Team:FSU/Engineering

Engineering

Overview


To combat the very sensitive and very complex issue of food insecurity and hunger in Tallahassee we delved deeper into a singular aspect that we could implement with integrated human practices and synthetic biology. It led to a rising awareness that fresh-food was lacking from lower income families, because the food’s shelf-life isn’t long enough to be transported fast enough. To extend shelf-life our research brought us to our ChiFresh project. It is the combination of a chitin biofilm and a chitosan spray. There was also the issue of expenses of producing chitin and chitosan. To decrease possible cost expenses, we also make a more efficient pathway in E Coli. C. to produce chitin.


Chitin Biofilm & Chitosan Spray

Iteration 1

Integrated Human Practices

Human practice reached out to local food banks and researched that there were multiple factors that could be improved upon. In this experiment the primary two factors that we fixated on were transportation storage and shelf life of foods. With back and forth conversations with multiple food banks and experts, our teammate Chase suggested Chitin for its preserving and biodegradable properties. Deeper we searched and found that Chitin has the characteristics to maintain a set environment like permeability and maintaining a temperature [12]. Chitosan, synthesized from Chitin, had the characteristics of having antimicrobial and antifungal properties [17,18]. Meta analysis of past works involving them, our team found plausible solutions that stakeholders would be interested in: a combination of a chitosan spray and a chitin film. Together as ChiFresh they would preserve the fresh-foods for a longer number of days by slowing down rates at which the vegetables decay and prevent them from bruising in transport.


A series of questions arised. Since other researchers' work has proven chitin biofilms characteristics on maintaining a set environment and extending food shelf-life [15,16,17], our team decided to focus towards the chitosan spray. This was done particularly because stakeholders expressed desire for tomatoes and green-beans, but past research with chitosan spray did not involve either of those vegetables. What could we do to improve the past sprays with our desired vegetables? Well we made the hypothesis that if a vegetable is sprayed with a chitosan spray its shelf life would be extended. The chitosan spray has been combined with many different combinations from thyme or cinnamon oil. We decided that cinnamon oil combined would be the best direction, since its results were more than just a singular day extension. However, we were also curious about what hasn’t been done yet. After researching different antimicrobial oils found in natural plants, Chokeberry was found. It is a native to Florida and has great characteristics to be a natural preservative. It was antimicrobial, anti-fungal and high in pectin [2,3]. We decided to make that one of our independent variables since it hasn’t been tested yet and is a native plant in Tallahassee.

Material

We purchased the chitosan spray Hyshield and the cinnamon essential oil from Amazon, and green beans and tomatoes from Publix. Big container from Walmart. Two 800mL beakers, stirrer, timer, weight-boats, and graduate cylinder were used.

Procedure

Washed vegetables with diluted water and dried. Sorted the tomatoes into equal four groups {one group was the control} of three tomatoes with size of small, medium, and large. And then we randomized the green beans into 24 per group. A big container was divided into three sections and its lid was divided into three sections as well. Labeling the sections control we placed the three tomatoes in the container and laid out the 24 green beans on the lid section. Then we prepared the chitosan solution by measuring out 300 mL of the Hyshield in two different beakers. Then added 3mL cinnamon oil into one of the beakers. Stirred the Hyshield and cinnamon oil solution for 15 minutes. One at a time from the next group of tomatoes was dipped in just the Hyshield spray for 2 minutes and then placed in the correctly labeled section. Repeat the dipping for the green beans. Then repeat the dipping with the remaining groups with the Hysyield and cinnamon oil solution. The containers were then set in a refrigerator at 40 degrees Fahrenheit. Every 3 days we checked on them and took note of any wrinkling, bruising, and mold growth. We also took photos for a visual comparison. We stopped the experiment on day 15.


Out of the four tomato groups there was only one that didn't mold and maintained its shape, firmness, and most of its vibrant coloring. That group was the solution of Hyshield with cinnamon oil. As compared to the other groups, the Hyshield Spray and Hyshield with chokeberry started molding at day 9. The control started molding at day 12. The solution of Hyshield and cinnamon oil had surpassed the controls shelf-life by three days. The tomatoes also still had very little bruising and maintained its shape. As for the green beans, overall there was no molding in the 15 day time period, but wrinkling, drying, and browning was experienced in all groups. Below is the results table that describes each vegetable, in each group, for every three days. There is also a visual comparison throughout the 15 day period.



Design cycle 2: Screening conditions and method

Iteration 2

Integrated Human Practices

There was a positive result with the Hyshield and cinnamon oil solution for the tomatoes, but overall there was no improvement on the extension of the green beans. Hysheld was made to speed up plant growth and not necessarily preserve food. Its exact formula was also kept confidential for commercial purposes. Rather than pursuing an already made chitosan spray we will replicate a past research protocol [40] that showed they were successful and only change which vegetables were used in the experiment. With known materials and their concentrations we would be able to better understand what in the spray plays a part in preserving the vegetables. More research will be done to examine the best project to replicate and see if the results improve.

Material

Chitosan (417963-100G), Tween 80 (59924-100G-F), Sodium triphosphate pentabasic (72061-100G), Glacial Acetic Acid (695092-500ML), Sodium Hydroxide (655104-25G), and weigh boats (Z708577-500EA) were all purchased from Sigma. The Cinnamon Essential Oil from Iteration 1 was also used in this experiment. Two 800mL beakers, stirrer, timer, weight-boats, graduate cylinder, centrifuge were used.

Procedure

Weight ratios of CS to CEO 1:1. Added dropwise of TPP solution into o/w emulsion (for CEO-CSN) or CS solution (for CSN) under constant stirring at room temperature for 60 mins. Nanoparticles were centrifuged at 27,000g for 14 mins. Prepared separately by dispersing 0.15% (w/v) of each one in distilled water and kept at 4 C for two coating solutions of CSN and CEO-CSN) with final concentration of 1.5 g/L. Wash and dry tomatoes and green beans. Separate the tomatoes by size: small, medium, and large. One of each size goes into one of the three groups. Separate the green beans by length: long and short. Split them equally between the three groups. Separate the container into three sections and its lid into three sections. Label one "control" for tomatoes on the container and "control" for green beans on the lid. Lay the vegetables for one group in their corresponding sections. Next label another section "CS" on both the lid and container. Dip the second group of vegetables into the Chitosan spray for 2 minutes each. Then lay them out in their sections. Label the last section "CS+COE" like before. Take the last group and also dip all the vegetables for 2 minutes each and lay them out in their sections. Place in the refrigerator at 40 degrees Fahrenheit. Every three days observe and change and take a photo. Repeat until 15 days are reached.

Though there are dents in the tomatoes they primarily maintained their vibrant red color and firmness. There was also no sign of mold growth on any of the tomatoes. There also were not any positive results for the green beans or improvements from the last iteration. Unfortunately, the experiment was not able to run to the full 15 day time period due to timing, so it can not be properly compared to the last iteration.



Project 2: Microbial Production of CO Secretion

Research & Imagination

Current production of chitin and chitosan involve three major steps: demineralization, deproteination, and decolorization. Chitosan requires a fourth step, deacetylation where the acetyl group is removed. These steps are often expensive, hazardous, and wasteful [18,19].


Furthermore, current chitin supply comes from, in large part byproducts of the fishing industry, such as crab shells and squid beaks [23-27], whose fisheries are depleting [20-22]. To realistically use chitin on a global scale, a scalable and sustainable source of chitin must be found that isn’t reliant on natural sources. We focused on how synthetic biology could produce this substance on an industrial scale.


To narrow down the complexity, we limited ourselves to only use single celled hosts for producing chitin. Two eukaryotic clades of natural chitin producers were evaluated: diatoms and fungi. Diatoms were eliminated because they require silicic acid as a key nutrient that they incorporate into their cellular structure. Providing this nutrient would be an unreasonable request given other potential producers do not require a silicon nutrient. Fungi where also eliminated for two reasons: 1) single cell fungi that produce chitin (yeast) produce chitin in very small amount compared to other polysaccharide structural materials; 2) chitin and chitosan are incorporated into the structural system of these organism [28]. This disqualifies fungi as suitable hosts for two reasons: first, there is a limit to how much chitin can be produced or we risk malformed, less competitive, chassis; second, these organisms incorporate chitin into their structural makeup and therefore complicated purification mechanisms would be required to separate chitin from undesired structural polysaccharides. To be a desirable industry option, we designed a scheme to avoid complicated purification procedures. The team has selected E. Coli as our chassis of choice for its simplicity to cultivate and transform.


Before we expressed chitin in a prokaryotic host, we investigated the chitin synthesis pathway. Chitin synthesis shares the synthesis pathway with peptidoglycan (PG) cell wall synthesis (Fig. 1) [29]. The chitin synthesis pathway shares a common substrate with PG synthesis, that being UDP-GluNAc, where we hypothesize that UDP serves as a suitable leaving group on the first carbon of substrate n where a glycosidic bond will form with the fourth carbon of the substrate n+1. This both simplifies and complicates the expression of chitin: the biosynthesis pathway already exists in most prokaryotic organisms (and E. Coli specifically), but direct competition with PG synthesis will limit the expression of chitin synthase (see our modeling report for more details on quantitative modelling).


Fig. 1 This is the Peptidoglycan (PG) synthesis pathway illustrated by Barreteau et al. [12]. UDP-GlcNAc would then be further metabolized in a collection of reactions into the substrate for PG synthesis. Chitin Synthases and related proteins like NodC use UDP-GlcNAc as the substrate. Most prokaryotes that have a cell wall can therefore be assumed to have the precursor for chitin and therefore also chitosan.


We then focused on chitin synthase itself. Chitin synthase is a challenging protein to express for many reasons: 1) Well studied Chitin Synthases are found in Eukaryotic organisms, which have post translational modifications that make it difficult to express in E. Coli. [30]; 2) Chitin Synthases in prokaryotic organisms are under-studied and uncharacterized; 3) Chitin synthases are transmembrane proteins from the glycosyltransferase family 2 [30,31]. This protein family is characterized by two main domains, a cytoplasmic catalytic domain that synthesizes the chain, and a transmembrane domain that transfers the chain out of the inner membrane into the periplasmic space; this process may or maynot be toxic [31,32]. Only one example of microbial chitin production has been found in the literature by this team: the chitin synthase that they used resulted in different morphologies in E. Coli from control [33]. To avoid a less competitive host, we instead looked towards producing chito-oligomers instead.


We have evaluated many examples of chito-oligomer production and we chose NodC to express as two iGEM teams have also expressed NodC [34,35]. NodC is considered a chito-ogliomer synthase, and not a true chitin synthase because it produces chito-oligomers between lengths of four and six residues (Fig. 2)[31,32]. Computer modeling and structure prediction from Dorfmueller et al. [32] suggest that this is due the the transmembrane domain of NodC is closed with a “stop-gap” that limits the size of the chitin chain to just a few residues. This allows NodC to be compatible with prokaryotic hosts.


chitin secretion cells

Fig. 2. This image was copied from DorfMueller et al. [32]. The left Structure is their predicted NodC (Rhizobium sp.) structure based on the conformation of other proteins in the GT-2 protein family. The rightmost structure is a known cellulose synthase (PDB code 1QGQ). Notice that the chain present in the NodC transmembrane domain is blocked by R349 and L19 whereas the chain in BcsA is allowed to flow freely. In a prokaryotic system, NodC is more compatible to then a true chitin synthase that would have the open translocation domain similar to cellulose synthase.

Using Darmstadt 2017 as inspiration, we expanded on the team’s design by incorporating proteins from the Nod operon, to which NodC is a part of [36]. The nod operon that we extracted NodC from also contains two other proteins of interest: NodI and NodJ [38,39] . Together they form an ABC Transporter Complex that secretes Nodulation Factors: Lipo-chito-oligomers with additional decorations (LCOs) [36,37] (see the modeling report for more information on the protein structure of the NodIJ complex). For our purposes, since the NodIJ complex secretes many different varieties of LCOs, and NodIJ has been shown to be efficient in secreting LCOs [37], we hypothesized that NodIJ might improve the secretion of Chito-oligomers.

pSB1K3 plasmid template was used to construct our template. The NodCIJ operon was constructed using three promoters of varying expression levels: Weak promoter: BBa_K2832184, Medium promoter: BBa_K2832161, and Strong promoter: BBa_K2832123. Then NodC, NodI, and NodJ (BBa_K2380000 ,BBa_K3792000, and BBa_K3792001 respectively) was expressed (gathered from the same organism Rhizobium leguminosarum with and RBS of BBa_B0034 for NodC and BBa_J61101 for NodI and NodJ. All operons were terminated with a double terminator sequence BBa_B0015. Control had all the same elements except NodI, NodJ, and their corresponding RBS’s were omitted.


Build and Test

The NodCIJ generator with weak, medium, and strong promoters were assembled and inserted into the plasmid pSB1K3 in one step using a NEBuilder HiFi DNA Assembly kit. The NodCIJ generator was ordered from IDT as three gBlocks, each with overlaps of 15 bp. The NodC generator with weak, medium, and strong promoters were also assembled in this way. The NodC generator was ordered from IDT as two gBlocks with 15 bp overlaps. The assembled plasmids were then transformed into NEB 5-alpha competent E. coli cells. The transformed cells were spread on LB agar plates with 50 ug/mL kanamycin and incubated overnight. Colonies formed on the plate containing the weak NodCIJ cells, and none of the other plates had any growth. The assembly, transformation, and plating was repeated. Colonies of the strong NodC cells grew after one day of incubation, and a colony was used to make liquid cultures. After 5 days of incubation, small colonies seemed to form on the other five plates. The small colonies were used to inoculate liquid media, but upon incubating overnight no cells seemed to grow in the liquid media.


In order to determine if the weak NodCIJ cells were secreting chitin, we purchased a Chitosan Assay Kit from Cell Biolabs (XAN-5126). This kit colorimetrically detects chitosan, producing a pink color that becomes more intense and absorbs more light at 540 nm when more chitosan is present in the sample. The NodCIJ culture was centrifuged, and the supernatant was collected. We believe NodI and J transport chitin outside the cells, so we expect most of the chitin to be in the supernatant. The NodC culture was not centrifuged. To ensure the concentration of chitin in the solution is high enough for the kit to detect, the samples were concentrated by being evaporated in a fume hood overnight and then resuspended in DI water. The NodCIJ sample was reduced by 1/12 and the NodC sample was reduced by 1/20. For each sample, an equal volume of 12.5 M NaOH was added, and the samples were placed in a 95 C water bath overnight. This step causes deacetylation of the chitin, converting it to chitosan. The chitosan assay was then performed on the samples. This involves reacting the samples with reagent A for 30 minutes and then reagent B for 20 minutes, both at 85 C. The absorbance of each sample was then measured at 540 nm using a plate reader.


Our calibration chart is shown below. These measurements are the relative absorption rate of chromogenic products produced by the chitin detection kit calibrated to different amounts.

chitin secretion cells

chitin secretion cells
More Information on Entry BBa_K3792006

This data reflects the absorbance of chitosan from the assay kit. From our data, we can see how the 400 ug/ml chitosan solution had an absorbance of 2.477 with the chitosan assay kit. However, the LB medium with the assay kit had a very high absorbance of 1.515. As a result, we suspect that our medium, LB, has chitosan. Furthermore, we can confirm there is chitosan in our chitosan solution, however, we cannot be sure if this is directly due to the cells or due to the medium LB. We hypothesize that there is chitosan in both our cells and the LB medium, as a result, we will further be testing our chitin secretion cells in a middle medium to confirm if our cells directly secret chitosan.


Learn & Improve


Based on the results of the assay kit, we cannot conclude the concentration of the chitin produced by the cell. We suspect that chitin can be found in the LB broth that we used to culture the cells since chitin can be found in the cell walls of yeast and other fungi [29]. To conduct a conclusive test, a test performed with agar instead of LB should be performed.


It is inconclusive whether the weak/medium promoter NodC pair plasmids transformed correctly. Because only the strong promoter NodC pair plasmid was successful to culture, we cannot conclude that NodC is a metabolic burden on the cell. Furthermore, we cannot conclude the effectiveness of the NodCIJ operon because NodCIJ cells were centrifuged and not NodC cells. A more conclusive test should be performed with the same treatment on both trials.


Furthermore, only the weak promoter NodCIJ operon was successfully cultured. Because the same procedure and the same personnel that transformed NodC was also used to transform the NodCIJ plasmids. Assuming that all NodCIJ transformations were successful, then we could conclude that the NodIJ complex was a metabolic burden on the cell.


To further improve the design of the cell, we came up with a possible design of L-form Bacillus Subtilis expressed with prokaryotic chitin synthases. L-form bacteria are bacteria that previously had a cell wall but lost the cell due to prolonged evolution in an environment with consistent osmotic pressure to the cell’s needs or genetic manipulation [41-43]. A previous team has developed and extensively characterized a device to induce L-form in the cell walls of Bacillus Subtilis [44]. Furthermore their device targets the murE gene [45]. This gene prevents the catalysis of a PG precursor [46]. This aids our project in three ways. As mentioned earlier, the only example this team found of a chitin synthase being expressed by E. Coli resulted in an unusually elongated polymorphism of E. Coli. “Bare Subtilis”, as Newcastle likes to call it, can therefore avoid three problems: 1) prevents uncompetitive polymorphisms as gram-positive organisms only have a single cell membrane and therefore the chitin chains cannot be entwined in the periplasmic space if there is no cell wall; 2) it lessens the metabolic burden of the chitin synthases as the synthases no longer competes with the PG metabolic synthesis pathway if PG synthesis is not necessary for the host to survive; 3) there is no need to also have an export protein complex, such as NodIJ, because true chitin synthases have a transmembrane domain that translocated the chitin chain as it is being produced, which further lessens the metabolic demand of the operon.




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