Appendix
Co-culture
Step 1: Optimizing Media for BC and Recombinant Protein Yield
Figure 1. BC Yield over a period of 3 days in grams at 30ºC in a static monoculture. The samples to measure BC mass were derived BC samples that have been blotted with a paper towel and measured on a scale. HS media, LB media, and HS Media enriched with 5 grams of tryptone were tested with BC seeds that all had masses of approximately 0.2 g ± 0.03 g. Data points are the average of one replicate.
Step 2: Impact of Extracellular Secretions on Growth of K. xylinus and E. coli
Figure 2. E. coli OD (595 nm) over a period of 3 days in absorbance 30ºC in a static monoculture. The samples to measure OD were derived from media surrounding the BC biofilm. Data points are the average of three biological replicates. Data is represented as an average OD over 3 days.
Figure 3. BC Yield over a period of 3 days in grams 30ºC in a static monoculture. The samples to measure BC mass were derived BC samples that have been blotted with a paper towel and measured on a scale. Data points are the average of three biological replicates. Data is represented as mean ± SD.
Step 3: Co-culture Between K. xylinus and E. coli
Figure 4. BC Yield over a period of 4 days in grams 30ºC in a static monoculture. The samples to measure BC mass were derived BC samples that have been blotted with a paper towel and measured on a scale. Data points are the average of two biological replicates. Data is represented as mean ± SD.
Figure 5. E. coli OD (595 nm) over a period of 4 days in absorbance 30ºC in a static monoculture. The samples to measure OD were derived from media surrounding the BC biofilm. Data points are the average of two biological replicates. Measurements at hour 23 and 71 have been excluded from the data set because of the high SD, indicating that the high degree of biological variance causes this information to be unreliable to base assumptions on and must be omitted. Data is represented as mean ± SD.
Intermittent Feeding
Figure 6. BC Yield over a period of 3 days in grams 30ºC in a static monoculture. The samples to measure BC mass were derived BC samples that have been blotted with a paper towel and measured on a scale. The 24-hour intermittent feeding sample was given 4.0 mL of HS media at 8:00 am every 24 hours. The 48-hour intermittent feeding sample was given 20 mL of HS media every 48 hours, or on day 2. Data points are the average of three biological replicates. Data is represented as mean ± SD.
BC Yield in Co Culture VS Monoculture
Figure 7. BC Yield over a period of 3 days in grams 30ºC in a static monoculture (orange line) and co-culture (yellow line). The samples to measure BC mass were derived BC samples that have been blotted with a paper towel and measured on a scale. Co culture BC samples were cultivated in a culture with K. xylinus and E. coli over a period of 4 days in a static culture at 30oC and given 4 mL of HS media every 24 hours. Monoculture BC samples were cultivated in a culture with only K. xylinus over a period of 4 days in a static culture at 30ºC and given 4 mL of HS media every 24 hours. Data points of the co culture samples are the average of three biological replicates. Data is represented as mean ± SD.
Functionalization
Distribution to GFP Throughout BC Film
Figure 8. The co-culture BC was autoclaved. Most of the GFP E. coli seen before autoclaving was in the surrounding edges, and once it was autoclaved most of the fluorescence was in the surrounding outer edges. Once autoclaved the fluorescence was more of a greenish/brown but remained on the outer edge, which can be assumed to be thermo-lysed bacterial cells.
Purification
Figure 9. BC grown in HS media and purified in either 0.5 M NaHCO3, 0.125M NaOH, or H2O for three days on stage 2 on the rocker then all air dried for 3 days were cut into multiple 2 mm x 2 cm strips and the transparency was measured at 595 nm with a spectrophotometer. The more transparent the material is, the lower the OD. Data is represented as a mean of 10 replicates, and the SD of the mean is represented as a graph in Figure 12.
Figure 10. The SD of the OD taken from BC grown in HS media and purified in either 0.5 M NaHCO3, 0.125M NaOH, or H2O for three days on stage 2 on the rocker then all air dried for 3 days were cut into multiple 2 mm x 2 cm strips and the transparency was measured at 595 nm with a spectrophotometer. The more homogeneous in appearance the BC from the three different purification was, the lower the SD it will have. Data is represented as a mean of 10 replicates from Figure 10, and the SD of the mean is represented Figure 11.
Figure 11. Images taken over the course of 3 days of BC purified in either 0.5 M NaHCO3, 0.125M NaOH, or H2O for three days on stage 2 on the rocker then all air dried for 3 days.
Determining Efficacy of NaHCO3 Bath on Longevity of BC Material
Figure 12. Picture of experimental set up. 0.5 x 20 mm slip of BC purified by either NaOH or NaHCO3 submerged in a solution 1.5 ml of 0.1 mg cellulase/0.01 g BC solution. The experiment was conducted with 10 replicates. This experiment intends to determine if NaOH (negative control) or NaHCO3 would hold up best in a commercial environment. The samples were placed at 30ºC in the incubator and agitated twice a day until the sample appeared dissolved in the solution within the tube and the solution was homogeneous. The NaOH sample began breaking down on day 2, while the NaHCO3 sample did not break down at all.
Drying
Figure 13. Time taken for BC that was originally 3 cm thick to dry using oven, vacuum, or air drying. The BC was grown in HS media and purified in 0.125M NaOH for 3 days. The oven drying occurred at 70ºC, vacuum drying occurred in a vacufuge at 60ºC, and the air drying occurred at room temperature at 24ºC. The oven drying consumed 7500 kW, the vacufuge consumed 4125 kW, and the air drying consumed 0 kW.
Figure 14. Images taken over the course of 3 days of BC that were originally 3 cm thick to dry using oven, vacuum, or air drying. The oven drying occurred at 70ºC and was lubricated with mineral oil, vacuum drying occurred in a vacufuge at 60ºC, and the air drying occurred at room temperature at 24ºC. The oven drying consumed 7500 kW, the vacufuge consumed 4125 kW, and the air drying consumed 0 kW.
Uniaxial Testing
Figure 15. Tensile stress-strain curves of BC samples cultured in PL 15 media
Figure 16. Tensile stress-strain curves of BC samples cultured in PL 30 media
Figure 17. Tensile stress-strain curves of BC samples cultured in PL 45 media
Figure 18. Tensile stress-strain curves of BC samples cultured in varying ratios of PL media (PL 15, PL 30, PL 45)
Figure 19. Tensile stress-strain curves of BC samples cultured in PU 15 media
Figure 20. Tensile stress-strain curves of BC samples cultured in PU 30 media
Figure 21. Tensile stress-strain curves of BC samples cultured in PU 45 media
Figure 22. Tensile stress-strain curves of BC samples cultured in varying ratios of PU media (PU 15, PU 30, PU 45)
Figure 23. Tensile stress-strain curves of BC samples cultured in JU 15 media
Figure 24. Tensile stress-strain curves of BC samples cultured in JU 30 media
Figure 25. Tensile stress-strain curves of BC samples cultured in JU 45 media
Figure 26. Tensile stress-strain curves of BC samples cultured in varying ratios of JU media (JU 15, JU 30, JU 45)
Figure 27. Tensile stress-strain curves of BC samples cultured in pure HS media
Figure 28. Tensile stress-strain curves of BC samples for comparison to PHB incorporated BC
Figure 29. Tensile stress-strain curves of PHB incorporated BC samples
Figure 30. Tensile stress-strain curves of BC samples purified with NaOH
Figure 31. Tensile stress-strain curves of BC samples purified with NaHCO₃
Figure 32. Tensile stress-strain curves of paper packaging samples
Figure 33. Tensile stress-strain curves of Juice (JU) 30 BC samples produced in a co-culture. This specific ratio of co-culture samples was selected for uniaxial tensile testing as the first fruit waste media BC test yielded JU 30 to have the highest ultimate tensile strength.
Figure 34. Average tensile stress-strain curves of JU 30 BC samples produced in a co-culture
Figure 35a. Bar graph comparing ultimate tensile strength of JU 30 BC, compared to JU 30 BC produced in a co-culture. The poorer mechanical properties of the JU 30 co-culture BC can be attributed to the poor growth observed in the samples. Next steps of accurately validating the effect of co-culture on mechanical properties of BC would be to set up another set of experiments to validate earlier results. Future experiments should put emphasis on controlling variables during the production of the BC samples, with the only manipulated variable being the type of culture.
Figure 35b. Bar graph comparing Young’s Modulus of JU 30 BC, compared to JU 30 BC produced in a co-culture
Figure 35c. Bar graph comparing maximum elongation of JU 30 BC, compared to JU 30 BC produced in a co-culture
Nisin
Figure 36. PCR amplification of pSB1A3 plasmid containing GB1, using T7 promoter and terminator specific primers (GB1 = 393 bp), indicating successful cloning.
Figure 37.1 Diagnostic gel for GB2-Xpress expression vector ("2021 backbone") miniprepped-samples (samples digested at 37oC for 60 minutes with no inactivation because of BamHI, therefore we expected the samples to not be fully digested). Lanes 4, 5, 6, and 7 show our expected band sizes. Lane 8 contains a negative control.
Figure 37.2 SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.
Figure 38 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.
Figure 39. His-tag purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1.
Table 1: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis, collected on July 5th. Each measurement (n=3) was recorded 24 hours after initial plating using a ruler to the nearest 0.1cm and converted to mm.
Table 2: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis, collected on July 6th. Each measurement (n=3) was recorded 24 hours after initial plating using a ruler to the nearest 0.1cm and converted to mm.
Table 3: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis over time, collected on August 17th. Each measurement (n=3) was recorded in pixels using ImageJ, then converted to mm (1mm = 3.3 pixels). Disc diameters were approximated to 210 pixels. Time 0:00 corresponds to measurements taken 24 hours after initial plating.
Table 4: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis over time, collected on August 30th, biological replicate #1. Each measurement (n=3) was recorded in pixels using ImageJ, then converted to mm (1mm = 3.3 pixels). Disc diameters were approximated to 210 pixels. Time 0:00 corresponds to measurements taken 24 hours after initial plating.
Table 5: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis over time, collected on August 30th, biological replicate #2. Each measurement (n=3) was recorded in pixels using ImageJ, then converted to mm (1mm = 3.3 pixels). Disc diameters were approximated to 210 pixels. Time 0:00 corresponds to measurements taken 24 hours after initial plating.
Table 6: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis over time, collected on September 21st, biological replicate #1. Each measurement (n=3) was recorded in pixels using ImageJ, then converted to mm (1mm = 3.3 pixels). Disc diameters were approximated to 210 pixels. Time 0:00 corresponds to measurements taken 24 hours after initial plating.
Table 7: Raw data for Kirby-Bauer disc diffusion tests against B. subtilis over time, collected on September 21st, biological replicate #2. Each measurement (n=3) was recorded in pixels using ImageJ, then converted to mm (1mm = 3.3 pixels). Disc diameters were approximated to 210 pixels. Time 0:00 corresponds to measurements taken 24 hours after initial plating.
Table 8: Raw data for ampicillin’s minimum inhibitory concentration tests against B. subtilis incubated overnight, collected on August 17. Each measurement (n=2) was recorded with a 96-well plate spectrophtometer. Each well contained 100 μL of B. subtilis LB broth culture (D100 dilution of an overnight culture) and 100 μL of antimicrobial agent. An empty well was set as a blank.
Table 9: Raw data for nisin’s minimum inhibitory concentration tests against B. subtilis incubated overnight, collected on August 17. Each measurement (n=2) was recorded with a 96-well plate spectrophtometer. Each well contained 100 μL of B. subtilis LB broth culture (D100 dilution of an overnight culture) and 100 μL of antimicrobial agent. An empty well was set as a blank.
Table 10: Raw data for minimum inhibitory concentration tests against B. subtilis incubated overnight, collected on August 17. Each measurement (uninhbited growth n=3, sterility n=2) was recorded with a 96-well plate spectrophtometer. Each well contained 200 μL of LB broth culture (D100 dilution of an overnight culture). An empty well was set as a blank.
PHB
Figure 40. Samples B0030 (1 and 4), B0034(2, 3, 4), B0035 (2), and DeadRBS (1) all had bands appear in the desired band size of ~7100 bp, indicating that for B0030, it has been successfully digested, ligated, and transformed into TOP 10 cells, while for the other samples it has successfully been transformed into BL21 cells. Due to time restrictions, the B0030 RBS phasin-HlyA plasmid (BBa_K4437502) was not transformed into BL21 E. coli cells.
Figure 41. The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E. coli strain autoinduced for 24 hours. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was stained with Coomassie blue. The gel was loaded as follows: (1) Ladder, (2) LanM positive control (BBa_K3945001), (3) Dead RBS upstream of phasin-Hlya in PHB construct (BBa_K4437500), (4) B0034 RBS upstream of phasin-Hlya in PHB construct (BBa_K226002), (5) B0035 upstream of phasin-Hlya in PHB construct (BBa_K4437504), (6) Tokyo 2012 PHB construct only (BBa_K934001). The two distinct bands at ~27 kDa and ~20 kDa in the three plasmids that contained the phasin-HlyA inserts and the bands faint in the controls (Lane 1, LanM BBa_K3945001 and lane 6, Tokyo 2012 PHB BBa_K934001) and the negative control then there is phasin-hlyA being produced.
Figure 42. The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E. coli strain autoinduced for 48 hours on a Western blot using Anti-FLAG antibodies and the complementary capture antibody. The gel was loaded as follows: (1) Ladder, (2) Tokyo 2012 PHB construct only (BBa_K934001) (3) Dead RBS upstream of phasin-Hlya in PHB construct (BBa_K4437500), (4) B0034 RBS upstream of phasin-Hlya in PHB construct (BBa_K226002), (5) B0035 upstream of phasin-Hlya in PHB construct (BBa_K4437504).
Figure 43. The ultimate tensile strength (MPa) of pure BC and composite PHB-BC.
Figure 44. The Young’s Modulus of pure BC and composite PHB-BC.
Figure 45. The maximum elongation of pure BC and composite PHB-BC.
Golden Gate
Figure 46. Lane 1 contains 2uL of Quick-Load Purple 2-log 100bp ladder. Each lane contains 10uL of the digested sample. Prepared on a 1.5% gel which ran at 100V for 50 minutes.
In regard to the second object of inserting our recombinant nisin insert into the RFP flipper, we were able to amplify and successfully insert what we predict to be our nisin insert from G-Block 2 (NisQ with an N-terminus NusA solubility factor and 6x His-tag) in Figure 2.
Figure 47. Lane 1 contains 2uL of Quick-Load Purple 2-log 100bp ladder. Each lane contains 10uL of each amplified G-block after Golden Gate Assembly.
Fruit Waste Media
Figure 48.
Glucose concentration of orange juice samples after enzymatic treatment. Each treatment was for 24 hours. Each measurement (n=3) was taken with a refractometer and converted to a g/mL value.
Figure 49.
Glucose concentration of orange peel samples after enzymatic treatment. Each treatment was for 24 hours. Each measurement (n=3) was taken with a refractometer and converted to a g/mL value.
Figure 50.
Glucose concentration of orange pulp samples after enzymatic treatment. Each treatment was for 24 hours. Each measurement (n=3) was taken with a refractometer and converted to a g/mL value.
Figure 51.
BC growth in a co-culture, grown in varying Juice FWM (15%, 30%, 45%) with daily feedings. Each measurement (n=3) was taken with a scale.
Figure 52.
BC growth in a co-culture, grown in varying Peel FWM (15%, 30%, 45%) with daily feedings. Each measurement (n=3) was taken with a scale.
Figure 53.
BC growth in a co-culture, grown in varying Pulp FWM (15%, 30%, 45%) with daily feedings. Triplicates used. Each measurement (n=3) was taken with a scale.
Figure 54.
E. coli growth in a co-culture, grown in varying Juice FWM (15%, 30%, 45%), with daily feedings of 1.767 mL of the respective FWM media. Each measurement (n=3) was taken with a spectrophotometer at OD600 of a 400μL media aliquot.
Figure 55.
E. coli growth in a co-culture, grown in varying Peel FWM (15%, 30%, 45%), with daily feedings of 1.767 mL of the respective FWM media. Each measurement (n=3) was taken with a spectrophotometer at OD600 of a 400μL media aliquot.
Figure 56.
E. coli growth in a co-culture, grown in varying Pulp FWM (15%, 30%, 45%), with daily feedings of 1.767 mL of the respective FWM media. Each measurement (n=3) was taken with a spectrophotometer at OD600 of a 400μL media aliquot. 
Figure 57. Overview of the costs analysis of several growth strategies. Legend - Juice (JU), Pulp (PU), Peel (PL)
Figure 58. The percent difference of the several production iterations of BC in our lab. Legend - Juice (JU), Pulp (PU), Peel (PL), Co-culture (C) and Monoculture (M)
Table 11. Glucose concentrations of orange samples treated with enzymes (cellulase, pectinase, and invertase). Each measurement (n=3) was taken with a Brix Refractometer.
Table 12. E. coli growth in coculture grown in Juice FWM samples. Initially, 8 mL of media was added on day 1 and 1.767 mL of media was added every day following for intermittent feeding. Each measurement (n=3) was taken via weight of BC with a scale.
Table 13. E. coli growth in coculture grown in Peel FWM samples. Initially, 8 mL of media was added on day 1 and 1.767 mL of media was added every day following for intermittent feeding. Each measurement (n=3) was taken via weight of BC with a scale.
Table 14. E. coli growth in coculture grown in Pulp FWM samples. Initially, 8 mL of media was added on day 1 and 1.767 mL of media was added every day following for intermittent feeding. Each measurement (n=3) was taken via weight of BC with a scale.
Table 15. E. coli growth in coculture grown in Juice FWM samples. Initially, 8 mL of media was added on day 1 and 1.767 mL of media was added every day following for intermittent feeding. Each measurement (n=3) was taken with a spectrophotometer at OD600 from a 400 μL aliquot of the sample’s media.
Table 16. E. coli growth in coculture grown in Peel FWM samples. Initially, 8 mL of media was added on day 1 and 1.767 mL of media was added every day following for intermittent feeding. Each measurement (n=3) was taken with a spectrophotometer at OD600 from a 400 μL aliquot of the sample’s media.
Table 17. E. coli growth in coculture grown in Pulp FWM samples. Initially, 8 mL of media was added on day 1 and 1.767 mL of media was added every day following for intermittent feeding. Each measurement (n=3) was taken with a spectrophotometer at OD600 from a 400 μL aliquot of the sample’s media.
KB-Perry
| File | Download |
|---|---|
| KB-Perry Engineering Drawings | Link |
| Bridge (L) | Link |
| Bridge (R) | Link |
| Legs | Link |
| Pegs | Link |
| Slider (L) | Link |
| Slider (R) | Link |
BioSculpting
| File | Download |
|---|---|
| Prototype 1/2 Box Moulds | Link |
| BC Cardboard Corrugation (Flute) Mould | Link |
| BC Cardboard Flat (Liner) Mould | Link |
| Prototype 3 Corrugation (Flute) Mould | Link |
| Prototype 3 Flat (Liner) Mould | Link |
| Prototype 4 Corrugated (Flute) Mould (L) | Link |
| Prototype 4 Corrugated (Flute) Mould (R) | Link |
| Prototype 4 Flat (Liner) Mould (L) | Link |
| Prototype 4 Flat (Liner) Mould (R) | Link |
Modelling
Co-culture Modelling
The python files for all implementations of the model can be found here
Protein Modelling
The python files for protein production in a monoculture and co-culture can be found here here
The protein structure files, docking complexes, and molecular dynamics output files of Nisin, PHB and BC can be found here
PHB Modelling
The modified MATLAB model can be found here