Coding

Part:BBa_K4294207

Designed by: Aristotelis Anastopoulos   Group: iGEM22_Athens   (2022-09-30)


36nt LuxI - sfGFP

This is the coding sequence of sfGFP, fused with the genetic context of luxI gene.

Design

To prove the reliability and context independence of the BCDs, Mutalik et. al engineered several fusions of the first 36 nucleotides of commonly used regulators and enzymes with superfolder Green Fluorescent Protein (sfGFP). These first 36 nucleotides are sufficient enough to introduce a similar genetic context of the whole sequence they originated to the RBS [1]. sfGFP is an engineered version of the green fluorescent protein that can be fused with poorly folded peptides without misfolding and losing its functionality [2]., making these 36nt fusions a simple and probably reliable way to characterize translation rates provided by the same RBS for different coding sequences. A similar strategy was recently deployed to determine the effect of the spacer sequence between a RBS and the start codon in the context dependency of the RBS [3].

Based on the above information, we built a fusion of the first 36 nucleotides of the LuxI synthase with sfGFP to characterize the relative strength of our deployed RBS in the context of the LuxI coding sequence. sfGFP maintained its fluorescent properties and allowed us to conduct the characterisation

Athens2022-Senders-Circuit-luxi36ntsfGFP.png

Illustration of the 36nt LuxI- sfGFP fusion reporter protein.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 454
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

[1] Kudla G, Murray AW, Tollervey D, Plotkin JB. Coding-sequence determinants of gene expression in Escherichia coli. Science. 2009 Apr 10;324(5924):255-8. doi: 10.1126/science.1170160. PMID: 19359587; PMCID: PMC3902468.

[2] Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006 Jan;24(1):79-88. doi: 10.1038/nbt1172. Epub 2005 Dec 20. Erratum in: Nat Biotechnol. 2006 Sep;24(9):1170. PMID: 16369541.

[3] Duan Y, Zhang X, Zhai W, Zhang J, Zhang X, Xu G, Li H, Deng Z, Shi J, Xu Z. Deciphering the Rules of Ribosome Binding Site Differentiation in Context Dependence. ACS Synth Biol. 2022 Aug 19;11(8):2726-2740. doi: 10.1021/acssynbio.2c00139. Epub 2022 Jul 25. PMID: 35877551.



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