Difference between revisions of "Part:BBa K3425031"

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	generates only one-nucleotide scar. Therefore, five nucleotides were added to the		generates only one-nucleotide scar. Therefore, five nucleotides were added to the
	<a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>		<a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</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. Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and ATG start codon (see iGEM resources 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 Type IIS iGEM Standard assembly.	+	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.
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		+	Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and ATG start codon (see iGEM resources 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 Type IIS iGEM Standard assembly.
	</p>		</p>
	<b>Sequence Context Dependence</b>		<b>Sequence Context Dependence</b>

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Revision as of 10:23, 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.

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.
Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and ATG start codon (see iGEM resources 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 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 [2], for example BCD2 BBa_K1114107 or BBa_J364100.

Characterization Issues

The ancestral RBS of this part, BBa_B0032, was characterized as weak. 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.

Aligned spacing is the distance between the centre of Shine-Dalgarno sequence and ATG start codon (see iGEM resourcesor [2]). 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
[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