RBS

Part:BBa_K4601132

Designed by: Doriane Blaise   Group: iGEM23_Evry-Paris-Saclay   (2023-09-23)


Synthetic RBS designed for LacZalpha

This part is a synthetic RBS specifically designed for the LacZ-alpha (pUC19 like) (BBa_K2448003) using the Salis Lab RBS Library Calculator v2.0 [1-3].

Usage and Biology

This RBS was used to drive the expression of LacZ-alpha (pUC19 like) (BBa_K2448003) under the control of the Na+ RiboSwitch v2 (BBa_K4601022) in the composite part BBa_K4601232. In this context, the predicted features of this RBS, according to Salis Lab RBS Calculator v2.1.1 [1,4-7], are: 

Translation Initiation Rate (au)	:	29191.35
dG_total (kcal/mol)	:	-7.03
dG_mRNA_rRNA (kcal/mol)	:	-32.17
dG_spacing (kcal/mol)	:	0.01
dG_stacking (kcal/mol)	:	-0.38
dG_standby (kcal/mol)	:	-0.00
dG_start (kcal/mol)	:	-2.76
dG_mRNA (kcal/mol)	:	-28.28
Warnings	:	none

This RBS was selected fortuitously during the cloning process from a library of 96 RBSes (ASWTTAATAATKTAKAGAGGVGGTATAK) for which the estimated Translation Initiation Rates (TIR) range from 4223.38 to 393272.53.

References

[1] Reis AC, Salis HM. An automated model test system for systematic development and improvement of gene expression models. ACS synthetic biology (2020) 9: 3145–3156.

[2] Farasat I, Kushwaha M, Collens J, Easterbrook M, Guido M, Salis HM. Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria. Molecular Systems Biology (2014) 10: 731.

[3] Ng CY, Farasat I, Maranas CD, Salis HM. Rational design of a synthetic Entner-Doudoroff pathway for improved and controllable NADPH regeneration. Metabolic Engineering (2015) 29: 86–96.

[4] Cetnar DP, Salis HM. Systematic quantification of sequence and structural determinants controlling mRNA stability in bacterial operons. ACS Synthetic Biology (2021) 10: 318–332.

[5] Espah Borujeni A, Cetnar D, Farasat I, Smith A, Lundgren N, Salis HM. Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences. Nucleic Acids Research (2017) 45: 5437–5448.

[6] Espah Borujeni A, Channarasappa AS, Salis HM. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Research (2014) 42: 2646–2659.

[7] Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology (2009) 27: 946–950.

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]


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Categories
Parameters
None