Part:BBa_J36849
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:
BBa_J36849 This image shows both the induced and uninduced cells for part 49 in varying levels of flourophore (0nM to 100nM). The cells were induced at 1mM IPTG. This data shows that there is no appreciable difference between the induced and uninduced cells at any given level of flourophore. All curves appear to have the same amount of fluorescence. We found similar results using a microscopy assay. For more info please see our [http://2009.igem.org/Team:Washington/Project/Display#Data iGEM 2009 Washington Display Wiki].
+ Control We used the sreptavadin coated to show us what the magnitude of fluorescence increase we should see with increased flourophore levels. We were able to see that as the level of fourophore was increased we could see increased retention between the beads and the flouophore. The black is beads with no flouophore, the red is with 10 nM, and the purple is 100 nM. These showed a clear difference between the beads without flourophore, and the beads with flourophore. The cells shown at right, matched the readings of the beads when they had no flourophore added.
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 J36849 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1179
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1188
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 609
Illegal AgeI site found at 1143 - 1000COMPATIBLE WITH RFC[1000]
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