Our team utilized the Sirius part from the 2018 iGEM Toulouse team composed of a carbohydrate binding module from family 3 (CBM3a), N/C terminal linkers, and red fluorescent protein (mRFP), part BBa_K2668020. The Toulouse team designed a versatile molecular binding platform as their product. With the addition of two linker proteins, they used CBM3a to build a base for attachment of molecules to cellulose that would allow for different biotechnological applications such as functionalized bandages, textile factories to develop cloth with new features, and the conductive paper industry. Our team is creating another application for carbohydrate binding modules and increasing their diversity within synthetic biology. We have designed a way to evaluate the desired concentration of CBMs needed to loosen cellulose fibrils enough for the addition of a plasticizing agent while also forming amorphous cellulose.
The Toulouse Team’s use of CBM3a and the assays they performed helped our team design a project to decrystallize bacterial cellulose in order to make a base for our cellulose-based plastic. Our project utilizes the attachment of CBMs to cellulose to understand the molecular dynamics of decrystallization of the cellulose fibril structure.
Through conversations with Dr. Oded Shoseyov, a CBM and biopolymer film expert, we learned about the potential binding inhibition caused by the mRFP and the linker proteins to CBM binding to cellulose. He also mentioned the use of the mRFP is not a reliable way to quantify the amount of CBMs bound to cellulose. Through our crystallinity testing, our team has learned that current agricultural films on the market have a crystallinity index of around 66% (for further information about this, refer to our Results page). Our team is interested in the quantification of CBMs bound to cellulose in order to reach the desired crystallinity of successful agricultural films being used on farms. In order to do this, we will improve on the Sirius fusion protein created by the Toulouse team in 2018. By removing the mRFP we can understand the binding affinity of CBMs to cellulose without a bulky protein in the way and design a new quantification process. We have designed these methods with guidance from Dr. Oded Shoseyov, and by referencing his research on cellulose binding protein A in the cellulolytic bacterium Clostridium cellulovorans . These methods are described extensively on our Engineering page.
Additionally, the CBM3a part from Toulouse served as a foundation for the design of our new part containing CBM2a, BBa_K3426000, that binds amorphous and crystalline cellulose with a slightly lower affinity than CBM3a, hypothetically allowing for the addition of plasticizer integration . In this way, we have improved upon the current CBM proteins in the iGEM registry while broadening the applications of CBMs.
Figure 1: Our plasmid design for CBM2a.
Our first step in CBM3a production was designing a plasmid that included the Sirius CBM3a-mRFP fusion protein. The Toulouse 2018 team cloned Sirius into plasmid backbone pSB1C3 first, then transitioned into pET-28a(+) before performing protein production and purification. We decided to clone the gene block into pET-28a(+) from the beginning, before protein production, using Golden Gate Cloning. Similarly to Toulouse, IDT assembled our gene block and primers. We chose to use pET-28a(+) as opposed to pSB1C3 because it would allow us to transition directly into protein production after growing successfully transformed DH5 alpha cells containing our Golden Gate product. In the process of implementing our design, we faced some difficulties in amplification of the pET-28a(+) backbone, using our designed primers with BsaI sites for Golden Gate. This could be due to the large size of it, around 5.4kb, in comparison with pSB1C3, which is only around 2kb. When more DNA needs to be amplified through the Polymerase chain reaction (PCR), it can create mutations and errors along the way that Toulouse 2018 may have not experienced with a smaller backbone. Due to this difficulty, we have considered fitting our gene block into another plasmid to more easily amplify the backbone, and improve the efficiency of Golden Gate Assembly.