Difference between revisions of "Part:BBa K5396010"
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The Nt-BaCBM2-Cys was created through PCR amplification and Gibson Assembly utilizing our composite parts: | The Nt-BaCBM2-Cys was created through PCR amplification and Gibson Assembly utilizing our composite parts: | ||
| − | *<partinfo> | + | *<partinfo>BBa_K5396009</partinfo> |
| − | *and <partinfo> | + | *and <partinfo>K5396010</partinfo> |
The product of the reaction was transformed into the ''E. coli'' strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing. | The product of the reaction was transformed into the ''E. coli'' strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing. | ||
Revision as of 18:16, 26 September 2024
T7-Nt-BaCBM2-Cys
This composite part codes for the N-terminal of Spidroin Nt2RepCt fused to our BaCBM2-Cys, controlled by T7-LacO promoter and is expressed in the presence of IPTG.
Usage and Biology
Nt2RepCt
Spidroins are the primary proteins that compose spider silk. This part contains the N-terminal domain, which is involved in the initial formation of silk fibers and is crucial for the protein's solubility and stability, and is fused to the BaCBM2 protein, that has the ability to bind to plastics.
BaCBM2-Cys
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.
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 the sensor is essential for amplifying the signal of microplastics in electrochemical measurements.
Part Generation
The Nt-BaCBM2-Cys was created through PCR amplification and Gibson Assembly utilizing our composite parts:
The product of the reaction was transformed into the E. coli strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing.
This Biobrick consists of the following basic parts:
Expression and Purification
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 96
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 30
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 96
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 96
- 1000COMPATIBLE WITH RFC[1000]