Difference between revisions of "Part:BBa J36848"

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We also ran 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. In this experiment we sought to see binding between the cells and the biotinylated flourophore. The results are summarized below.
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<td><p style="font-size:18px"> Positive Control </p> </td>
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<td><p style="font-size:18px"> Negative Control </p> </td>
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Image:M_beads1.png|'''Positive Control''' ''This image shows the streptavidin-coated beads mixed with a 10nM concentration of flourohphore. These beads where allowed to incubate for an hour, then were spun down and diluted into one milliliter of water. We then analyzed these images using imageJ to calculate the intensity profiles along a line drwan through the beads. As we expected, this allowed us to see appreciable binding in comparison to the beads without any flouorphore (show to the right). This binding was characterized by the halo of fluorescence, or the two peaks shown on the line plot. ''
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<td rowspan="4">These images were analyzed using imageJ <sup>9</sup>. The image on the left shows an image of the beads with fluorophore added to them. These beads were diluted and spun diluted and spun down until the background level of fluorescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in fluorescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated fluorophore binds to streptavidin in a detectable manner.  When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right). This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent. In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytometry.</td>
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Image:M_beads2.png|'''Negative Control''' ''This image shows the streptavidin-coated beads without any flourohphore. These beads where allowed to incubate for an hour, then were spun down and diluted into one milliliter of water. We then analyzed these images using imageJ to calculate the intensity profiles along a line drawn through the beads. As we expected these beads did not show the same intensity spikes due to the presence of the flourophore around the edges of the beads.''
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Image:M_cells1.png|'''J36848: Induced''' ''These cells were mixed with 10nM flourohphre, and then spun down. Next they were re-suspended in one milliliter of water, and measured under the microscope. Because these cells were induced we expected a similar result to to the positive control shown above. However, after imaging the cells, we were barely able to see any florescence. Measuring a line plot with imageJ showed that the induced cells matched the same intensity profile as the beads without flourophore. In addition there were no appreciable differences between the induced and uninduced cells shown at right.''
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Image:M_cells2.png|'''J36848: Uninduced''' ''These cells were mixed with 10nM flourohphre, and then spun down. Next they were re-suspended in one milliliter of water, and measured under the microscope. These uninduced cells showed no appreciable levels of fluorescence after imaging and measurement under the microscope. However they also showed similar levels of florescence to the induced cells.''
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<td><p style="font-size:18px"> Induced Cells </p></td>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 17:41, 20 October 2009

Lac-inducible generator of Lpp-OmpA(46-66)-Streptavidin wild-type + His6tag

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 wild-type + 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.

Possible error in Spring 2008 distribution information

The sequence data for this construct in the 2008 Spring Distribution suggest it's on plasmid backbone pSB1A3, not pSB1A2 as the stated in the documentation. The bases following the PstI site are 5'-tccggcaaaaaa-3' which matches pSB1A3, while the same locus on pSB1A2 reads 5'-gcttcctcgctc-3'.

Also, the 'inconsistent' sequence data is due to the fact that, in order to conform to the composite parts format, an 8 base scar is shown in the 'get selected sequence' readout. The sequencing data is checked against this sequence with the 8-base scars, not the 6-base in-frame scars that are part of the sequencing data from the actual plasmid. --robere, University of Washington iGEM team, 11 Sept 2009


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 (FL1-A: 488 nm excitation, 515-545 nm emission) of the cells/beads:

  • BBa_J36848 This image shows both the induced and uninduced cells for part 48 incubated in three levels of flourophore: 0M, 10nM, and 100nM. Expression of the surface display streptavidin in the cells was induced with 1mM IPTG. This data shows that there is no appreciable difference between the induced and uninduced cells at any given level of fluorophore. 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].

  • + Control We used streptavidin-coated beads (SVP-15-5 1.5-1.9 μm polystyrene spheres, [http://www.spherotech.com/coa_pol_par.htm Spherotech]) as a positive control for binding of the biotinylated fluorophore (biotin (5-fluorescein) conjugate, [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=53608|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma Aldrich]) 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.

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.

  • Positive Control This image shows the streptavidin-coated beads mixed with a 10nM concentration of flourohphore. These beads where allowed to incubate for an hour, then were spun down and diluted into one milliliter of water. We then analyzed these images using imageJ to calculate the intensity profiles along a line drwan through the beads. As we expected, this allowed us to see appreciable binding in comparison to the beads without any flouorphore (show to the right). This binding was characterized by the halo of fluorescence, or the two peaks shown on the line plot.

  • Negative Control This image shows the streptavidin-coated beads without any flourohphore. These beads where allowed to incubate for an hour, then were spun down and diluted into one milliliter of water. We then analyzed these images using imageJ to calculate the intensity profiles along a line drawn through the beads. As we expected these beads did not show the same intensity spikes due to the presence of the flourophore around the edges of the beads.

  • J36848: Induced These cells were mixed with 10nM flourohphre, and then spun down. Next they were re-suspended in one milliliter of water, and measured under the microscope. Because these cells were induced we expected a similar result to to the positive control shown above. However, after imaging the cells, we were barely able to see any florescence. Measuring a line plot with imageJ showed that the induced cells matched the same intensity profile as the beads without flourophore. In addition there were no appreciable differences between the induced and uninduced cells shown at right.

  • J36848: Uninduced These cells were mixed with 10nM flourohphre, and then spun down. Next they were re-suspended in one milliliter of water, and measured under the microscope. These uninduced cells showed no appreciable levels of fluorescence after imaging and measurement under the microscope. However they also showed similar levels of florescence to the induced cells.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 432
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 474
    Illegal AgeI site found at 525
  • 1000
    COMPATIBLE WITH RFC[1000]