Difference between revisions of "Part:BBa J36848"
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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. | 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. | ||
| − | <gallery heights=300px widths= | + | <gallery heights=300px widths=425> |
Image:48.png|We have | Image:48.png|We have | ||
Image:Beads cytometer.png|This shows our controls. Here we used | Image:Beads cytometer.png|This shows our controls. Here we used | ||
Revision as of 03:25, 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.
We have
This shows our controls. Here we used
For more info please see our [http://2009.igem.org/Team:Washington/Project/Display#Data iGEM 2009 Washington Display Wiki].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 432
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 474
Illegal AgeI site found at 525 - 1000COMPATIBLE WITH RFC[1000]