Difference between revisions of "Part:BBa K3425032"
 
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<partinfo>BBa_K3425026 short</partinfo>
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<partinfo>BBa_K3425032 short</partinfo>
  
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BBa_K3425025 is an RBS (<ahref="https://parts.igem.org/wiki/index.php/Part:BBa_B0030">BBa_B0030</a>) modified for Type IIS iGEM Standard assembly (RFC1000) by adding a five-nucleotide spacer (TAGTA) to the end of the sequence.  
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<html>
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<p>
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BBa_K3425032 is an RBS (<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) modified for <a href="https://parts.igem.org/Help:Standards/Assembly/Type_IIS">Type IIS iGEM Standard assembly</a> (RFC1000) by adding a five-nucleotide spacer (TAGTA) to the end of the sequence.
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The distance between an RBS and the ATG start codon is an important factor affecting the strength of an RBS. Whereas BioBrick assembly generates six-nucleotide scar between the RBS and the ATG, Type IIS iGEM Standard assembly generates only one-nucleotide scar. Therefore, five nucleotides were added to the BBa_B0030 for its use in Type IIS assembly. However, note that the nucleotides are different.
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</p>
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<p>This part is part of iGEM Uppsala 2020's <a href="https://parts.igem.org/Collections/iGEM_Type_IIS_Collections">iGEM Type IIS standard collection</a>.</p>
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<br>
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Generally, the strength of an RBS depends heavily on 1) the sequence of the RBS 2) the aligned spacing and 3) the upstream and downstream genetic context. Ancestral RBS <ahref="https://parts.igem.org/wiki/index.php/Part:BBa_B0030">BBa_B0030</a> was characterized as strong, however due to the dependence on the sequence context, the results are necessarily affected by the choice of the reporter. In line with that, previous characterizations report different relative strength of <ahref="https://parts.igem.org/wiki/index.php/Part:BBa_B0030">BBa_B0030</a> and other members of [<a href="https://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection">RBS Community Collection</a>]. Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and ATG start codon (see <a href="https://parts.igem.org/Ribosome_Binding_Sites/Design">here</a> or refer to Ma <i>et al.</i> (2002)). Aligned spacing generally varies from 5 to 13 bases Ma <i>et al.</i> (2002) and optimal distance varies for organisms. Final aligned spacing of BBa_K3425025 when Type IIS iGEM Standard assembly is used is 10 nucleotides.
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<b>Aligned Spacing</b>
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<p>
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In conclusion, the performance of BBa_K3425025 can be only approximated by the characterization available for BBa_B0030 and it will always differ for expression of different genes.
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The distance between an RBS and a start codon is an important factor affecting the strength of an RBS. Whereas BioBrick assembly generates six-nucleotide scar between the RBS and the start codon,
 +
<a href="https://parts.igem.org/Help:Standards/Assembly/Type_IIS">Type IIS iGEM Standard assembly</a>
 +
generates only one-nucleotide scar. Therefore, five nucleotides were added to the
 +
<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>
 +
for its use in Type IIS assembly. However, note that the nucleotides of the spacer are different from the BioBrick scar, which might influence the performance of RBS.
 +
</p>
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<p>
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Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and the start codon (see
 +
<a href="https://parts.igem.org/Ribosome_Binding_Sites/Design">here</a>
 +
or reference [1]). Aligned spacing generally varies from 5 to 13 bases [1] and optimal distance varies for organisms. Final aligned spacing of BBa_K3425032 is 10 nucleotides when assembled using
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<a href="https://parts.igem.org/Help:Standards/Assembly/Type_IIS">Type IIS iGEM standard assembly</a>.
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</p>
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<b>Sequence Context Dependence</b>
  
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<p>
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Generally, the strength of an RBS depends heavily on <i><b>1)</b> the sequence of the RBS <b>2)</b> the aligned spacing and <b>3)</b> the upstream and downstream genetic context.</i> Therefore, the same RBS element performs differently in different sequence context. This means, that identical RBS element results in different expression levels for different genes. An interesting solution to this problem might be implementation of bicistronic architecture [1], for example BCD2
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<a href="https://parts.igem.org/Part:BBa_K1114107">BBa_K1114107</a> or
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<a href="https://parts.igem.org/Part:BBa_J364100">BBa_J364100</a>.
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</p>
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<b>Characterization Issues</b>
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<p>
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The ancestral RBS of this part,
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<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>
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was characterized as medium strong. However, due to the inherent dependence on the sequence context described above, the results are necessarily affected by the choice of the reporter. 
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<br>
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In line with that, previous characterizations report different relative strengths of <a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and other members of <a href="https://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection"> RBS Community Collection</a>.
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</p>
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<b>Conclusion</b>
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<p>
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In conclusion, the performance of BBa_K3425032 can be only approximated by the characterization available for <a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>. The actual performance will be affected by the choice of spacer and the sequence context including the expressed gene. By using BBa_K3425032 and Type IIS iGEM standard assembly, the spacer is defined.
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</p>
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</html>
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<br><br>
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<b>Refereces</b>
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<br>
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[1] Ma, J., Campbell, A., and Karlin, S. (2002) Correlations between Shine-Dalgarno Sequences and Gene Features Such as Predicted Expression Levels and Operon Structures. J. Bacteriol. 184, 5733–5745
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<br>
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[2] Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., and Endy, D. (2013) Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods. 10, 354–360
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<br>
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<br><br>
 
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===Usage and Biology===
 
===Usage and Biology===
  
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><b>Sequence and Features</b></span>
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<partinfo>BBa_K3425026 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3425032 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3425026 parameters</partinfo>
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<partinfo>BBa_K3425032 parameters</partinfo>
 
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Latest revision as of 21:24, 25 October 2020


RBS for Type IIS iGEM Standard Assembly

BBa_K3425032 is an RBS (BBa_B0034) modified for Type IIS iGEM Standard assembly (RFC1000) by adding a five-nucleotide spacer (TAGTA) to the end of the sequence.

This part is part of iGEM Uppsala 2020's iGEM Type IIS standard collection.


Aligned Spacing

The distance between an RBS and a start codon is an important factor affecting the strength of an RBS. Whereas BioBrick assembly generates six-nucleotide scar between the RBS and the start codon, Type IIS iGEM Standard assembly generates only one-nucleotide scar. Therefore, five nucleotides were added to the BBa_B0034 for its use in Type IIS assembly. However, note that the nucleotides of the spacer are different from the BioBrick scar, which might influence the performance of RBS.

Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and the start codon (see here or reference [1]). Aligned spacing generally varies from 5 to 13 bases [1] and optimal distance varies for organisms. Final aligned spacing of BBa_K3425032 is 10 nucleotides when assembled using Type IIS iGEM standard assembly.

Sequence Context Dependence

Generally, the strength of an RBS depends heavily on 1) the sequence of the RBS 2) the aligned spacing and 3) the upstream and downstream genetic context. Therefore, the same RBS element performs differently in different sequence context. This means, that identical RBS element results in different expression levels for different genes. An interesting solution to this problem might be implementation of bicistronic architecture [1], for example BCD2 BBa_K1114107 or BBa_J364100.

Characterization Issues

The ancestral RBS of this part, BBa_B0034 was characterized as medium strong. However, due to the inherent dependence on the sequence context described above, the results are necessarily affected by the choice of the reporter.
In line with that, previous characterizations report different relative strengths of BBa_B0034 and other members of RBS Community Collection.

Conclusion

In conclusion, the performance of BBa_K3425032 can be only approximated by the characterization available for BBa_B0034. The actual performance will be affected by the choice of spacer and the sequence context including the expressed gene. By using BBa_K3425032 and Type IIS iGEM standard assembly, the spacer is defined.



Refereces
[1] Ma, J., Campbell, A., and Karlin, S. (2002) Correlations between Shine-Dalgarno Sequences and Gene Features Such as Predicted Expression Levels and Operon Structures. J. Bacteriol. 184, 5733–5745
[2] Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., and Endy, D. (2013) Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods. 10, 354–360


Sequence and Features


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]