Difference between revisions of "Part:BBa K733008:Design"
Line 3: Line 3:
 
===Layout===
 
===Layout===
 
<partinfo>BBa_K733008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K733008 SequenceAndFeatures</partinfo>
−
 
  
 
===Design Notes===
 
===Design Notes===
−
Not available at this moment.
+
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.
  
 
===Source===
 
===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.
  
−
Not available at this moment.
+
The amino acid sequence of the FLAGTM 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===
 
===References===

Revision as of 09:41, 23 September 2012

lytC + linker + FLAG

Layout


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 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 + 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.

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 FLAGTM 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