Part:BBa_K733007:Design
Pveg + spoVG RBS + lytC + linker + RPMrel + consensus RBS + GFP + double terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Part Source
Pveg, spoVG (RBS), the cell wall binding domain of lytC and the helical linker are all components of Imperial College London's 2010 team's detection module. These components allow high expression of any tags subsequently attached to the linker on the cell wall of Bacillus subtilis.
The coding sequence of the screened phage display peptide 'RPMrel' was produced via codon optimization of RPMrel's amino acid sequence for Bacillus subtilis. This amino acid sequence (n-CPIEDRPMC-c) came out of work conducted by [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550331/pdf/neo0505_0437.pdf Kelly & Jones (2003)] to isolate colon tumor specific binding peptides from New England Biolabs' 'PhD-CX7C' phage display peptide library.
The B. subtilis consensus RBS used to express GFP was originally submitted to the Registry by Cambridge University’s 2008 team.
The GFP coding sequence used is derived from that naturally encoded in the Aequeora victoria genome and is further modified by amino acid substitution.
Our selected termination sequence was designed by Registry staff as a combination of a pair of hairpin sequences.
Design Considerations
Construction of this part in submission form was performed in the manner detailed below.
1) PCR amplification of consensus RBS + GFP + double terminator region using BBa_E0240 as the template.
2) Ligation into standard backbone pSB1C3.
3) PCR amplification of Pveg + spoVG RBS + lytC + linker + RPMrel region using BBa_K316037 as the template.
Forward primer design: 5’ – [6bp cap] [20bp overlap with standard prefix] – 3’
Forward primer sequence: 5’ – GATCATGAATTCGCGGCCGCTTCTAG – 3’ (26bp)
Reverse primer design: 5' - [8bp cap] [7bp SpeI restriction site] [6bp reverse-complementary double stop codon] [27bp reverse-complementary sequence of codon optimized RPMrel] [15bp reverse-complementary overlap with linker] - 3'
Reverse primer sequence: 5’ - GTTTCTTCACTAGTATTATTAACACATCGGGCGATCTTCGATCGGACAGGCCGCGGCTTTCGC - 3’ (63bp)
4) Ligation into consensus RBS + GFP + double terminator in standard backbone (from step 2).
Following successful PCR the PCR product was digested with EcoRI and SpeI, and the RBS + GFP + double terminator construct in backbone was digested with EcoRI and XbaI. The two digestion products were then ligated together.