Difference between revisions of "Part:BBa J36848"

Latest revision as of 03:00, 22 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:

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  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].

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  + 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. These beads have similar dimensions as a cell of E. coli (beads are 1.5 -1.9 micrometers in diameter, compared to 2*0.5 micrometers for a standard E. coli). 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 visualized the part-expressing cells using a fluorescence microscope after incubation with biotinylated fluorophore (as for the flow cytometry experiment described above). 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 positive control for streptavidin-biotin binding. In this experiment we sought to visualize 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 incubated with 10nM biotinylated flourophore. These beads were incubated for an hour, then were spun down and diluted into one milliliter of water. Fluorescence was then visualized using the fluorescence microscope. We then analyzed these images using [http://rsbweb.nih.gov/ij/ ImageJ] to calculate the intensity profiles along a line drawn 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 and the two peaks shown on the line plot.

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  Negative Control This image shows fluorescence microscopy of streptavidin-coated beads that had not been incubated in flourophore. We analyzed these images using [http://rsbweb.nih.gov/ij/ ImageJ] to calculate the intensity profiles along a line drawn through the beads. As we expected these beads did not show fluorescence intensity spikes like those seen with beads that had been incubated with the biotinylated fluorophore.

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  J36848: Induced We induced the expression of surface-display streptavidin in cells harboring J36848, incubated them with 10 nM biotinylated flourophore, pelleted cells by centrifugation, and resuspended in one milliliter of water. Fluorescence was then visualized using the fluorescence microscope. If cells expressed the streptavidin cell-surface construct, we expected a similar result to the positive control shown above. However, after imaging the cells, we did not visualize any significant fluorescence encircling the cells. Measuring a line plot with [http://rsbweb.nih.gov/ij/ 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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  J36848: Uninduced These cells were incubated with 10nM flourophore and then pelleted by centrifugation, and resuspended in one milliliter of water. Fluorescence was then visualized using the fluorescence microscope. Like the induced cells, these uninduced cells showed no appreciable levels of fluorescence after imaging and measurement under the microscope.

Sequence and Features