Difference between revisions of "Part:BBa J36851"

(Usage and Biology)
 
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===Usage and Biology===
 
===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:
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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:
  
 
<gallery heights=300px widths=425>
 
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Image:51.png|This image shows both the induced and uninduced cells for part 51 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].
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Image:51.png|'''BBa_J36851''' ''This image shows both the induced and uninduced cells for part 48 in varying levels of flourophore (0nM to 100nM). Expression of the surface display streptavidin in the cells was 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. There is an increase in fluorescence that increases with increased concentration of incubation - we believe this is because there is due to residual fluorophore present in solution after washing. Fluorescence retention was minimal compared to streptavidin-coated beads (see Control). For more info please see our [http://2009.igem.org/Team:Washington/Project/Display#Data iGEM 2009 Washington Display Wiki].''
Image:StreptBead cyto.png|This shows our controls. 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.
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Image:StreptBead cyto.png|'''+ Control''' ''We used streptavidin-coated beads as a positive control for binding of the biotinylated fluorophore to streptavidin.  For this experiment, we treated beads as we treated the cells - incubation in biotinylated fluorophore followed by washing and fluorescence measurement by flow cytometry. As the concentration of flourophore was increased we could see increased retention between the beads and the flouophore. The black line is beads with no flouophore, the red is with 10 nM, and the blue is 100 nM. These showed a clear difference between the beads without flourophore and the beads with flourophore. See [http://2009.igem.org/Team:Washington/Notebook iGEM 2009 Washington Protocols] for details.''
 
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'''We also visualized these cells under a microscope. We used the same streptavidin-coated beads (SVP-15-5 1.5-1.9 &mu;m 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 [https://parts.igem.org/Part:BBa_J36848 BBa_J36848] for representative images.'''
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 03:02, 22 October 2009

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 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 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]