Generator

Part:BBa_J36851

Designed by: Perry Tsai   Group: iGEM06_Harvard   (2006-10-29)
Revision as of 17:46, 20 October 2009 by Dwhite (Talk | contribs) (Usage and Biology)

Lac-inducible generator of Lpp-OmpA(46-159)-Streptavidin single-chain dimeric + His6 tag

This device contains a lac promoter and strong ribosome binding site for lac-inducible expression of the fusion protein of Lpp signal peptide, OmpA aa46-159, and streptavidin single-chain dimeric + His6 tag. This expression should display streptavidin on the cell surface of E. coli.

NOTE ABOUT THE SEQUENCE: The mixed site between parts is 'only' six base pairs, ACTAGA. There is no spacer T or G nucleotide. These spacer nucleotides have been placed in the results for "get selected sequence" as an automatic composite-parts addition for the BioBricks mixed site between assembled parts. However, this does not apply for the two spacer nucleotides betweeon R0010 and B0034, and the one spacer nucleotide after B0034, because those were standard BioBricks.

Usage and Biology

Characterized by [http://2009.igem.org/Team:Washington Washington 2009 iGEM team]. We sought to use these parts to display streptavidin on the surface of the cell. We confirmed the expression of these proteins by Western blot using an anti-His detection reagent. We then assayed each part for biotin binding using flow cytometry. Our assay was to incubate cells with a biotinylated fluorophore, wash cells, and then monitor by flow cytometry the retention of fluorophore on the surface of cells that had this part induced with IPTG. In this experiment, increased florescence would indicate binding interactions between the streptavadin and the biotin. Our results are described below in the histogram, the y-axis is the event frequency (equivalent to the number of cells counted) and the x-axis is the fluorescence intensity (FLA-1) of the cells/beads:


We also ran these cells under a microscope. We used the same streptavidin-coated beads (SVP-15-5 1.5-1.9 μm polystyrene spheres, [http://www.spherotech.com/coa_pol_par.htm Spherotech]) as a control. In this experiment we sought to see binding between the cells and the biotinylated flourophore. The results are summarized below.

  • Note: Images shown are not from cells with part J36851, but these results were identical for all cells tested

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1458
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1467
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 888
    Illegal AgeI site found at 1422
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None