Amplification of Gene Block and Backbone:
Goal: To PCR amplify pET28a(+) backbone #2 and the new CBM3a gene block we received from IDT to attach Bsa1 recognition sites for construction of our plasmid through Golden Gate Assembly.
Result: Long range amplification of the pET28a(+) backbone required troubleshooting. We were working with two different pET28a(+) backbones (denoted as #1 and #2). Throughout our amplification process, we learned pET28a(+) backbone #1 might have some mutation or off target annealing causing issues with sizing of the amplification, which would come out to be around a 1.3 kb band, shown in Figure 1.
Figure 1: pET28a(+) Backbone #1 amplification results showing a band at around 1.3 kb, illustrating the off targeting annealing of our primers. This revealed that there may be a mutation with this backbone.
We eventually started using pET28a(+) backbone #2 and the correct conditions under which the plasmid amplifies without off target annealing sites: 27 cycles without Touchdown PCR. This amplification product was saved and later used in Golden Gate Assembly.
We ordered the CBM3a gene block from IDT. The size of this sequence is roughly 1.3 kb. Therefore, we were expecting a single band at 1.3 kb after amplification. The original CBM3a gene block from IDT was truncated, so we were seeing multiple bands at 0.2 kb, 0.9 kb, and 1.3 kb. This is shown in Figure 2.
Figure 2: Gel Electrophoresis results of the original CBM3a amplification that gave 3 bright bands instead of 1 (lane 3 - "Original CBM3a Amp").
We ordered a new CBM3a gene block from IDT, which we can denote as CBM3a #2. CBM3a #2 amplified easily, although three bands at 0.2 kb, 0.9 kb, and 1.3 kb still showed up on the gel. The band at 1.3 kb, however, was much brighter in comparison, indicating that there’s more specific primer binding to the region of interest. This amplification was used alongside pET28a #2 for Golden Gate Assembly. Figure 3 shows the successful amplification of the CBM3a #2 and pET28a backbone #2.
We ordered a new CBM3a gene block from IDT, which we can denote as CBM3a #2. CBM3a #2 amplified easily, although three bands at 0.2 kb, 0.9 kb, and 1.3 kb still showed up on the gel. The band at 1.3 kb, however, was much brighter in comparison, indicating that there’s more specific primer binding to the region of interest. This amplification was used alongside pET28a(+) #2 for Golden Gate Assembly. Image 3 shows the successful amplification of the CBM3a #2 and pET28a(+) backbone #2.
Figure 3: Amplification of the new IDT order, CBM3a #2, and pET28a(+) backbone #2. This was the last amplification done before successful Golden Gate Assembly results.
Construction of CBM3a Plasmid:
Goal: To begin the process of making CBM3a protein, our team needed to construct a plasmid containing the CBM3a gene block using Golden Gate Assembly with Bsal Type IIS enzyme.
Result: The amplification products pET28a(+) backbone #2 and CBM3a #2 were used for Golden Gate Assembly. Prior to Golden Gate Assembly, we selected for the amplified backbone with DPN1, which cuts dsDNA at methylated sites to get rid of any template DNA in our mixture. After Golden Gate Assembly, we transformed into DH5 alpha cells and ran colony PCR with T7 primers to confirm our construct size. Our insert was constructed inside the T7 promoter and terminator. The gel proved to be successful, showing a band around 1.3 kb. This proved our insert was correctly constructed into pET28a(+) backbone #2.
Figure 4: Colony PCR gel of the Golden Gate Construct using T7 primers. A band around 1.3 kb is shown for colonies 2 and 4.
Amplification of Gene Blocks and Backbone:
Goal: To PCR amplify gene blocks containing CBM2a and mRFP to attach BsaI recognition sites for Golden Gate Assembly in pET28(+) backbone.
Result: The CBM2a gene block that we ordered from IDT was amplified resulting in the gel in Figure 5. The gel reveals two faint bands for CBM2a amplification in both lanes where the PCR product was loaded, one at approximately 1.7 kb and the other near 1 kb. The second band at 1 kb was an incorrect size, since our gene block is 1,689 bp. So, we performed a gel extraction on the upper, correct band at 1.7 kb to isolate the correct band size in order to continue forward with Golden Gate Assembly.
Figure 5: CBM2a amplification gel with 2 bands in both loads. The 1.7 kb band was extracted and amplified more.
We then PCR amplified the gel extraction, resulting in Figure 6. This gel revealed a much brighter band at the correct size. These samples were saved for Golden Gate Assembly.
Figure 6: Gel from the amplified extraction product that was at 1.7 kb.
The second part of our CBM2a fusion protein contained the mRFP, which came separate from the CBM2a gene block. We PCR amplified this gene block which showed good results on the gel. This PCR product showed a band at about 0.7kb, which aligns with the correct size of the gene block, 681bp. This PCR product was thus saved for Golden Aate Assembly.
Figure 7: Gel of the amplified mRFP product that shows a band at 0.7 kb (lane 2).
Amorphous Cellulose Production:
Goal: In order to integrate our plasticizers more effectively into cellulose, we needed to find a chemical method to make amorphous cellulose that would replicate the effect CBMs would have on the cellulose.
Result: For our plasticizer work, we started off by finding a dissolution method to decrystallize our microcrystalline cellulose before adding in our plasticizer. We made our first amorphous cellulose by mixing NaOH with MCC and titrating it to a pH of 10 with glacial acetic acid. The NaOH caused the MCC to swell and form a thick, white “snowy” mixture. This method was successful in creating amorphous cellulose.
Another method of cellulose dissolution that we explored early on was the use of ZnCl22 to make amorphous cellulose. This dissolved the microcrystalline cellulose and formed a thick, clear solution of the hydrated zinc chloride and the dissolved MCC.
Figure 8: NaOH treated amorphous cellulose
Goal: To find the identity and proper concentration of plasticizing molecules that would bring characteristics like flexibility to our cellulose to achieve plastic-like qualities.
Result: We used amorphous cellulose from ZnCl2 or NaOH treatment to begin testing our plasticizers. We first tried using our ZnCl2-dissolved cellulose with the addition of citric acid and glycerol as plasticizers. We made four plasticizer tests with glycerol and water and citric acid and water, as well as the two alone in order to understand the effects water has on the structure of the compounds. When we mixed these four different plasticizers with the ZnCl2-MCC, glycerol showed the most promising results as it thickened the dissolved cellulose solution, while the plain citric acid didn’t bring any changes to the solution. Both the citric acid and glycerol with water added only made our cellulose more liquidy. We also tried treating our ZnCl2-treated MCC with a combination of both glycerol and citric acid and then baking it for hours. After removing it from the oven, the samples showed to have gained thicker structures, but when we left it to sit at room temperature for a day, it became a very watery substance again. We believe this was due to the citric acid being somewhat unstable when exposed to light, causing it to bind to water molecules from the air and create a more liquidy solution.
We also tried a citric acid/glycerol mixture on the NaOH treated amorphous cellulose. We found a ratio of 70% (dry weight) plasticizer mixture to 30% (dry weight) amorphous cellulose to give the best results. After mixing the amorphous cellulose with the plasticizer mixture, we dehydrated the mixture in a dehydrator set at 65C for 2 hours to remove any excess water. We then moved our mixture to the oven set at 175C and let it bake for 45 minutes. This resulted in a thick gel that remained stable even at room temperature, a promising result.
To test whether or not these results with citric acid and glycerol were reproducible, in addition to testing other plasticizers, we made three mixtures: 1. Plasticizer and Zinc Chloride; 2. Plasticizer, MCC, and Zinc Chloride; and 3. Plasticizer, Glycerol, MCC, and Zinc Chloride
Using this experiment plan, we tested the following compounds: citric acid, glycerol, fructose, sucrose, mannose, and glutaraldehyde. The tubes containing just zinc chloride and a plasticizer served as controls to ensure that cellulose is the determining factor in the formation of a gel. If a gel was formed without cellulose, this would mean the plasticizer-ZnCl2 mixture produces a solid regardless of the presence of cellulose and the plasticizer will not incorporate into cellulose. As expected, no gels were formed in the control tubes. Certain plasticizers, like glycerol and glutaraldehyde, formed a gel in the presence of MCC. The mixtures containing sugars (fructose, sucrose, and mannose) did not form a gel even when combined with MCC. The mixture of glutaraldehyde, MCC and zinc chloride heated up, indicating an exothermic reaction, and the result was a viscous gel. Although lutaraldehyde acts as a cross-linking agent, it is a toxic substance commonly used as a disinfectant. For this reason, we ceased investigation into this chemical. Mixtures containing glycerol all formed a gel, confirming that glycerol is a promising plasticizer regardless of the second plasticizing compound. This preliminary testing led us to believe that glycerol does incorporate into amorphous cellulose, and may be a good plasticizer to include in our film.
Figure 9: A 70:30 ratio of plasticizer mixture (glycerol/citric acid) to amorphous cellulose after being baked.
Figure 10: Solution of MCC, ZnCl2 · 3H2O, and glycerol, before baking.
Goal:To understand how copolymer integration affects our film. Copolymers bring characteristics like tensile strength to polymers that are desired qualities in our film. Additionally, xanthan gum specifically has been proven to strengthen soil   .
Result: We tested the use of copolymers as an additive to cellulose. We followed a protocol that uses ionic liquid BMIMCl to dissolve the microcrystalline cellulose. We used ionic liquids diisopropyl imidazolium and diisobutyl imidazolium in place of BMIMCl. These samples formed composite ion gels with xanthan gum and cellulose, and then went through Soxhlet extraction with ethanol for 12 hours. This process was used to extract diisopropyl imidazolium from our cellulose-xanthan gum mixture, allowing us to reuse this ionic liquid. This experiment resulted in a fine, thin paste of xanthan gum and cellulose that was pressed between 2 glass plates and left to dry for 4 days at room temperature. The extracted diisopropyl imidazolium mixture did not form a film, but left a powdery mixture of xanthan gum and microcrystalline cellulose. We believe this was due to the ionic liquid as it was a symmetrical molecule, thus it could not effectively dissolve biomass as well as BMIMCl.
Figure 11: The Diisopropyl Imidazolium composite ion gel. This gel was formed from a mixture of DIPI, MCC, and xanthan gum baked in an oven for 9 hours.
Figure 12: Diisopropyl Imidazolium (left) and diisobutyl imidazolium (right) with cellulose and xanthan gum before baking.
Figure 13: The Soxhlet extraction set-up being used on the diisopropyl imidazolium composite ion gel.
Cleaning and Drying Methods
Goal:To find cleaning and drying methods to produce films for pure BC samples and future CBM and plasticized BC samples.
Result: After cleaning a purified BC sample in 70% ethanol, 0.1M sodium hydroxide, and water, our BC became cleaned as seen in Figure 14.
Figure 14: The cleaned cellulose and exposed fibers on the exterior.
Figure 14 shows cleaned BC where the fibrils begin to come undone from the cellulose, making the sample appear larger than it was before treatment. This may be caused by the cellulose taking up water during the cleaning process, creating a swollen appearance. Once cleaned, we implemented drying techniques on this sample to see if any films could be made.
Drying techniques included room-drying and freeze-drying to see if the sample films could be produced and in Figure 15 we see that the both techniques produced films.
Figure 15: Roomdried (left) and Freeze-dried films (right).
Further testing will be conducted on films to see which techniques offer properties such as tensile strength, biodegradability, etc. This will allow us to choose an optimal drying method for the CBM/plasticized molecule.
XRD Baseline Results Based on Agricultural Films
Goal:To set a baseline crystallinity index from agricultural films that are currently used in farms to compare our samples to.
Result: After performing X-ray Diffraction (XRD) tests on the four films provided by Dr. Husein Ajwa as seen in Figure 16, we received graphs for each film as seen in Figure 17.
Figure 16: XRD testing on A. Expoly film, B. Green/TIF film, C. Raven™ film, D: Sample of reflective film.
Figure 17: XRD graphs. A: Expoly film, B: Green/TIF film, C: Raven™ film, D: Sample of reflective film
From analysis of these results we made a table, as seen in our Proof of Concept page, consisting of all the crystallinity index values. We also found an average crystallinity value of 66% across the standards. This average will be the target index value for our CBM/Plasticized film in order to compete with current films on the market.
XRD Results of Dried BC
Goal:To find the starting crystallinity of BC, which we can compare our samples to once they have CBMs and plasticizers attached.
Result:The crystallinity index of room-dried BC was calculated to be 76% from the graph shown in Figure 18.
Figure 18: XRD graph of room dried BC film.
From analyzing the crystallinity index of BC film, we have a starting point of 76% and we want to decrease the crystallinity to, at most, 66% with our CBM and plasticizer additions to get to the level of crystallinity most successful agricultural films on the market are currently at.