Cellulose-Binding Modules of Type 3a (CBM3a)-Improved
Usage and Biology
CBM3a is a cellulose binding module isolated from the protein, scaffoldin CipA, in the organism Clostridium thermocellum. The organism Clostridium thermocellum, is thermostable allowing for this protein to withstand extreme temperatures. CBM3a will bind to cellulose, resulting in de-crystallization of the structure. Once de-crystallized, additional small molecules such as plasticizers can be added. This specific part is an adapted form of part BBa_K2668020, Sirius Fusion Protein from 2018 Toulouse iGEM. The mRFP gene was used to analyze attachment of CBM3a to cellulose. We are improving the part by removing the mRFP gene and the N and C linkers. Through our research and talking with experts, we learned that the mRFP gene is a bulky protein that can possibly interfere with binding of the CBM to cellulose. With this in mind, we have built a separate selection system that doesn't require the use of the mRFP gene to understand binding affinity without a large protein in the way. Additionally, the mRFP does not actually allow us to quantify the amount of CBM binding, but rather provides only a baseline detection method to see if any CBMs are bound to cellulose.
We designed a part that is an improvement on the Toulouse 2018 iGEM team part, Sirius. This part includes the CBM3a protein gene, endogenous N and C terminus linkers, and an mRFP gene. The mRFP gene was used to analyze attachment of CBM3a to cellulose. We are improving the part by removing the mRFP gene and the N and C linkers.
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, part BBa_K3426000, 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.
Sequence and Features
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- 1000COMPATIBLE WITH RFC