![](https://parts.igem.org/images/partbypart/icon_coding.png)
Part:BBa_K2273104:Design
AmyERFC25 version of B. subtilis alpha-amylase without its native signal peptide
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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