With COVID-19 restricting lab access and creating other limitations, it is more important than ever to thoroughly plan and design each piece of our project so that it may come together smoothly despite time restrictions. As we progress in our research, the design of our project will evolve and become more advanced. We have achieved engineering success through the following the cycle:
Discover → Research and Design → Build → Assay → Learn → Improve
Plastic waste is a widely discussed issue. We specifically focused on learning about the huge plastic waste problem in the agricultural industry. Although plastic mulches are a necessity in farming practices, they produce acres of plastic waste that have little chance of making it to a recycling facility due to soil contamination on the plastic and expensive shipping costs.
As our team learned more about this issue, a light bulb went off: we should engineer a biodegradable thermoplastic using a cellulose producing microorganism that can save growers money and protect the earth! With this solution in mind, we began designing our research plan and our product goals.
Research and Design
Upon starting our research, our team planned to test a range of chemical plasticizers and copolymers. We began by modeling amorphous (non-crystalline) cellulose by testing microcrystalline cellulose with various ionic liquids, producing cellulose that is similar to the expected results of CBM binding. This model of our bacterial cellulose was used in order to select plasticizers and copolymers in a timely fashion, since bacterial cellulose can take up to ten days to form. Thanks to this work, once CBM expression begins we will have a better idea of the plasticizer, copolymers, and cross-linking agents to incorporate. We tested various chemical plasticizers such as sorbitol, glycerol, and more. Additionally we have experimented with the inclusion of cross-linking agents such as citric acid and glutaraldehyde in concurrence with chemical plasticizers. These concepts are described in our Results page. Our team also met with professional chemists and polymer scientists, namely Dr. Peter Weiss, Dr. Rebecca Braslau, and Dr. Doug Hayes, for advice on finding effective plasticizing and decrystallizing agents. Due to COVID-19 and the fires in California, our ability to continually do lab work was hindered so saving time was a top priority. Using ionic liquids that mimic the effects of CBM binding allowed us to understand the importance of the decrystallization of cellulose, even without stable lab access.
Carbohydrate binding modules (CBMs) are regions on protein complexes such as cellulase that attach to the fibrils of cellulose and induce structural modifications . CBMs have been shown to increase cellulose affinity to dyes, amplify tensile strength, and produce other qualities like filtration abilities . There are 80 families of CBMs, with multiple members of specific CBMs in each family . There is an impressive range of CBMs available to experiment with, and previous work has shown that individual CBMs can add very different qualities to cellulose. There is little published work on using a CBM to create a plasticized cellulose, making our initial experimentation novel.
Despite the overwhelming variety of CBMs, we decided to test just two CBMs to begin with: CBM3a and CBM2a. CBM3a was chosen since it is part of the 2018 Toulouse iGEM team’s Sirius fusion protein, part BBa_K2668020. We also selected Carbohydrate Binding Modules of Type 2a (CBM2a), because it has a slightly lower binding affinity to cellulose, which we theorize will allow greater plasticizer incorporation . CBM2a binds to both amorphous and crystalline cellulose irreversibly, but its low binding affinity allows it to traverse along the strands while never fully dissociating from the cellulose matrix.
|Part Name||Part Number||Part Type||Description||Designer||Length|
|Sirius: CBM3a - mRFP1 fusion (Sirius)||BBa_K2668020||Coding region||CBM3a, mRFP, N- and C-Terminus linkers.||Younes Bouchiba||1,349 bp|
|Carbohydrate Binding Modules of Type 2a (CBM2a) Fused to mRFP1 (CBM2a)||BBa_K3426000||Coding region||CBM2a, mRFP, N- and C-Terminus linkers.||Kyra Eyerman||2,371 bp|
It is vital that we not only account for the production of our plasticizer, but also its integration with bacterial cellulose. Cellulose is a highly crystalline compound which is composed of a dense fibril network that is held together by intra and intermolecular hydrogen bonding. CBMs can interrupt these bonds and allow for the incorporation of our plasticizing molecules. This is why it is important for us to find a CBM that can facilitate the attachment of our plasticizing molecules.
Design for the Future
Though we weren’t able to get to our desired final step, our team designed an experimental process that will get us to our goal: a simple co-culture between our bacterial cellulose producer and the organism producing our plasticizing chemical and CBM. Together these organisms will synthesize our thermoplastic. E. coli will produce our CBM as well as the plasticizing molecule of interest while K. rhaeticus produces the bacterial cellulose.
We decided on creating a co-culture because the plasticizing agent can attach to nascent cellulose strands as K. rhaeticus synthesizes them, effectively creating a plastic factory in the media without the need for external interference. We theorize that the attachment of a CBM and plasticizer right as cellulose strands are extruded will result in more thorough incorporation throughout the cellulose matrix, in comparison with adding CBMs in solution with fully formed crystalline cellulose.
While designing this co-culture, we understood the possible roadblocks such as competition between organisms for nutrients in the media. The co-culture would function using quorum sensing between the two organisms. Quorum sensing is the ability of one organism to detect and respond to cell population density of the other organism by gene regulation. A signaling molecule is used to send and receive signals in the culture, allowing the two strains to live harmoniously without using up eachother’s carbon sources. This ensures one organism doesn’t outcompete the other.
A co-culture is ideal for our project because it creates a sustainable biofactory of our desired product. Our two strains can live harmoniously and actively produce our plastic bed mulch without exhausting their resources in the media. Once formed, we can extract our plastic and cure it without killing our culture in the process. This idea was inspired by the 2016 Imperial College iGEM team, since they were able to create a co-culture of two engineered E.coli. Their project included a quorum sensing, circuit-like interaction between their two strains. The Imperial College of London was also able to create a co-culture between two engineered K. rhaeticus strains that communicated using pSender and pReceiver plasmids, which our team received from the college as a stepping stone to eventually create communication between our bacteria. Imperial College’s contribution to iGEM also helped us build our co-culture design.
After the design was outlined, we transitioned into heavily researching carbohydrate binding modules as a method of decrystallizing cellulose and attaching our plasticizer molecule. Bacterial cellulose is extremely rigid, so decrystallizing it is necessary in order to do the proper modifications to create a thermoplastic.
At this point, our team branched into two main areas of interest: plasticizing molecules and CBMs. We first explored the field of biopolymer additives including plasticizers, cross-linking agents, and copolymers. Plasticizers such as glycerol and sorbitol are known to increase tensile strength in cellulose-based polymers. Cross linking agents, such as citric acid and glutaraldehyde are molecules that contain two or more reactive ends capable of chemically attaching to specific functional groups. This would serve as a method to increase linkage of a plasticizing molecule to cellulose. Additionally, citric acid has been proven to reduce water vapor permeability, a characteristic we strive for in bed mulch films  . Copolymers are produced by the copolymerization of two or more different monomers. This allows for them to take on properties of their constituent parts. So far, we have explored xanthan gum and PHAs. Xanthan gum has been proven to increase tensile strength which will help to counteract the brittleness of cellulose  .
In order to produce CBMs, we built off of the work of the 2018 Toulouse iGEM team, Cerberus. We designed a pET28a plasmid containing their fusion protein, Sirius, or in the registry, BBa_K2668020. This part contains a gene for CBM3a as well as a red fluorescent protein (mRFP). Our plasmid was designed for Golden Gate Assembly using BsaI recognition sites to construct our plasmid. We ordered our gene blocks from IDT and devised a plan to use CBMs as a method of cellulose strand separation, allowing for incorporation of a plasticizing molecule.
Our team also created a new part containing CBM2a fused to mRFP1, part number BBa_K3426000. We designed this protein part based on the Sirius part, since the 2018 Toulouse iGEM team blueprinted an assaying method utilizing the mRFP. Our part contains the genes for CBM2a, an mRFP, and N- and C-terminus linkers. We designed this part to be assembled into a pET28a plasmid using Golden Gate Assembly to achieve scarless cloning with BsaI recognition sites. Our research design accounts for comparison of CBM3a and CBM2a incorporation into cellulose using colorimetric assaying of the mRFP with plain bacterial cellulose (BC) as our control. We plan on incorporating our plasticizing molecules as well and studying how this may affect the CBM3a or CBM2a binding affinities. We have been successful in cloning the Sirius part we ordered, but are still in the process of cloning our new CBM2a part.
Assays and Future Work
Since we weren’t able to assay the binding affinity of our CBMs and potential plasticizers to cellulose, we designed what these experiments may have looked like. Once the CBM has been expressed in BL21 cells, we plan to begin large scale protein production and purification.
We will perform a binding assay using the cellulose pull down assay method the 2018 Toulouse iGEM team used on their CBMs to illustrate CBM binding to cellulose. Fluorescence will be analyzed to understand the presence of molecular interaction between cellulose and the CBM.
In addition, our team explored the use of bacterial polyesters, polyhydroxyalkanoates (PHA), as a potential plasticizer for our future work. PHAs are a large family of biodegradable polymers produced by bacteria. They possess the capability of acting as a plasticizing agent to bacterial cellulose and are considered to be “microbial plastics”. PHAs can be synthesized from E. coli, thus making it a good contender for the plasticizing molecule in co-culture design. The 2018 Edinburgh iGEM team, Valeris.ED, utilized PHAs for their project to make a “biobased plastic” by constructing PHA synthesis pathways by adding specific genes to E. coli to facilitate PHA synthesis. Edinburgh also constructed a thermoplastic as an alternative to petroleum based plastic, but through the use of by-products from whisky distilleries and pot ale (containing dead yeast, proteins from barley, and mainly water), to produce PHA plastic in E. coli. Opening an avenue of potential future collaboration between our team and Edinburgh to understand PHA synthesis mechanisms within E.coli, but most importantly tackle plastic pollution, is helpful to the creation of our own “biobased” thermoplastic.
Our team was able to interview Dr. Oded Shoseyov, an expert in CBM attachment and cellulose film production. When telling him about the gene blocks we made for our CBMs, he told us to think twice about adding an mRFP gene because they can hinder binding affinity of CBMs to cellulose. He told us it is because the mRFP gene is a large protein that interferes with binding. He additionally pointed out that the fluorescence will not allow us to quantify the amount of CBM binding, but rather can only be used as a baseline detection method. He instructed us to remove the mRFP and follow a different selection and quantification, which we have laid out in the next section, Improvement.
Our cellulose-based film will be installed in farms around growing crops, so it is necessary that the bacteria we are using in the production of the film do not contaminate crops or soil. Due to our biology and engineering backgrounds, we know all the recombinant DNA used in our project is not harmful to soil or humans because CBM and bacterial cellulose DNA already exist in nature. However, we understand that GMO production is a sensitive topic and many growers feel uncomfortable knowing our product could contain live bacteria and recombinant DNA. With this in mind, we have developed a proposed curing process that involves dehydration of the samples and conditions that should not allow the bacteria to survive. We will also ensure that all recombinant DNA present in the film is denatured via UV radiation, enzymatic degradation, or temperature exposure.
Although we were not able to conduct the experiments related to the improvement of the Sirius part, we did design the Golden Gate construct without the mRFP using Geneious.
|Part Name||Part Number||Part Type||Description||Designer||Length|
|Carbohydrate Binding Modules of Type 3a (CBM3a) - Improved||BBa_K3426001||Coding region||CBM3a Sirius part from the 2018 Toulouse Team improved to remove the mRFP, N- and C-Terminus linkers.||Kyra Eyerman||480 bp|
We also designed the protocols required for testing this plasmid design against bacterial cellulose. The following is the method of selection and quantification Dr. Shoseyov instructed us to go forward with instead of using the mRFP, starting with protein production:
The advantage of this selection and quantification assay is it allows for accurate determination of the maximum amount of CBM saturation of cellulose. Through this, we will be able to experiment with varying amounts of CBMs and plasticizing molecules to achieve the optimum ratio between the two. Additionally, this assay will help us understand the differences in affinity between CBM2a and CBM3a that wouldn’t be observable simply using the cellulose pull down method used by the Toulouse 2018 team. This is why it was also necessary for us to remove the mRFP, since it will not offer benefits to our assays and will possibly hinder plasticizer incorporation.