Difference between revisions of "Part:BBa J36848"

Revision as of 04:05, 19 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 suggest it's on plasmid backbone pSB1A3, not pSB1A2 as the 2008 Spring Distribution states. The bases following the PstI site are 5'-tccggcaaaaaa-3' which matches pSB1A3, while the same section of 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. --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 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 hisogram in which the x-axis is the count and the the y-axis is the fluorescence level (FLA-1) of the cells/beads:

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  This image shows both the induced and uninduced cells for part 48 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].

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  This shows our controls. 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.

Sequence and Features