Part:BBa_K3425031

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.
Ancestral RBS BBa_B0032 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 strengths of BBa_B0032 and other members of RBS Community Collection. Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and ATG start codon (see iGEM resourcesor [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 Type IIS iGEM Standard assembly.

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

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]