Difference between revisions of "Part:BBa K2273057:Design"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2273057 short</partinfo>
 
<partinfo>BBa_K2273057 short</partinfo>
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<partinfo>BBa_K2273057 SequenceAndFeatures</partinfo><br>
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<partinfo>BBa_K2273057 SequenceAndFeatures</partinfo>
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===Design===
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This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix:<br>
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<table>
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===Design Notes===
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<tr>
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tba
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<td width="70">Prefix with</td>
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<td width="200"><span style="color:blue">EcoRI</span>, <span style="color:green">NotI</span>, <span style="color:red">XbaI</span> and <span style="color:silver">SD</span></td>
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<td><span style="color:blue">GAATTC</span><span style="color:green">GCGGCCGC</span>T<span style="color:red">TCTAGA</span>T<span style="color:silver">AAGGAGG</span>TCAAAA</td>
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</tr>
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<tr>
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<td>Suffix with</td>
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<td><span style="color:orange">AgeI</span>, <span style="color:red">SpeI</span>, <span style="color:green">NotI</span> and <span style="color:blue">PstI</span></td>
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<td><span style="color:orange">ACCGGT</span><u>TAA</u>T<span style="color:red">ACTAGT</span>A<span style="color:green">GCGGCCG</span><span style="color:blue">CTGCAG</span>A</td>
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</tr>
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</table>
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Sites of restriction enzymes generating compatible overhangs are indicated by sharing one color. (<span style="color:blue">EcoRI</span> and <span style="color:blue">PstI</span> are marked in blue, <span style="color:green">NotI</span> in green, <span style="color:red">XbaI</span> and <span style="color:red">SpeI</span> in red and <span style="color:orange">AgeI</span> in orange. Additionally, the <span style="color:silver">Shine-Dalgarno sequence</span> is marked in silver and the stop codon is underlined.)<br><br>
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This part was designed to meet the needs of the Signal Peptide Toolbox which was created by the [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)].<br><br>
 
===Source===
 
===Source===
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The PhrA signal peptide of <i>B. subtilis</i> Phosphatase RapA inhibitor was amplified via PCR from the <i>B. subtilis</i> wild type W168 genome using the primers listed below.
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tba
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<br>
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<table>
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<tr>
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<td width="150">PhrA_SP fwd</td>
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<td>gatcGAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAAATGAAATCTAAATGGATGTCAG</td>
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</tr>
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<tr>
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<td>PhrA_SP rev</td>
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<td>gatcTCTGCAGCGGCCGCTACTAGTAttaaccggtTGCATGAACCATCACCTGAG</td>
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</tr>
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</table>
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Following amplification, the signal peptide was digested using EcoRI and PstI and ligated into pSB1C3.<br><br>
 
===References===
 
===References===
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Ulf Brockmeier, Michael Caspers, Roland Freudl, Alexander Jockwer, Thomas Noll and Thorsten Eggert "Systematic Screening of All Signal Peptides from <i>Bacillus subtilis</i>: A Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-positive Bacteria" Journal of Molecular Biology 362 (2006): 393-402. [https://www.ncbi.nlm.nih.gov/pubmed/16930615 PubMed]<br><br>
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Jan Maarten van Dijl and Michael Hecker "<i>Bacillus subtilis</i>: from soil bacterium to super-secreting cell factory" Microbial Cell Factories 12:3 (2013): 1-6. [https://www.ncbi.nlm.nih.gov/pubmed/23311580 PubMed]<br><br>
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Ling lin Fu, Zi Rong Xu, Wei Fen Li, Jiang Bing Shuai, Ping Lu, Chun Xia Hu "Protein secretion pathways in <i>Bacillus subtilis</i>: Implication for optimization heterologous protein secretion" Biotechnology Advances 25 (2007): 1-12. [https://www.ncbi.nlm.nih.gov/pubmed/16997527 PubMed]

Revision as of 13:08, 30 September 2017

PhrA signal peptide of B. subtilis Phosphatase RapA inhibitor


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

This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix:

Prefix with EcoRI, NotI, XbaI and SD GAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAA
Suffix with AgeI, SpeI, NotI and PstI ACCGGTTAATACTAGTAGCGGCCGCTGCAGA

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 in red and AgeI in orange. Additionally, the Shine-Dalgarno sequence is marked in silver and the stop codon is underlined.)

This part was designed to meet the needs of the Signal Peptide Toolbox which was created by the [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)].

Source

The PhrA signal peptide of B. subtilis Phosphatase RapA inhibitor was amplified via PCR from the B. subtilis wild type W168 genome using the primers listed below.

PhrA_SP fwd gatcGAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAAATGAAATCTAAATGGATGTCAG
PhrA_SP rev gatcTCTGCAGCGGCCGCTACTAGTAttaaccggtTGCATGAACCATCACCTGAG

Following amplification, the signal peptide was digested using EcoRI 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

Jan Maarten van Dijl and Michael Hecker "Bacillus subtilis: from soil bacterium to super-secreting cell factory" Microbial Cell Factories 12:3 (2013): 1-6. PubMed

Ling lin Fu, Zi Rong Xu, Wei Fen Li, Jiang Bing Shuai, Ping Lu, Chun Xia Hu "Protein secretion pathways in Bacillus subtilis: Implication for optimization heterologous protein secretion" Biotechnology Advances 25 (2007): 1-12. PubMed