Generator

Part:BBa_J36849

Designed by: Perry Tsai   Group: iGEM06_Harvard   (2006-10-29)
Revision as of 03:01, 22 October 2009 by Swanson (Talk | contribs) (Usage and Biology)

Lac-inducible generator of Lpp-OmpA(46-66)-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 aa46-66, 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 for our protein secretion system. We used a western blot to confirm the expression of the proteins. Then we decided to test each part using flow cytometery. Using a biotinylated flourophore we hoped to visualize these cells by checking for increased florescence due to the binding interactions between the streptavadin and the biotin. Our results are described below in the histogram, the y-axis is the event frequency and the x-axis is the fluorescence intensity (FLA-1) of the cells/beads:


We also visualized 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. Cells containing induced part J36851 were also tested for cell-surface-displayed streptavidin by incubation with a biotinylated fluorophore and visualization of cell fluorescence using fluorescence microscopy. In this experiment we sought to visualize binding between the cells and the biotinylated flourophore. No appreciable fluorescence was seen. See BBa_J36848 for representative images.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1179
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1188
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 609
    Illegal AgeI site found at 1143
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None