Coding
AmyE_RFC25

Part:BBa_K2273104:Design

Designed by: Henri Deda   Group: iGEM17_TU_Dresden   (2017-09-26)

AmyERFC25 version of B. subtilis alpha-amylase without its native signal peptide


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design

This part has the following prefix and suffix which includes a strong Shine Dalgarno Sequence (SD):

Prefix with EcoRI, NotI, XbaI and SD 5'-GAATTCGCGGCCGCTTCTAGAGAAGGAGTGTCAAGA-3'
Suffix with SpeI, NotI and PstI 5'-TACTAGTAGCGGCCGCTGCAG-3'

Sites of restriction enzymes generating compatible overhangs are indicated by sharing one color. (EcoRI and PstI are marked in blue, NotI in green, XbaI and SpeI are red. Additionally, the Shine Dalgarno Sequence is marked in silver.)

This part was designed to meet the RFC25 criteria and is used in the 2017 TU Dresden iGEM project [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)].

Source

The AmyERFC25 of B. subtilis alpha-amylase without its native signal peptide was amplified via PCR from the B. subtilis wild type W168 genome using the primers listed below.

AmyE_RFC25 I fwd gatcTCTAGAGAAGGAGTGTCAAGAGAAACGGCGAACAAATCGAATGAG
AmyE_RFC25 III rev gatcCTGCAGCGGCCGCTACTAGTATCAATGGGGAAGAGAACCGCTTAA

Additionally, a range of RFC25 opposing restriction sites was removed via PCR mutagenesis. Following PCR amplification, the AmyERFC25 was digested using XbaI and PstI and ligated into pSB1C3.

References

Ulf Brockmeier, Michael Caspers, Roland Freudl, Alexander Jockwer, Thomas Noll and Thorsten Eggert "Systematic Screening of All Signal Peptides from Bacillus subtilis: A Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-positive Bacteria" Journal of Molecular Biology 362 (2006): 393-402. PubMed