Difference between revisions of "Part:BBa K733008:Design"
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Construction of this part in submission form was performed in the roundabout manner detailed below. | Construction of this part in submission form was performed in the roundabout manner detailed below. | ||
| − | '''1) PCR amplification of Pveg + spoVG RBS + | + | '''1) PCR amplification of Pveg + spoVG RBS + LytC + linker + FLAG™ region using BBa_K316037 as the template.''' |
''Forward primer design:'' | ''Forward primer design:'' | ||
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5’ – GTTTCTTCACTAGTATTATTATTTATCATCATCATCTTTATAATCGGCCGCGGCTTTCGC – 3’ (60bp) | 5’ – GTTTCTTCACTAGTATTATTATTTATCATCATCATCTTTATAATCGGCCGCGGCTTTCGC – 3’ (60bp) | ||
| − | It was discovered that the transposon insAB had been inserted within the | + | 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 + FLAG™ sequence separately after which overlapping PCR would be used to join them. |
| − | '''2) PCR amplification of 1-954bp | + | '''2) PCR amplification of 1-954bp LytC from the B. subtilis genome.''' |
''Forward primer design:'' | ''Forward primer design:'' | ||
| − | 5’ – [6bp cap] [7bp XbaI restriction site] [30bp overlap with | + | 5’ – [6bp cap] [7bp XbaI restriction site] [30bp overlap with LytC] – 3’ |
''Forward primer sequence:'' | ''Forward primer sequence:'' | ||
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''Reverse primer design:'' | ''Reverse primer design:'' | ||
| − | 5’ – [27bp reverse-complementary overlap with | + | 5’ – [27bp reverse-complementary overlap with LytC] – 3’ |
''Reverse primer sequence:'' | ''Reverse primer sequence:'' | ||
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''Forward primer design:'' | ''Forward primer design:'' | ||
| − | 5’ – [31bp overhang overlapping with | + | 5’ – [31bp overhang overlapping with LytC] [19bp overlap with linker] – 3’ |
''Forward primer sequence:'' | ''Forward primer sequence:'' | ||
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'''5) Ligation into standard backbone pSB1C3.''' | '''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. | + | Following successful PCR the PCR product and pSB1C3 were digested with XbaI and SpeI. The two digestion products were then ligated together. |
| − | + | ||
== '''Part Source''' == | == '''Part Source''' == | ||
Revision as of 14:38, 26 September 2012
lytC + linker + FLAG
- 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 Notes
Construction of this part in submission form was performed in the roundabout manner detailed below.
1) PCR amplification of Pveg + spoVG RBS + LytC + linker + FLAG™ 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 FLAG™] [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 + FLAG™ 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 + FLAG™ 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 FLAG™ 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.
Part Source
The 1-954bp region of lytC presented in this BioBrick was amplified from the Bacillus subtilis genome using specific primers.
The helical linker is a component of Imperial College London's 2010 team's detection module.
The amino acid sequence of the FLAG™ peptide and its derivative peptides exist in the public domain. The DNA coding sequence we submit here was generated by codon optimization for expression in the B. subtilis chassis.
References
Yamamoto, Hiroki, Shin-ichirou Kurosawa, and Junichi Sekiguchi. "Localization of the Vegetative Cell Wall Hydrolases LytC, LytE, and LytF on the Bacillus subtilis Cell Surface and Stability of These Enzymes to Cell Wall-Bound or Extracellular Proteases." Journal of Bacteriology 185.22 (2003): 6666-6677. Print.