Latest revision as of 01:17, 2 October 2024

R0010-BaCBM2-Cys

This composite part codes for the BaCBM2 protein with an additional amino acid (cysteine), expressed by the R0010 promoter in the presence of IPTG (regulated by lacI).

Usage and Biology

This CBM2, or Carbohydrate-Binding Module 2, is a protein sourced from Bacillus anthracis. It belongs to a broader family of carbohydrate-binding modules that are crucial for the degradation of polysaccharides. These modules are important to break down complex carbohydrates, enabling microorganisms to convert them into usable energy sources.

Recent study has shown that CBM2 has the ability to bind to certain types of plastics, especially those derived exhibiting similar structural features of polysaccharides. This binding ability is largely due to the protein's carbohydrate-binding properties, which facilitate interactions with specific functional groups found on plastic surfaces. [1]

Figure 1. 3D simulation of BaCBM2-Cys.

The cysteine modification allows a strong interaction between the protein and our sensor surface, due to the affinity between the SH group and the Au(111) surface. This increase in interaction with our sensor is essential for amplifying the signal of microplastics in electrochemical measurements.

Part Generation

The BaCBM2-Cys was generated by PCR using as template the BBa_K5396000

The reverse primer adds the cysteine at the end of the sequence. Our plasmid was assembled using the Golden Gate Assembly with the following parts:

• BBa_J428341(linear, digested with BsaI separately and purified from agarose gel)
• BBa_R0010
• BBa_J435345
• BBa_K5396003
• and BBa_J428069

We transformed the plasmids through electroporation into the E. coli strain DH5α and confirmed the correct assembly by Sanger sequencing.

We did not proceed with the purification, characterization of BaCBM2-Cys, or sensor testing. Instead, we shifted our focus to the third cycle of our project, which also involves BaCBM2-Cys, but fused to the N-terminal of spidroin: BBa_K5396010

Sequence and Features