Difference between revisions of "Part:BBa J36851"

Revision as of 17:46, 20 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:

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  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].

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  + 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.

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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  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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  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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  J36851: 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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  J36851: 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.

• Note: Images shown are not from cells with part J36851, but these results were identical for all cells tested

Sequence and Features