Difference between revisions of "Part:BBa K339010:Design"
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===Source===
 
===Source===
 
Designed from literature sequences by Kraft et al., 2007.
 
Designed from literature sequences by Kraft et al., 2007.
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<h2>Derivation of the promoter</h2>
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This promoter is found naturally in <i>E. coli</i>. This promoter is comprised of two naturally occuring promoters.
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 +
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Kraftpaperimage.png" border="0" alt="Kraft et al idea"></img>
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This image shows the plasmid schematic from paper by Kraft et al.
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 +
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In order to derive the promoter that is submitted, ibpAB (native) was blasted in order to locate the location in the  sequence provided in the paper.
 +
 +
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-clustal.png" border="0" alt="IbpAB"></img>
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Clustal W alignment with native promoter region in front of ibpAB operon(P2_ibpAB)and the sequence provided in the paper (P1_proposed)
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Similarly, the FxsA promoter (native) was also aligned with the entire sequence in the paper.
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 +
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/FxsA.png" border="0" alt="FxsA"></img>
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 +
These two alignments allowed us to derive the important DNA parts. There were parts eliminated. For example an internal PvoII site was eliminated as well at a T7 ribosome binding site was removed to use standard ribosome binding site. (B0034)
 +
 +
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Protein%20man/finalsequence.png" border="0" alt="ibpAB"></img>
  
  
 
===References===
 
===References===
 
Kraft, M., Knupfer, U., Wenderoth, R., Pietschmann, P., Hock, B., & Horn, U. (2007). An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of escherichia coli. Applied Microbiology and Biotechnology, 75(2), 397-406.
 
Kraft, M., Knupfer, U., Wenderoth, R., Pietschmann, P., Hock, B., & Horn, U. (2007). An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of escherichia coli. Applied Microbiology and Biotechnology, 75(2), 397-406.

Revision as of 06:36, 30 October 2010

ibpAB


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

Source

Designed from literature sequences by Kraft et al., 2007.

Derivation of the promoter

This promoter is found naturally in E. coli. This promoter is comprised of two naturally occuring promoters.

<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Kraftpaperimage.png" border="0" alt="Kraft et al idea"></img>

This image shows the plasmid schematic from paper by Kraft et al.


In order to derive the promoter that is submitted, ibpAB (native) was blasted in order to locate the location in the sequence provided in the paper.

<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-clustal.png" border="0" alt="IbpAB"></img> Clustal W alignment with native promoter region in front of ibpAB operon(P2_ibpAB)and the sequence provided in the paper (P1_proposed)

Similarly, the FxsA promoter (native) was also aligned with the entire sequence in the paper.

<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/FxsA.png" border="0" alt="FxsA"></img>

These two alignments allowed us to derive the important DNA parts. There were parts eliminated. For example an internal PvoII site was eliminated as well at a T7 ribosome binding site was removed to use standard ribosome binding site. (B0034)

<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Protein%20man/finalsequence.png" border="0" alt="ibpAB"></img>


References

Kraft, M., Knupfer, U., Wenderoth, R., Pietschmann, P., Hock, B., & Horn, U. (2007). An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of escherichia coli. Applied Microbiology and Biotechnology, 75(2), 397-406.