Difference between revisions of "Part:BBa K3425031"
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<b>About Sequence Context Dependence</b> | <b>About Sequence Context Dependence</b> | ||
<p> | <p> | ||
− | 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 | + | 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 |
<a href="https://parts.igem.org/Part:BBa_K1114107">BBa_K1114107</a> or | <a href="https://parts.igem.org/Part:BBa_K1114107">BBa_K1114107</a> or | ||
<a href="https://parts.igem.org/Part:BBa_J364100">BBa_J364100</a>. | <a href="https://parts.igem.org/Part:BBa_J364100">BBa_J364100</a>. | ||
<br> | <br> | ||
− | + | <b>Characterization Issue</b> | |
− | + | <p> | |
− | In line with that, previous characterizations report different relative strengths of <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a> and other members of <a href="https://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection"> RBS Community Collection</a>. | + | The ancestral RBS of this part, |
+ | <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>, | ||
+ | was characterized as strong. However, due to the inherent dependence on the sequence context described above, the results are necessarily affected by the choice of the reporter. | ||
+ | <br> | ||
+ | In line with that, previous characterizations report different relative strengths of | ||
+ | <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a> | ||
+ | and other members of | ||
+ | <a href="https://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection"> | ||
+ | RBS Community Collection</a>. | ||
+ | </p> | ||
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">iGEM resources</a>or [1]). Aligned spacing generally varies from 5 to 13 bases [1] and optimal distance varies for organisms. Final aligned spacing of BBa_K3425031 is 9 nucleotides when assembled using<a href="https://parts.igem.org/Help:Standards/Assembly/Type_IIS"> Type IIS iGEM Standard assembly</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">iGEM resources</a>or [1]). Aligned spacing generally varies from 5 to 13 bases [1] and optimal distance varies for organisms. Final aligned spacing of BBa_K3425031 is 9 nucleotides when assembled using<a href="https://parts.igem.org/Help:Standards/Assembly/Type_IIS"> Type IIS iGEM Standard assembly</a>. |
Revision as of 10:08, 19 October 2020
RBS for Type IIS iGEM Standard Assembly
BBa_K3425031 is an RBS (BBa_B0032) modified for Type IIS iGEM Standard assembly (RFC1000) by adding a five-nucleotide spacer (TACTA) to the end of the sequence.
About 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_B0032 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.
About 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 Issue
The ancestral RBS of this part,
BBa_B0032,
was characterized as 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_B0032
and other members of
RBS Community Collection.
In conclusion, the performance of BBa_K3425031 can be only approximated by the characterization available for BBa_B0032 and it will always vary when used for expression of different genes.
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]