Part:BBa_K733007:Design

Pveg promoter + spoVG RBS + lytC + helical linker + RPMrel peptide + consensus RBS + GFP + double terminator

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 roundabout manner detailed below.

1) PCR amplification of Pveg + spoVG RBS + lytC + linker + FLAGTM 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] [24bp reverse-complementary sequence of codon optimized FLAGTM] [15bp reverse-complementary overlap with linker] - 3'

Reverse primer sequence: 5’ – GTTTCTTCACTAGTATTATTATTTATCATCATCATCTTTATAATCGGCCGCGGCTTTCGC – 3’ (60bp)

It was discovered that the transposon insAB had been inserted within the lytC coding region of the K316037 plasmid we had used for amplification. Thus it was decided to amplify a ‘safe’ copy of the 1-954bp region of lytC and the linker + FLAGTM sequence separately after which overlapping PCR would be used to join them.

2) PCR amplification of 1-954bp lytC from the B. subtilis genome.

Forward primer design: 5’ – [6bp cap] [7bp XbaI restriction site] [30bp overlap with lytC] – 3’

Forward primer sequence: 5’ – GATCATTCTAGAGTTGCGTTCTTATATAAAAGTCCTAACAATG – 3’ (43bp)

Reverse primer design: 5’ – [27bp reverse-complementary overlap with lytC] – 3’

Reverse primer sequence: 5’ – TACAACTGGATTCTTTAGCTGATTAGC – 3’ (27bp)

3) PCR amplification of linker + FLAGTM from existing construct.

Forward primer design: 5’ – [31bp overhang overlapping with lytC] [19bp overlap with linker] – 3’

Forward primer sequence: 5’ – GGTTGCTAATCAGCTAAAGAATCCAGTTGTAAGCAGAGGCTCACGCGCAC – 3’ (50bp)

Reverse primer design: 5’ – [8bp cap] [6bp BamHI restriction site] [7bp SpeI restriction site] [31bp overlap with FLAGTM and linker sequence] – 3’

Reverse primer sequence: 5’ – GTTTCTTCGGATCCACTAGTATTATTATTTATCATCATCATCTTTATAATCG – 3’ (52bp)

NB. This reverse primer was originally intended to allow PCR addition of a BamHI site to the 3’ end of the construct. This would allow it to be ligated into the multiple cloning site of integration vector pDG1661. That function was never pursued and the primer was recycled for use here.

4) Overlapping PCR linking products from steps 2 and 3.

5) Ligation into standard backbone pSB1C3.

Following successful PCR the PCR product and pSB1C3 were digested with XbaI and SpeI. The two digestion products were then ligated together.