Coding

Part:BBa_K4197011

Designed by: Guillaume Gomez   Group: iGEM22_Toulouse_INSA-UPS   (2022-09-21)
Revision as of 21:42, 8 October 2022 by Charline baraban (Talk | contribs)


OmpA_DARPin

Gene fusion to express the DARPin-sfGFP fusion protein at the surface of E.coli.

Introduction

This part is composed of the gene coding for the DARPin E2_79 protein (see BBa_K4197019) in fusion with the lpp-ompA-N gene (see BBa_K1694002). This part was designed to link IgE at the surface of E. coli with the sfGFP as a reporter of the localisation of the fusion proteins.

The DARPin E2_79 protein has a strong affinity for the constant part of IgE (Baumann et al., 2010). It was merged to the membrane protein Lpp-OmpA-N of E. coli (see BBa_K1694002) to display the DARPin on the surface of E. coli. This lipoprotein is the most abundant in the membrane of E. coli with 100,000 copies per cell (Ortiz-Suarez and al. 2016) and is often used to display proteins on the surface of bacteria (Yang and al. 2016). The sfGFP was chosen because it keeps its fluorescence even in extra-cellular conditions (see BBa_K1694002).

Construction

The brick was contructed from the BBa_K1694002).To delete the sfGFP fragment, pET-21 b (+)_OmpA_DARPin-sfGFP was linearized by PCR using the high fidelity Phusion DNA polymerase with primers “exclusion sfGFP by In-Fusion (F)” and “exclusion sfGFP by In-Fusion (R)”, which excluded the sfGFP sequence (see Primers List). The expected size of the amplicon was 6202 bp. Amplification product sizes were checked on EtBr stained agarose gel. Amplicons were further purified from the gel. Figure 26 shows an amplification product matching the expected size.

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  • Forward : TAAGAAGGAGATATACCATGGCGGAAGCGGGTATCACC
  • Reverse : CTCGAGTGCGGCCGCAAGCTTCGGATCGTCCTATGATGGAGG

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  • CForward : CGCGGCCGCTTCTAGAGCGGAAGCGGGTATCACC
  • Reverse : AGCGGCCGCTACTAGTCGGATCGTCCTATGATGGAGG

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References

  1. Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, Bayer EA, Shoham Y: Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Applied and Environmental Microbiology 1995, 61:1980-1986.
  2. Nogueira ES, Schleier T, Durrenberger M, Ballmer-Hofer K, Ward TR, Jaussi R: High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis. Protein Expr Purif 2014, 93:54-62. DOI: 10.1016/j.pep.2013.10.015.
  3. Young TS, Schultz PG: Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem 2010, 285:11039-11044. DOI: 10.1074/jbc.R109.091306.
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Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 130
    Illegal NheI site found at 92
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 130
    Illegal BamHI site found at 124
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
    Illegal AgeI site found at 832
  • 1000
    COMPATIBLE WITH RFC[1000]


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