Part:BBa_K3425033

RBS for Type IIS iGEM Standard Assembly

BBa_K3425033 is an RBS (BBa_B0030) 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. Sequencing results of this part cloned in pSB1C00 can be found in the Sequence Analysis section and the trace files are in the collection page as well as our Parts page.

Aligned Spacing

The distance between an RBS and the 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_B0030 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 ATG 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_K3425033 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_B0030 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_B0030 and other members of RBS Community Collection.

Conclusion

In conclusion, the performance of BBa_K3425033 can be only approximated by the characterization available for BBa_B0030. The actual performance will be affected by the choice of spacer and the sequence context including the expressed gene. By using BBa_K3425033 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]