Part:BBa_K2273103:Design

AmyE_EV 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 was generated in a modified version of RFC25 and has the following prefix and suffix:

Prefix with	EcoRI, NotI, XbaI, AgeI and NgoMIV	5'-GAATTCGCGGCCGCTTCTAGAACCGGTGCCGGC-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 AgeI and NgoMIV are marked in orange. NgoMIV was underlined for clarity.)

This part was designed to meet the RFC25 criteria and is used as gene of interest for proof of concept of the Signal Peptide Toolbox of [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)].

Source

The AmyE_EV of B. subtilis alpha-amylase without its native signal peptide was amplified via PCR from AmyE_RFC25 (BBa_K2273104) using the primers listed below.

AmyE_EV fwd	gatcGAATTCGCGGCCGCTTCTAGACCGGTGCCGGCGAAACGGCGAACAAATCGAATGAG
AmyE_RFC25 III rev	gatcCTGCAGCGGCCGCTACTAGTATCAATGGGGAAGAGAACCGCTTAA

Following PCR amplification, the AmyE_EV was digested using EcoRI 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