Difference between revisions of "Part:BBa J36850"

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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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'''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 J36850 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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<p>Improvement by HZAU-China 2018</p>
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<p>Part name : [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2632008 BBa_K2632008]</P>
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Image:M_beads1.png|'''Positive Control''' ''This image shows the streptavidin-coated beads mixed with a 10nM concentration of flourophore. 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 flourophore (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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Image:M_beads2.png|'''Negative Control''' ''This image shows the streptavidin-coated beads without any flourophore. 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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Description : We removed the CDS of Streptavidin and inserted the RGD motif (a peptide of 11 amino acids) into OmpA. After our improvement, this part was given a new function. We used a surface display system Lpp-OmpA from this part to display an RGD motif on the outer membrane. This motif can specifically target alpha v beta 3 (αVβ3), a biomarker on the surface of tumor cells. Therefore, with the improved part, bacteria can target to tumor cells through displaying RGD motif. Finally, we demonstrated the tumor targeting ability through co-culturing <i>E. coli</i> containing this part with αvβ3-positive and αvβ3-negative cells in our experiment. </p> <br>
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Image:M_cells1.png|'''J36850: Induced''' ''These cells were mixed with 10nM flourophore, 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|'''J36850: Uninduced''' ''These cells were mixed with 10nM flourophore, 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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*Note: ''Images shown are not from cells with part J36850, but these results were identical for all cells tested''
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_J36850 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_J36850 SequenceAndFeatures</partinfo>
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Latest revision as of 01:09, 18 October 2018

Lac-inducible generator of Lpp-OmpA(46-159)-Streptavidin wild-type + 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 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.

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 J36850 This image shows both the induced and uninduced cells for part 50 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 J36850 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.

Improvement by HZAU-China 2018

Part name : BBa_K2632008

Description : We removed the CDS of Streptavidin and inserted the RGD motif (a peptide of 11 amino acids) into OmpA. After our improvement, this part was given a new function. We used a surface display system Lpp-OmpA from this part to display an RGD motif on the outer membrane. This motif can specifically target alpha v beta 3 (αVβ3), a biomarker on the surface of tumor cells. Therefore, with the improved part, bacteria can target to tumor cells through displaying RGD motif. Finally, we demonstrated the tumor targeting ability through co-culturing E. coli containing this part with αvβ3-positive and αvβ3-negative cells in our experiment.


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 711
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 753
    Illegal AgeI site found at 804
  • 1000
    COMPATIBLE WITH RFC[1000]