Part:BBa_K2524011:Design
Mambalgin-1 in E. coli Improvement
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 763
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 160
Design Notes
Add info regarding why this part is an improvement. Strategies related to previous issues during detection - bands lost in dye front.
Previous Mamba construct in E. coli [ Insert link to 2015 part page]
Why we decided to do generator construct - Previous attempts with 2015 construct yielded questionable results. Protein of interest was ~9 kDa, which tended to get lost within the dye-front during SDS PAGE.
How the CDNA sequence differs from 2015 construct [ picture of alignment ]
How the portions of the sequence benefit our construct strategy.
History outline of construct development
DNA research through NCBI blast
- comparisons between the old sequence and original from snake
Uniprot research of Mambalgin-1 protein
- Determine functional portion of protein sequence --> trim down unnecessary amino acids
- Original protein sequence contains an initial signal sequence which was not included
CDNA sequence design in snapgene
IDT plasmid
Primers to add restriction sites
Part into PGEX
PCR to create official biobrick prefix site proximal to Tac promoter
Generator part into PSB1C3
Mutagenesis to remove second EcoRI site upstream of Mamba CDS
Sequencing
Protein Expression Protein purification - GST sepharose resin Protein Detection - western blot with monoclonal Anti-GST antibodies
Source
Protein sequence from UNIPROT NCBI Blast