Difference between revisions of "Part:BBa K2524011:Design"

 
(→‎Design Notes)
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Add info regarding why this part is an improvement. Strategies related to previous issues during detection - bands lost in dye front.
 
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===
 
===Source===

Revision as of 22:53, 3 October 2017


Mambalgin-1 in E. coli Improvement


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 763
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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

References