Difference between revisions of "Part:BBa J36848"

(Usage and Biology)
(Usage and Biology)
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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 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.
 
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.
  
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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. ''
 
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. ''
 
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.''
 
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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</gallery>
  
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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.''
 
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.''
 
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.''
 
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.''

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:

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.


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]