Part:BBa_K2524011:Design

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

To improve our previous Mambalgin-1 part

We improved our previous Mambalgin-1 construct (https://parts.igem.org/Part:BBa_K1110003) for E. coli this year by removing extra base pairs that coded for a portion of the signal peptide segment. The new sequence produces the Mambalgin-1 protein without any signal peptide. Next, we added a TAC promoter, LAC operon, GST-Tag, and an HRV-3 protease site upstream of the Mambalgin-1 coding sequence. This increased the size of our target protein by 26 kDa, which made protein band detection much easier on SDS PAGE. Subsequently, the strategy solved our issues related to losing our protein band in the SDS PAGE dye-front.

The GST tag also allowed for affinity gravity column chromatography purification. In fact, the HRV-3 protease site made cleavage of Mambalgin-1 from the GST tag possible in our lab (we don’t have access to FPLC).

Uniprot research of Mambalgin-1 protein conducted:

  - Determined functional portion of protein sequence --> trim down unnecessary amino acids
      - Original protein sequence contains an initial signal sequence which was not included 

The construct was submitted to IDT in minigene format.

Results:

http://2017.igem.org/wiki/thumb/6/69/T--Georgia_State--Mamba_IDT.png




Primers were designed to add restriction sites in order to remove the sequence from the plasmid.

Mamba EcoRI Forward TCTAAGAATTCATGCTGAAATGTTACCAACATGG

Mamba NotI Reverse ATTATGCGGCCGCCTATTTGTTGCATCTGTCTGTTGAG

The restriction sites made the sequence compatible for insertion into the PGEX 6p-3t backbone.


PCR to create official biobrick prefix site proximal to Tac promoter

Mamba Add XbaI Forward GCAATATCTAGAGTTGACAATTAATCATCGGCTCG

Mamba add SpeI & PstI Reverse CTGCAGCGGCCGCTACTAGTACTATTTGTTGCATCTGTCTGTTG


Generator part into PSB1C3


Mutagenesis to remove second EcoRI site upstream of Mamba CDS

EcoRI Mutagen Forward-sense CCCTGGGATCCCCGAATCCGATGCTGAAATGT

EcoRI Mutagen Antisense ACATTTCAGCATCGGATTCGGGGATCCCAGGG





Protein Expression Protein purification - GST sepharose 4B resin Column protease cleavage with HRV-3 protease -> elute pure Mambalgin-1 afterward Protein Detection - western blot with monoclonal Anti-GST antibodies f

Source

Protein sequence from UNIPROT NCBI Blast

===References===