary Garbage Day app. Craig was also very adamant that bioplastics are not accepted for composting, and that he does not anticipate them being accepted in the near future. This is both due to the risk of perceived contamination previously mentioned by Natalia, and because conventional bioplastics are difficult for consumers to differentiate from regular plastics. Given the prevalence of contamination, by accepting bioplastics, the CWM opens the door to more confusion and contamination with regular plastics. Again, this consideration was an important one, and will inform the way we design our prototypes.

## Dry Lab

**Week 1**

## Whole Team

During our first week of spring research term, we began by ideating and developing our drylab subprojects; we identified drylab’s key overall objectives, focus areas, and potential paths forward for each of our software, hardware, and modeling components. We solidified a workflow, meeting schedule, and constant stream of communication, especially between wetlab and drylab. Finally, we also began reviewing some specific coding and program development concepts and began working towards our software subprojects, with an especially strong emphasis on our SAMARA subproject. However, by the end of the week, we decided to divert our attention away from SAMARA and focus on objectives that would directly inform wetlab design.

**Week 2**

## Whole Team

At the beginning of the week, we presented drylab ideation/subprojects. We received feedback from wetlab about their priorities and project feasibility, and used this to iterate our initially ideated projects that we proposed in order to better inform wet lab processes and drive the Cellucoat narrative and overall project. We attended a collaborative meeting with AFCM Egypt, and learned about their neural network model for predicting aptamers suitable for their project. From the ideation, we conducted literature reviews and discussed with advisors on how to move forward. We compiled a guide to using current antimicrobial peptide (AMP) machine learning tools to assist wet lab with their AMP selection and design goals.

**Week 3**

## Uniaxial Testing

Biaxial testing method was explored as a way to mechanically quantify bacterial cellulose (BC). While conducting the necessary literature reviews on the preparation, testing methods, and analysis of uniaxial tensile testing, University of Calgary professors in the Biomedical Engineering department were contacted to locate the equipment for biaxial testing.

## Modelling

After brainstorming ways we could adequately describe and validate the co-culture we wanted to start with some sort of proof of concept. This involved using a system of ordinary differential equations to describe the kinetics of the system. This week we derived the first set of equations used in modelling biomass and substrate consumption. After which we were able to plot them and communicate the results with the wet lab team. Also understanding that there could exist other extracellular substances secreted that could have an effect on bacteria growth, we set out to model them. From literature we discovered two acidic substances and then modelled the production of these acidic substances that could alter the pH of the growth media of K.xylinus and E.coli.
Began literature review for our molecular dynamics. Downloaded and began exploring the necessary software tools and understanding how to use them. Researched and understood the file format of ‘PDB’ - the file that contains the proteomic data required to perform molecular dynamic modeling - and how to use it.

## SAMARA

During this week, we finished Version One of the iGEMScraper component of SAMARA and deployed it to the iGEM Calgary Github page. We managed to successfully scrape an excess of 3000 software and modeling pages. However, the output file had many false-positives, empty pages, and broken, unusable text, especially around equations. Thus, while a good start, further development had to continue to get the text to a more usable format.

## INN

A team member had previously proposed this project using a different study. Once we decided to focus on antimicrobial peptides (AMPs), we looked into how machine learning had been used specifically to make research involving AMPs easier. We compiled a frontend guide to different AMP prediction AIs.

**Week 4**

## Uniaxial Testing

Pivoted our focus from biaxial testing to uniaxial testing, as the uniaxial tensile testing method involved a more simplified testing that can also be used to verify if the samples behaved differently in all directions. As a result, we reached out to professors to locate the equipment for this testing.

## Modelling

Continued literature review on molecular dynamics. Started modeling and got initial results for the structure visualization. Performed molecular dynamics analysis on a super computer to process the PDB file found and decided on based on wetlab’s specifications for nisin that they plan to use experimentally. Super computer returned two files - .xtc and .gro - which contain the trajectories and molecular structure of our protein respectively. Began researching different analytics that can be performed on the protein and how it can best support wetlab efforts.
Continuing from the graph that showed the production of acidic substances from our coculture we produced a graph that shows how pH would change during the growth of K. xylinus and e.coli. We then compared this graph to literature and got similar results.

## SAMARA

Development continued on the iGEMScraper component and version two was released this week. Existing code was modified in order to utilize Scrapy Items and Item Pipelines. Establishing this framework now allows for quicker, easier processing later down the line. Specifically, the establishment of the Item Pipeline allowed for items to be “dropped” before being printed to a file. This saves on post-processing and allows for a quicker run overall. This also reduced the number of valid pages from >3000 to less than 2300. Furthermore, incorporated processing elements to remove equations from the text, allowing it to later be summarized with greater ease. While imperfect, it does provide direction for the scraper moving forwards.

## INN

We looked at the literature and front-ends compiled earlier, and identified a study that we wanted to replicate using involution. We reached out to the author, who provided us with the code and models. Wet lab ran FASTA sequences for potential inserts through this model, and got nisin as a top hit.

**Week 5**

## Uniaxial Testing

Connected with Dr. Elena Di Martino, a professor in the department of Civil Engineering at the University of Calgary, with a research focus on Biomedical Engineering and tissue mechanics. She connected us to her graduate student who will give some of the team members training on the uniaxial testing equipment in the following week. A member who took a course on tissue mechanics in the previous academic year gave a tutorial to the drylab members who are involved in the subproject.

## Modelling

Molecular dynamics docking began, continuing analysis. Explored more potential molecular dynamics projects suggested by the team - evaluating feasibility. Began analyzing a single cellulose chain and docking analysis of cellulose with nisin to see if it would be likely to bind if BC is soaked in nisin filled liquid.

## SAMARA

Development of SAMARA’s front-end deployment continued this week. Django was used to create a multitude of pages to allow for rapid, near autonomous deployment in the future. While CSS usage on the pages remains ‘experimental’ for the time being, integration between the files was achieved, laying the groundwork for more formal web development in the future.

## INN

We started to run into problems getting the original author’s code to run, as it was written in a deprecated version of Python (Python 2) and so were its associated packages. Our devices couldn’t run the original model, as it couldn’t install the requirements.

**Week 6**

## Uniaxial Testing

Dr. Elena Di Martino’s student, Louise Neave, gave us training on the uniaxial testing equipment. We conducted initial tests on the BC materials developed at the lab, but determined that our samples were too thin and brittle to do proper testing. One of the wet lab members attended the training with the dry lab members and took notes on the characteristics of samples that will make it ideal for mechanical testing (~2 mm in thickness).

## Modelling

Nisin molecular dynamics and docking results produced, processed, and shared results with wet lab. Brainstormed further things to look into to assist their experimental workflow.

## SAMARA

Development of the SAMARA front-end began, and integration between the Scrapy scraper and the Django deployment started. Using scrapy-django items, conversion between the two formats was achieved and a database was created. Further development, however, is needed to create a proper website deployment. We also ran the scraped software pages on our summarizing model from GTP-3 and got accurate, meaningful results.

## INN

We attempted to use Colabs to run the model, but it no longer supported the version of Python it was written in. We attempted to use virtual environments, but were not able to get it to run either. We decided that we could either run the model on an old device/VM set up to run Python 2, or port the code to Python 3 and use the next closest versions of software we could find.

## BioSculpting

Began the BioSculpting subproject, and started researching potential ways to shape the BC into a box-like format. Extensive literature review was conducted and the wet lab was consulted to try to establish parameters for box design. This will further be established by the uniaxial testing and HP-inspired design.

**Week 7**

## Uniaxial Testing

The engineering graduate student that we communicated with suggested for us to consider confocal microscopy; advised us to examine tissue microstructure, confocal and/or multiphoton microscopy as it is a good starting point. After communicating with the lab technician, we completed the required safety training to access the engineering lab to conduct uniaxial testing. Uniaxial testing was postponed until the BC sample is grown to a sufficient thickness and size.

## Modelling

No further modifications were made to the coculture model. However, we discussed with the wet lab as to getting values from the lab experiments that can be used to improve the model. Also started working on helping them automate the process of graphing growth curves of some of the bacteria they have growing.
Efforts also began in the scaling of bacterial cellulose production. In order to produce sufficient quantities for experiments and testing, we need more than a few petri dishes worth of BC. To help combat this, we began to experiment towards the development of a larger growth environment for the BC. While formal construction has yet to begin, steps were taken to begin the procurement of the necessary materials.

## SAMARA

This week, the backend of the final version of SAMARA was completed; the summarizer was incorporated into the scraper workflow and the deployment, while lacking in css, was fully functional. The final run of the scraper has yet to be completed due to a lack of computational time, though we plan to have this completed by next week. Further work on developing the page’s aesthetics, as well as on the final deployment, is necessary in the coming days.

## INN

Running a VM or older PC seemed unfeasible given the timeframe we had to complete the project, so we decided to move the original model’s code to versions that we could run on our devices.

## BioSculpting

Efforts also began in the scaling of bacterial cellulose production. In order to produce sufficient quantities for experiments and testing, we need more than a few petri dishes worth of BC. To help combat this, we began to experiment towards the development of a larger growth environment for the BC. While formal construction has yet to begin, steps were taken to begin the procurement of the necessary materials.

**Week 8**

## Uniaxial Testing

Members completed safety courses and an orientation by a Lab technician to gain access to an engineering lab. BC samples (air dried samples, oven dried samples) were tested using a CellScale UniVert biomaterials testing equipment. The data collected from the uniaxial testing were analyzed. Some data were omitted due to the lack of validity in the collected measurement. A 3D-printed dog bone mold was created to assist in the creation of consistent uniaxial shapes.

## Modelling

No updates.

## SAMARA

SAMARA’s final run was completed over the weekend. Modifications were made to the code to allow for the summarization model to use CUDA/GPU-based processing. This substantially sped up times when ran on an NVIDIA RTX 3060ti-based computer. Per-item summarizing times went from ~2 minutes each to 15-30 seconds, dramatically speeding up the code execution time. Files were successfully read by Django and displayed properly. The CSS still needs work, however.

## INN

We moved the original model’s code to versions that we could run on our devices. Next, we started trying to convert the convolution to involution. However, we ran into an unanticipated problem: the INN was built to take two-dimensional data, while the CNN took one-dimensional data. We weren’t sure how to fix this without having to write our own framework, which we weren’t confident we could do.

## BioSculpting

Our BC scaling process began this week; a large 8x12” glass container was procured to allow for a larger interface between our K. xylinus media and the oxygen. This produced promising results in terms of BC production, allowing us to produce large quantities of BC in a relatively short time. This helps in providing a consistent supply of BC for our BioSculpting subproject.

**Week 9**

## Uniaxial Testing

Worked on documentation for the first uniaxial testing. Considered a project pivot in manufacturing cellulose acetate for increased strength, compared to pure bacterial cellulose. Conducted literature review on the process of making cellulose acetate, specifically a protocol that we can replicate in the lab. Revised the project plan to consider the project pivot.

## Modelling

Co-culture modeling verged into new ideas this week. Firstly, bacterial cellulose production was modeled adding on to our previous models. This would be used to quantify K.xylinus’ growth. Investigated errors in a graph used to show pH change and determined we didn’t account for other substances that might have a buffer effect. Looked into this and found that there actually was no buffer so we would continue researching as to how the pH changes. A new idea was added onto co-culture modeling and this involved kinetic modeling of the rate and amount of PHB secretion.

## BioSculpting

BioSculpting had its first major successes this week. Two cellulose boxes were created with dimensions of 3x3cm and 6x6cm, respectively. Both boxes withstood stress testing using improvised weights and both were able to hold a large amount of mass relative to their sizes. Good results show potential in this method of box creation.

**Week 10**

## Uniaxial Testing

Conducted uniaxial testing on samples from 2 BC boxes we created in the previous week (samples from box 1, samples from box 2). The data collected from the uniaxial testing were analyzed. Similar to the first uniaxial testing, some data were omitted due to the lack of validity in the collected measurement. Worked on documentation for the second uniaxial testing. For the project pivot of producing cellulose acetate for increased strength, we had another meeting with one of our HP contacts for feedback on our experimental protocol.

## Modelling

Co-culture modelling did not explore many new areas this week. However, we continued exploring literature and previous iGEM webinars to validate a means of modeling Nisin expression.
In terms of Molecular Dynamics, we were able to collaborate with the wet lab to come up with their needs using this protein modeling technique. We established that they specifically were in need of quantifying the binding strength of Nisin and PHB with the BC.In order to accomplish this we would be implementing docking to determine binding strength and molecular dynamics to observe their behavior in water under several conditions.

## INN

We were able to reshape the data to fit into the 2D INN. We trained it and tested it, and then tuned hyperparameters. For all of these, we used the same datasets as the original model.

## BioSculpting

BioSculpting began work on creating a cardboard-like piece of cellulose; we created a mold to create the internal corrugation (the cardboard “flute”) and managed to successfully produce it. The molds for the flat pieces (the cardboard “liners”) were designed and printed this week in preparation for the next BC harvest.

**Week 11**

## Uniaxial Testing

After conducting literature review, we selected an experimental protocol to replicate in the lab in the following week for the cellulose acetate alternative. Regarding the uniaxial testing, different materials of similar thickness will be explored to compare to the mechanical properties of BC.

## Modelling

We were able to get both monoculture and co-culture values from the wetlab that can be used to fit our model and get parameters to improve the model. From the discussions with the wet lab, they have decided to make use of BC mass as the means of quantifying the biomass of K.xylinus. This change was reflected in the model and the graphs were made.
No updates from molecular dynamics.

## SAMARA

Development of the SAMARA front-end began, and integration between the Scrapy scraper and the Django deployment started. Using scrapy-django items, conversion between the two formats was achieved and a database was created. Further development, however, is needed to create a proper website deployment. We also ran the scraped software pages on our summarizing model from GTP-3 and got accurate, meaningful results.

## INN

We ran our nisin insert through the INN, which predicted that it retained its antimicrobial properties. This aligns with the prediction made by the CNN our model was based on.

## BioSculpting

BioSculpting managed to successfully produce the cellulose “liners” and combine them with our corrugated “flute” to produce BC cardboard. The result was relatively stiff and strong, though testing has not been conducted yet.
Scaling up of the BC production continued, and we explored the possibility of using kombucha as a cost effective way of producing large quantities of BC. The amount of BC currently produced is insufficient for many rounds of iterative design, and current production quantities are not enough to supply both the wetlab and drylab with as much BC as we require. Supplies necessary for kombucha production were procured and growth is planned to begin next week.

**Week 12**

## Uniaxial Testing

Communicated with the wetlab members working on fruit waste media (FWM) BC samples for uniaxial tensile testing. Prepared samples for uniaxial tensile testing by purifying and air drying FWM BC samples and BC grown in kombucha media. Conducted uniaxial tensile testing of paper bags used for packaging for comparison and started documentation of the collected data.

## Modelling

We explored several means of using non-linear regression for fitting our model. However, we encountered a roadblock as the lack of experimentally measured glucose values or a means of reference did not allow us to fit this model. Therefore we sought to create a data set to use as a means to fit our model.We also found experimental models to replicate to use as a basis for modeling Nisin production and PHB secretion.
We performed docking on both Nisin and PHB as well as Nisin and BC. The results would be analyzed to give the wet lab a sense of the strength of how Nisin would interact in our final BC packaging.

## SAMARA

Further development on creating a nice front-end for SAMARA continued.

## BioSculpting

BioSculpting was put on hold this week due to a lack of large amounts of BC. However, in its stead, we began to explore the colouring of BC samples as a way to increase its consumer appeal. We successfully extracted dye from a red cabbage and began colouring experiments with it, using its pH indicator properties to prototype many colours with the same dye.

**Week 13**

## Uniaxial Testing

Prepared air dried BC samples by cutting the samples into dog bone shapes. Plastic and compostable packaging material that was around the same thickness or thinner than BC samples was also cut for uniaxial tensile testing. Uniaxial testing of plastic and compostable packaging material, FWM BC samples, and BC purified with NaOH and NaHCO₃ were conducted.

## Modelling

We found a model using literature that thoroughly describes the PHB production pathway. Hence, we started working on replicating the model and modifying it to fit our purpose as well as include the modifications made in our own constructs.
To be more reflective of wet lab’s experiments, we looked into creating an algorithm that can describe the feeding strategy that’s used in the wet lab. We found one that could describe it and worked on incorporating it into the rest of the system of equations.

## BioSculpting

Our BC colouring continued this week, and we tested a variety of ex-situ and in-situ methods of dye incorporation in order to determine our ideal colouring method. While all methods work, our current “lean” is towards the usage of ex-situ absorption-based colouring, as it helps to mitigate the dye degradation that occurs in the autoclaving process.

**Week 14**

## Uniaxial Testing

Data collected through uniaxial testing of plastic and compostable packaging material, FWM BC samples, and BC purified with NaOH and NaHCO₃ were analyzed. Following analysis, force-displacement graphs and stress-strain curves were documented. Bar graphs for easier comprehension of the strength of tested materials were generated to target a broader audience for the Faculty Talk.

## Modelling

The model used to describe the lab’s feeding strategy was incorporated into the change in volume. We also started work in terms of modeling Nisin production as we found a model and started working on modifying it to meet our experimental needs.

## BioSculpting

Created a BC cardboard box through the usage of 3D-net style molds; the sheets were created, layered on top of one another, and water-glued together to create a single uniform sheet of BC cardboard. Then, we folded it into shape to create a box. Very crude, hideous colour, but structurally sound.

**Week 15**

## Uniaxial Testing

Reached out to a lab technician regarding scanning electron microscope (SEM) and transmission electron microscopy (TEM) imaging for our material to understand its structure and its relevance to the mechanical properties, to better understand the results from uniaxial testing. Given the time constraint of securing an appropriate lab for the cellulose acetate experiment and reconsidering the feasibility, this aspect of the subproject was not pursued further.

## Modelling

Created script to run MD on a Nisin and cellulose complex as received from our docking results. However, we ran into issues as the software gromas could not recognize the cellulose chains. Hence, we decided to run MD on only Nisin.
Worked intensively on modifying the model found that describes PHB production by performing parameter optimization and adding onto it other pathways not captured by it. Also continued on testing out several strategies that can be used to incorporate the feeding strategy model into the rest of the co-culture model including the protein production.

## BioSculpting

Waiting for BC growth to continue the subproject; no further updates.

**Week 16**

## Uniaxial Testing

Discussed with wetlab members on the remaining uniaxial testing required for BC, and started finalizing data analysis to be presentable for the Wiki.
Considering the feedback from the Faculty Talk, raw data was reorganized to be better presentable for the Wiki. Wiki content was put together to be uploaded to the server. Discussion of future tests to be held in September were discussed with wetlab members before the end of the summer research term.

## Modelling

Ran the first molecular dynamics simulation on Nisin. This would be used as a control to compare against other runs of Nisin with different simulation conditions to simulate the environmental conditions Nisn would be put under during production and use.
Final modifications were made onto the Nisin production model and simulation plots were curated. Also the PHB model was completed as all necessary pathways were added.
To the PHB production model, we looked into ways to change it to describe how changes in several ribosome binding strengths would change the production and secretion of PHB. Made progress in terms of incorporating the feeding strategy model into the rest of the system of equations. However, the simulations did not reflect what should be expected so we decided to take a pause and move forward with it as a future direction.

## BioSculpting

Waiting for BC growth to continue the subproject; no further updates.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Summaries of how the iterative engineering design process informed various subprojects{% endblock %}
{% block sidebar\_content %}

## Engineering Success

[Overview](#header1)
[Nisin](#header2)
[Co-culture](#header3)
[BC Costs](#header4)
[Mechanical Properties](#header5)
[INN](#header6)
{% endblock %}
{% block page\_content %}

## Overview

Many components of Cellucoat went through iterations of the design cycle before coming together in the finished product you see today. After feedback on the difficulty of protein secretion from certain bacteria, we decided to integrate our recombinant proteins through a co-culture between *E. coli* and *K. xylinus*, as supported our proof of concept with GFP. To mitigate the costs associated with by 60% BC, our team made supplemented our bacterial media with fruit waste media. To further alleviate the shortcomings of raw BC, we incorporated PHB and used unaxial testing to further improve its mechanical strength. To support the functionalization of our BC with antimicrobial peptides, we developed INN to take FASTA sequences of antimicrobial peptides and predict its properties.

## Nisin Production and Characterization

We successfully cloned our composite part, NisQ-His (BBa\_K4437001), into a pSB1A3 backbone, but had difficulty with protein expression because our expected band size (7kDa) was so small. We pivoted our strategy and successfully cloned NusA-NisQ-His (BBa\_K4437002) into the Xpress expression vector (BBa\_K3945014), forming a new composite part GST-His-NusA-NisQ-His (BBa\_K4437003) which allowed us to successfully produce protein from BL21 *E. coli*. We visualized bands at 91kDa on our SDS-PAGE and Western blot gels which corresponded to the expected band size of GST-His-NusA-NisQ-His.

![](https://static.igem.wiki/teams/4437/wiki/nisin/western-nisin.png)

***Figure 1.1***
*Western blot of the whole cell lysate of GST+NusA+NisQ auto-induced in overnight express. A his-tagged positive control was also included. The protein ladder used was the Novex sharp pre-stained protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (Abcam) for the primary antibody and Goat Anti-Mouse:HRP (Abcam) for the secondary antibody.*

Our future steps will involve cleaving NisQ from NusA and GST using enterokinase, and purifying our protein with Ni-NTA. Once purified, we planned to run a Kirby-Bauer antimicrobial test against *B. subtilis* and compare zone of inhibition sizes to our characterization tests with bought nisin.

We successfully characterized nisin against both Gram-positive bacteria and several fungi strains specific to fruit. Our partnership with U of Alberta yielded results for nisin’s effectiveness against fungi, which showed that nisin could not inhibit the growth of Mucor ramanpianus, Penicillium claviforme, Rhizopus, Penicillium duclouxii, Penicillium commune, or Trichoderma viride. Based on this information, we decided to no longer classify nisin as antifungal. Our characterization continued with our series of Kirby-Bauer disc diffusion tests, where we tested nisin against other common antimicrobial agents (thymol and phenol) against *B. subtilis*. Nisin effectively inhibited growth, and while not to the same extent as thymol and phenol, a visible zone of inhibition was present.

![](https://static.igem.wiki/teams/4437/wiki/nisin-kb-b-sub.png)

***Figure 1.2.***
*Nisin’s effectiveness against *B. subtilis*. Red arrow indicates the zone of inhibition, showing that nisin can effectively inhibit bacterial growth.*

![](https://static.igem.wiki/teams/4437/wiki/nisin/untitled-design.png)

***Figure 1.3.***
*Average zone of inhibition sizes for Kirby Bauer disc diffusion test over time results against *B. subtilis*. *B. subtilis* was plated at time 0.00 hours and incubated at 37oC, and treatment rounds were applied every 2 hours for 6 hours total. Lab-grown BC paper discs were autoclaved and soaked in either bought nisin (20,000 - 40,000 IU/mL), thymol, phenol, or water and placed in a separate quadrant of the plate. Diameter measurements (n=3) were taken 24 hours later using Fiji ImageJ software.*

For our application as a preservative packaging, we also tested nisin’s effectiveness against *B. subtilis* over time. We demonstrated that nisin could inhibit *B. subtilis* after 2, 4, and 6 hours of growth at optimal conditions (ie. 37oC on LB agar) - and because fruit transportation trucks are often much cooler, thus slowing down bacterial growth, we predict that our packaging would be effective for much longer. We also demonstrated our packaging’s success in preventing deterioration on fruit, as we set up a spot test with our lab-grown bacterial cellulose soaked in nisin, on the surface of grapes. We recorded a timelapse for 12 days and found that our packaging noticeably protected the grape’s surface against shriveling.

## Using a Co-culture Between *E. coli* and *K. xylinus* to Integrate Recombinant Proteins

In the field of molecular biology Escherichia coli (*E. coli*) is one of the ideal model organisms especially when it comes with dealing with recombinant proteins. However, even with all of the advantages, one of the greatest disadvantages *E. coli* has is its difficulty with secreting proteins. Dr. Brianne Burkinshaw, a professor in the department of biochemistry at the University of Calgary confirmed the difficulty with the secretion of proteins from *E. coli*. Initially, we had designed our project in which *E. coli* would release our antimicrobial protein however, after talking to the difficulties involved due to the complexity of *E. coli* secretion systems we proceed to look for a new way of integration for our recombinant protein. We chose a co-culture system.

A co-culture is when two bacteria are able to grow together in the same media. In our co-culture system we have *K. xylinus* producing bacterial cellulose (BC), and *E. coli* producing our recombinant protein. In this system *E. coli* integrates itself within the BC fibers. Therefore, instead of secreting the protein we decided to lyse the *E. coli* cells using an autoclave at 121ºC and 15psi. This releases our antimicrobial protein -Nisin- into the BC fibers.

To model this we decided to use green fluorescence protein (GFP) which has been inserted into *E. coli*. This recombinant GFP*E. coli* was grown in a co-culture with *K. xylinus* to visualize the attachment and the effects of sterilization using an autoclave to the protein and BC. Initial tests using GFP *E. coli* included making a co-culture system inside a test tube. The BC produced in the test tube were green as outlined in Figure 2.1.

![](https://static.igem.wiki/teams/4437/wiki/prelim-coculture.jpg)

***Figure 2.1.***
*Preliminary co-culture in a test tube with *K. xylinus* producing bacterial cellulose and GFP *E. coli* expressing green fluorescence protein.*

To further analyze the distribution of the GFP *E. coli* on the BC we proceeded to set up our co-culture systems in a larger surface area such as a petri dish as shown in Figure 2.2. However, the BC sample was very thin and the concentration of GFP was hard to visualize. Henceforth, another approach we took to visualizing GFP integrated into BC was folding it to better see the actual concentration of GFP integrated into the BC.
![](https://static.igem.wiki/teams/4437/wiki/petri-coculture.jpg)

***Figure 2.2.***
*Figure 2: Co-culture in a petri dish. Petri dish on the left is a control with bacterial cellulose containing no GFP *E. coli*. Petri dish on the right contains GFP *E. coli* integrated within the bacterial cellulose fibres.*

![](https://static.igem.wiki/teams/4437/wiki/bc-coculture.jpg)

***Figure 2.3.***
*Folded bacterial cellulose produced in a co-culture.*

This allowed us to reach the conclusion that protein producing *E. coli* -GFP *E. coli* in our case- is able to integrate itself within the BC fibers and remain there.

However, our BC serves the purpose of a fruit packaging and customers do not want bacterial cellulose with bacteria still present on it. For this reason all bacteria needs to be removed from the packaging. We used the autoclave to sterilize the BC sample which effectively removed all bacterial cells present on the BC by lysing them. This means our GFP *E. coli* would have been lysed by the GFP would theoretically remain on the BC. This process of using an autoclave to lyse *E. coli* cells containing our recombinant protein is deemed effective for our antimicrobial protein- Nisin. Advantageously, Nisin is a thermostable protein which has a denaturation temperature of 160ºC.

In doing so we were able to show the co-culture is an adequate way of integrating recombinant proteins secreted by *E. coli*.

![](https://static.igem.wiki/teams/4437/wiki/gfp-bc.jpg)

***Figure 2.4.***
*A control bacterial cellulose which has not been autoclaved and does not have any GFP *E. coli* present. On the right is an autoclaved bacterial cellulose sample consisting of lysed *E. coli* cells and GFP remaining on the BC.*

## Cost of Bacterial Cellulose Production

One major factor that has limited the BC industry from making headway is it being capital intensive. This was the motivation for us utilizing the fruit waste media as through HP, we discovered that for Cellucoat to be implemented as a produce packaging, it has to be cost-attractive. To determine if our modified media made from fruit waste reduced the production costs of Cellucoat, we conducted a cost analysis. We performed the analysis by calculating the cost of producing BC based on the costs of the media for several iterations of the experiments.

Here is the summary of the cost analysis on a lab scale:

![](https://static.igem.wiki/teams/4437/wiki/bc-costanalysis.png)

***Figure 3.1.***
*Summary of a cost analysis performed on several monoculture experiments for the production of bacterial cellulose*

From the analysis, we discovered that glucose makes up about 33% of media costs. Hence, if we could make the glucose cost zero, we would reduce production costs by 33%. However, after running experiments from this analysis we realized that we were able to reduce the cost of production by 60% instead of 33%. This was from modified media consisting of 45% FWM and 55% HS. We can also hypothesize that since this media still contains HS media, reducing the amount of HS and maximizing BC yield could help us further reduce the costs of BC production. Ultimately, reducing the costs would make bolster the affordability of Cellucoat.

We also conducted a financial analysis to determine the costs needed to implement Cellucoat in the real world. Through this analysis, we found that the total start-up capital needed to launch Cellucoat is $19,201,035.56.
More detailed information on the breakdown of our costs can be found on the Entrepreneurship page.

## BC and PHB Mechanical Properties

After conducting HP interviews and testing the mechanical properties and strength of the BC our team was planning on using as the material for Cellucoat, it was found that BC is very weak. For comparison, when less than 2 mm thick, is 500x weaker than plastic of the same thickness, and has a strength akin to that of paper.

To rationalize the benefit of adding PHP to BC to create a composite that has improved mechanical properties, our team decided to use pure solid PHB that came as granules and dissolve it into a 0.25M solution with 100% Acetic acid. 1.5 mL of the 0.25 g PHB per 1 mL of acetic acid solution (0.25 g/mL) was poured into BC palettes that have been culturing for three weeks at 30 degrees and being fed using a 24 hour schedule with 4 mL of HS media. After both samples have grown, they were autoclaved, purified using NaHCO3 solution and air dried.

After drying, the samples were cut to size for uniaxial tests. The uniaxial testing looked at the tensile strength, stiffness, and stretchability of the BC and BC and PHB composites.

![](https://static.igem.wiki/teams/4437/wiki/appendix/ap-39.png)

***Figure 4.1:***
The ultimate tensile strength (MPa) of pure BC and composite PHB-BC.

![](https://static.igem.wiki/teams/4437/wiki/appendix/ap-40.png)

***Figure 4.2:***
The Young’s Modulus of pure BC and composite PHB-BC.

![](https://static.igem.wiki/teams/4437/wiki/appendix/ap-41.png)

***Figure 4.3:***
The maximum elongation of pure BC and composite PHB-BC.

Based on these results, BC showed an ultimate tensile strength 3.780MPa higher than PHB-BC, which means that BC only could withstand a larger force before tearing. The Young’s Modulus of pure BC was also 758.2MPa higher than composite PHB-BC. This value indicates that pure BC is the stiffer of the two polymers, and is less flexible. Finally, the maximum elongation of composite PHB-BC was 0.02868 higher than pure BC, meaning the PHB and BC composite was more flexible and able to be stretched. The reduction in strength of the PHB BC composite can be attributed to the uneven distribution of PHB within the BC due to the ex situ method used to incorporate PHB and acid hydrolysis causing the BC to break down when exposed to 100% glacial acetic acid.

Taken together, these results contribute to the iteration of the engineering design cycle. With this test, it has revealed the need for PHB as an additive to BC to improve its stretchability and flexibility. Furthermore, these tests have also demonstrated the need for the use of a co-culture to recombinantly integrate PHB into BC, as dissolving the hydrophobic PHB granules in acid solutions severely compromises the integrity of the material.

## Using Involution to Predict Protein Properties

As we started exploring ways to functionalize our BC, we realized that it would be helpful to have a quick way to check whether proposed inserts actually had the antimicrobial functions we wished to use them for, given that not all proposed inserts had been experimentally verified. We compiled a user guide to different antimicrobial peptide prediction models so that the wet lab could begin preliminary experiments while we constructed our own network. We aimed to investigate whether involution was an effective way to make predictions about biological data, specifically FASTA sequences.

Once our network was complete, it was able to predict that the nisin fragment we aimed to clone and insert in the wet lab indeed retained its antimicrobial properties. This was then experimentally validated by Kirby-Bauer tests.

![](https://static.igem.wiki/teams/4437/wiki/inn-nisin-predic.jpg)

***Figure 5.1.***
*Prediction of antimicrobial properties of the proteins in the nisin fragment inserted into our BC.*

Our INN accurately made predictions about antimicrobial properties when given a FASTA sequence, performing with the same accuracy as the convolutional model we based it on. Detailed information about the performance of the INN and the metrics used to evaluate it are discussed on the [INN page](https://2022.igem.wiki/calgary/swm-inn).

![](https://static.igem.wiki/teams/4437/wiki/inn-performance-1.jpg)

***Figure 5.2.***
*Comparison of sensitivity, specificity, and accuracy between CNN and INN*

![](https://static.igem.wiki/teams/4437/wiki/inn-performance-2.jpg)

***Figure 5.3.***
*Comparison of Maxwell correlation coefficient (MCC) and area under the receiver operating characteristic (AUROC) between CNN and INN*

These results are promising, but in following the engineering design process, we know that our work is not complete, and we can extend it in future iterations; we’d like to attempt hyperparameter tuning again and construct the network using a different method than data reshaping to see whether this can make the network even more accurate. As well, we’d like to collect data on the computational load of our network to examine how its performance matches up to other models. In following the iterative engineering process, we’ve taken the first steps to apply a new machine learning technique in the synthetic biology context, laying the framework for more investigation into the uses of involution and its performance.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}A full list of our goals, results, and future directions{% endblock %}
{% block sidebar\_content %}

## Results

[IHP](#header1)
[Nisin](#header2)
[PHB](#header3)
[Co-culture](#header4)
[Golden Gate](#header5)
[FWM](#header6)
[Entrepreneurship](#header7)
[Education](#header8)
[INN](#header9)
[Bio Sculpting](#header10)
[SAMARA](#header11)
[Co-culture Modelling](#header12)
[Uniaxial Testing](#header13)
[Docking and Molecular Dynamics](#header14)
{% endblock %}
{% block page\_content %}

## Integrated Human Practises

**Goal:** We aimed to develop meaningful connections with stakeholders in all parts of the produce packaging pipeline, and to use their expertise to shape our project design into a solution that reflects the needs and values of those it will impact.

**Result:** The feedback and perspectives of industry and community stakeholders have been integral to our project, and have shaped every iteration of our design. Their input informed multiple key project pivots, including the transition from a wrapping to a box, the type and integration approach of our antimicrobial peptide, and the abandonment of a proposed detection system. Our HP contacts also gave important technical recommendations, such as how to conduct antimicrobial tests and identify microbe types. The conception and refinement of our PHB subproject was also thanks to HP input. Finally, and perhaps most importantly, our stakeholders validated the need for our proposed solution, which is critical to a responsible and effective project.

**Future Directions:** While the current design of our project has been shaped by integrated feedback from these stakeholders, the need for community input does not end here. As we develop further iterations of Cellucoat, our aim is to connect with more retailers and distributors to test and validate our solution at the industrial level. Importantly, we plan to enter conversations with the International Fresh Produce Association to explore the potential for Cellucoat to meet emerging needs for sustainable packaging solutions. To develop production feasibility, a future direction includes testing the integration of our antimicrobial material with current production systems. Finally, our aim moving forward would be to integrate more feedback about the prototype design of Cellucoat at both the consumer and end-user level, with regards to factors such as color, shape and size.

## Nisin Expression and Characterization

**Goal:** Nisin is an antimicrobial peptide consisting of 34 amino acids. It is a food safe and thermo stable protein which is currently being used as a preservative in canned foods. Our goal was to incorporate nisin Q (NisQ) into our bacterial cellulose using a co-culture. We designed multiple gene fragments with varying primer cut sites to be inserted in varying plasmid backbones. This plasmid with the nisin gene fragment inserted into it would be transformed into the BL21 strain of **E. coli*.*

**Result:** We were able to insert our gene fragment 1 which consists of XbaI and EcoRI cut sites. This was successfully cloned and inserted into a pSB1A3 backbone. A sequencing of our pSB1A3-nisin plasmid was sent in to analyze how well of a match our inserted sequence is in comparison to our original nisin sequence. We had accurate sequencing results indicating successful cloning. We also cloned and transformed *E. coli* BL21 cells with our GST-NusA-NisQ-His composite part and successfully produced protein, as indicated by our SDS-PAGE and Western gel results. The GST solubility tag increased our NisQ protein size from 7kDa to 91kDa, making it easier to visualize on a gel.

To characterize our antimicrobial peptide we also ran Kirby Bauer disc diffusion tests against *B. subtilis* using co-cultured bacterial cellulose paper discs immobilized with purchases nisin. The tests showed that nisin can inhibit bacterial growth for up to 6 hours at optimal conditions.

**Future Directions:** To further our nisin characterization and production results, our team will purify NisQ from the GST and NusA solubility tags for Ni-NTA protein purification and Kirby-Bauer tests against *B. subtilis*. Once successful protein expression has been accomplished, we will use our strain of BL21 *E. coli* which can express nisin, in a co-culture with *K. xylinus* where it will begin to produce protein and functionalize the bacterial cellulose.

## PHB Improvement and Expression

**Goal:** We aimed to improve the strength and durability of our bacterial cellulose (BC) product by fortifying it with polyhydroxybutyrate, a biodegradable polymer produced in bacteria under nutrient-limiting conditions. Using parts designed by previous iGEM teams, we designed a recombinant PHB-production and secretion system in *E. coli* with a variable expression system of phasin, an associated protein which plays a role in secretion. Ideally, by modifying phasin expression levels with variable ribosomal binding site strengths, the resulting system can be integrated into our co-culture model to produce a PHB-BC composite with fine-tunable mechanical properties.

**Result:**The purpose of experimenting with different strengths of RBS upstream of the phasin-hlyA gene was to create a set of constructs that can be used to fine-tune PHB expression and secretion by up or down-regulating phasin translation. Our Western blot results indicate that the stronger the RBS upstream of the phasin-hlyA gene will upregulate the amount of phasin produced.

**Future Directions:** Having successfully modified phasin expression levels using ribosomal binding strengths, and subsequent PHB secretion levels, this subproject represents a promising tool for modifying relative concentrations of PHB incorporated into a PHB-BC composite material. As PHB has an effect on BC’s mechanical properties, including flexibility and tear resistance, this represents a means to meet a variety of packaging material needs beyond hard plastic. As such, future directions include testing the effect of varying PHB concentrations on PHB-BC mechanical properties, and validating the applications of the materials in the packaging industry.

## Co-culture

**Goal:** Bacterial cellulose (BC) is a promising material because of its potential to be functionalized. A method to functionalize BC would be to integrate the molecules simultaneously as the BC is being produced through a co-culture. Multiple factors come into play when generating a co-culture, so this subproject focuses on how to both optimize co-culture conditions for BC and recombinant protein production and post-production treatments of the BC material.

**Result:** To determine the optimal media to grow the *E. coli* and *K. xylinus* monocultures, the results indicate that using either HS media or HS media enriched with tryptone will have similar BC yields. Next, assessing the impact of *K. xylinus* and *E. coli* extracellular secretions on the other organism’s growth indicated that the extracellular secretions of *E. coli* increase BC yield by 51.61% and have no significant impact on *E. coli* cell density. Lastly, the final BC yield from the co-culture is 27.05% lower than the BC yield from a monoculture.

In regards to the post-production treatments, the results indicate that a four-day 0.5 M sodium bicarbonate purification treatment, 20-minute boiling water bath, and leave to air dry for two to three days results in a material with the greatest transparency, homogeneity in appearance, and retained strength compared to a 0.125 M NaOH bath.

**Future Directions:** Future experimentation with these two strains of *E. coli* and *K. xylinus* in a co-culture will help determine how byproducts of each population’s growth impacts the growth of the other populations, and how each changes the properties of the final BC product. Furthermore, if the co-culture would be used on an industrial scale to produce BC, PHB, and the antimicrobial peptide for weeks at a time, antibiotics must be added to the co-culture to create a selective environment. Hence, *K. xylinus* will be given antibiotic resistance to chloramphenicol with the IBMc396 plasmid.

## Golden Gate Assembly

**Goal:** The drawback to using nisin is its narrow spectrum of antimicrobial activity, as seen through how nisin is a potent agent only against certain fungi and gram-positive bacteria.To give our stakeholders in industry the option to customize their antimicrbial peptide, this subproject aims to insert nisin into our RFP flipper expression vector through Golden Gate assembly.

**Result:** We were able to successfully insert our nisin insert into our RFP flipper, however, were unable to complete sequencing our construct to confirm that the nisin insert was added.

**Future Directions:** After being able to insert nisin into our RFP flipper device, next steps include swapping nisin with one or more antimicrobial peptide inserts (done through overlapping overhangs). Candidates for potential antimicrbial peptides to further this proof of concept include Iturin A, Bac5, and Pumilacidin.

## FWM

**Goal:** The conventional *K. xylinus* growth media, Hestrinn-Schramm (HS) media, represents up to 65% of BC production costs. Given the comparatively low costs of plastic, this represents a key focus for efforts to increase the economic viability of our proposed solution. Inspired by Julianna Schneider and a scoping of current literature, the goal of this subproject is to supplement HS media with enzymatically hydrolysed orange peel. If successful, this serves as a proof-of-concept for the use of other fruit waste in BC production.

**Result:** Based on the successful growth of BC in a co-culture supplemented with our fruit waste media (FWM), we were able to show that FWM can sufficiently replace part of the HS media. In our competitive cost analysis, we demonstrate that the 45% pulp mixture was the most cost-effective FWM composition, which reduced BC production cost by a staggering 60%. Significantly, uniaxial tests confirmed that this cost reduction did not compromise the mechanical strength of the BC product. In fact, the supplementation of HS with some FWM compositions resulted in increased strength: compared to the sample grown in 100% HS media, the 30% juice sample and the 45% pulp samples showed 2.5 and 1.4 times increase in strength, respectively.

**Future Directions:** This subproject successfully demonstrated that fruit waste media made from navel oranges is a viable, and even beneficial, supplement to HS media. For future production initiatives, this concept can be expanded to include other types of fruit waste, which are prolific in the produce industry. Future experiments could also incorporate a wider range of enzymes for a more complete breakdown of the waste components into glucose.

## Entrepreneurship

**Goal:** The entrepreneurship subgroup aimed to build a business case for commercializing Cellucoat as it takes the next step to becoming a venture.

**Result:** We conducted and analyzed customer discovery interviews for Cellucoat in the development of a business plan, cost analysis, and a discourse around the implications and future of Cellucoat through a legal, ethical, and social analysis.

**Future Directions:** Before Cellucoat can become a widely used product, two key areas need to first be developed: 1. The ability to reduce the cost of production and 2. Optimizing the growth conditions of both *K. xylinus* and *E. coli*, we can maximize the BC yield. To aid in the development of this, we plan to keep refining the entrepreneurship of Cellucoat through pitching competitions and launchpad programs.

## Education and Public Engagement

**Goal:** We aimed to introduce synthetic biology to grade school students across Calgary through presentations and demonstrations. We wanted to introduce them to the basic concepts of genetic engineering, spark interest in research and synthetic biology, and engage students in relevant hands-on activities.

**Result:** We visited three schools across Calgary, engaging with approximately 250 students. We received feedback from over 150 students and educators that indicated these presentations were effective and engaging. 80.8% of respondents found the workshop enjoyable, and 58.7% felt that the workshop increased their understanding of synthetic biology. 78.4% of respondents expressed potential interest in a future synthetic biology class integrated into their curriculum.

**Future Directions:** After receiving overwhelmingly positive feedback, we adapted our presentations into a toolkit for educators looking to introduce synthetic biology to their students. This included a video introduction to synthetic biology, a video for educators explaining how to do the demo we performed, and a video for students demonstrating the demo. The toolkit also included additional resources and FAQs for teachers. We plan to disseminate this toolkit to educators at the schools we visited, and continue gathering feedback about how this toolkit can be bettered and expanded.

## Involutional Neural Network

**Goal:** We sought to replicate the AMPScanner, the CNN used by Veltri et al. as an Involutional Neural Network (INN). INNs approach their data in a spatial-specific, channel-agnostic way, and have a lower computational cost than CNNs on generic image datasets while maintaining equal or greater accuracy. Our replication aimed to investigate whether INNs perform comparably to traditional CNNs on biological data (FASTA sequences).

**Result:** Our INN replication performed comparable to the Veltri et al. CNN prior to hyperparameter tuning, using measures of sensitivity, specificity, and accuracy. (Table of measures). After hyperparameter tuning, the accuracy of the INN remained the same.

**Future Directions:** Hyperparameter tuning did not result in a notable increase in performance for the INN. In the future, it would be valuable to test whether different approaches to tuning, or a different method of fitting our data to our INN, would result in performance increases. As we had to make significant changes to the Veltri et al. paper in order to run the original CNN, and the original paper did not report measures of computational load, we were not able to determine whether INNs perform better in this regard. Fully recreating Veltri et al. using the same versions of software as our INN in the future will enable us to accurately compare measures of computational load (such as computation time and memory use).

As the INN still performed as accurately as the conventional CNN, it may be used in the future combined with Golden Gate in order to modularize Cellucoat; the INN can be used to identify peptide sequences that are antimicrobial, and can be expressed using Golden Gate.

## BioSculpting

**Goal:** We aimed to develop and iterate a production method for clamshell-like BC packaging that incorporated many of the strengths of the material while mitigating many of its shortcomings. The project also aimed to find ways to mechanically strengthen BC without the incorporation of other materials.

**Result:** The subproject resulted in the creation of multiple BC box prototypes, ranging from small, 64 cm³ boxes to large 720 cm³ boxes. We also developed a workflow and the appropriate hardware to produce BC cardboard, allowing for our unmodified BC to gain crucial mechanical strength.

**Future Directions:** Naturally, our future directions will be influenced towards the creation of larger prototypes to test our workflow on larger scales. We aim to further iterate the development of BC cardboard by testing more effective and efficient methods of production, hoping to mimic the rapid production found in other packaging materials. We also aim to reduce the amount of wasted BC used in the process, finding alternative uses for scrap pieces and, if not possible, incorporating the wasted BC into compositing workflows.

## SAMARA

**Goal:** SAMARA aimed to serve as a comprehensive, modular replacement for 2018 Calgary’s SARA subproject. Specifically, we aimed to solve SARA’s issue of extensive manual summarization while simultaneously providing a comprehensive scraping framework for future teams to build off of.

**Result:** We successfully produced SAMARA in two components. First, the iGEMScraper, a Scrapy CrawlSpider designed to crawl through iGEM wiki pages, parse the data, filter and clean it, and then pass it to a summarizer to extract the most valuable information from the text. Second, the front-end deployment, constructed using Django, allows for end-users to easily access the summarized information without needing any programming ability.

**Future Directions:** SAMARA, like many web scrapers, suffers from difficulties when scraping non-standard pages and may incorrectly flag them as empty or not relevant. Though iGEM’s pages maintain relative standardization, it remains an issue when scraping hundreds of independently-created pages. This is being somewhat mitigated in other industries through the development of AI-based web scrapers, but the technology remains immature. SAMARA’s summarizer, the sshleifer distilbart cnn 12-6 model, is also imperfect. Though it produces acceptable results, the quality is not yet comparable to those of modern, GPT-3 based models. The main drawback of the GPT-3 models was the cost associated with their use, one that we avoided to remain as open source as possible. However, in the future, should these models be made open source and free to use, updating the summarizer would be a substantial upgrade in the quality of the summarized text.

## Co-culture Modelling

**Goal:** The aim of the modeling project was to have an understanding of and validate the creation of a co-culture between *K. xylinus* and *E. coli*, inherently giving us explorations into the conditions that can allow for the optimal production of BC, PHB and Nisin.

**Result:** After creating mathematical models that consisted of a system of differential equations, we were able to run simulations that derived plots of the biomass concentration, substrate consumption, production of BC , Nisin and other extracellular substances that *K. xylinus* secretes which are acetic and gluconic acid. Ultimately, this helped inform wet lab experiments by giving us an understanding of how to regulate growth and environmental factors that influence production of our desired substances.

**Future Directions:** The implemented model falls short in some areas which can be resolved by further steps. One of such steps would involve conducting experiments to get data and use it to fit the model and improve it. Another improvement would involve considering other factors in the model that influence the behavior of the co-culture such as oxygen levels, surface area, and addition of other nutrients apart from the substrate consumed. Lastly, the model can be modified to implement more optimization techniques like Flux balance analysis to understand how we can make the maximum yield of our co-culture.

## Uniaxial Testing

**Goal:** Uniaxial tensile testing method was used to explore BC as a viable alternative to existing packaging materials and provide valuable feedback to the wetlab design. Through uniaxial testing, mechanical properties of BC were quantitatively analyzed as the characteristic stress-strain curves of BC were generated to assess its competitive strength.

**Result:** As the purpose of the uniaxial tensile testing was to explore BC as a viable alternative as a packaging material, we can conclude from the collected data and analyzed mechanical properties that BC’s mechanical properties are customizable depending on the conditions of its culture and how it is treated.

**Future Directions:** Another important consideration is to validate our BC material using different data analysis. By utilizing different data analysis, we can investigate how the material will behave under different conditions, predict its failures, and improve the mechanical properties. Instead of solely testing its behavior by subjecting it to constant increase in strain until failure, we could also explore stress relaxation and cyclic loading to simulate different loading conditions.

## Docking and Molecular Dynamics

**Goal:** The aim of conducting docking simulations was to understand the interactions and binding dynamics Nisin would have to both PHB and BC. Molecular dynamics simulations enabled us to understand the stability and conformational changes Nisin would undergo when put under several environmental conditions from production to use.

**Result:** Through docking simulations conducted on Nisin with BC, and Nisin with PHB, we found that Nisin has good affinity for these polymers. However, its affinity to BC is higher, thus validating that Nisin would immobilize well unto BC. We also discovered that the binding area of BC to Nisin is not the same as amino acid residues in Nisin that are effective against bacteria. Empirically, Nisin would be able to still perform its function after being immobilized unto BC.

**Future Directions:** Moving forward, molecular dynamics simulations on Nisin-BC and Nisin-PHB complexes that stem from docking simulations would be conducted so as to determine stability of the peptide when in this conformation. Also, conducting molecular dynamics will help us understand how these complexes change when several environmental conditions are in place such as changing temperatures, pressure or pH.

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