Difference between revisions of "Part:BBa K1741005"

Revision as of 05:40, 27 September 2015

sfGFP under rhamnose promoter with removed EcoRI site

RhamnoseWT promoter has been copied from E. coli genome: ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTCaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt then Eco RI site was removed by PCR to obtain Rha1 in a biobrick standard: ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTTaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt The only one point mutation makes the promoter slightly stronger and more sensitive to rhamnose.

Characteristic and Results

Legend:

RhaWT - rhamnose wild type Rha1 - [BBa_K1741005] Rha2 - [BBa_K1741006]

https://parts.igem.org/File:Teamuampoznanrhamnosem9graph.png The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.

All three rhamnose promoters are induced by rhamnose and repressed by glucose. Turns out that single point mutation in EcoRI restriction site in Rha1 significantly increased the activity of the promoter as comapred to the wild type construct. Rha2 is a weak promoter, perfect for people who would like to manufacture small amount of the protein of interest.

We also checked the tightness of our promoters.

All of our promoters are induced only by their respective sugars with a small exception of xylose promoters being slightly induced by arabinose and arabinose promoters being slightly induced by xylose. All of our promoters are repressed by glucose.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 199

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1741005 short</partinfo>
-rhaBAD promoter has been copied from E. coli genome: ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTCaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt
+RhamnoseWT promoter has been copied from E. coli genome: ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTCaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt
 then Eco RI site was removed by PCR
 to obtain Rha1 in a biobrick standard:
 ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTTaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt
 The only one point mutation makes the promoter slightly stronger and more sensitive to rhamnose.
+===Characteristic and Results===
+Legend:
+RhaWT - rhamnose wild type
+Rha1 - [[https://parts.igem.org/Part:BBa_K1741005 BBa_K1741005]]
+Rha2 - [[https://parts.igem.org/Part:BBa_K1741006 BBa_K1741006]]
+https://static.igem.org/mediawiki/parts/9/92/Teamuampoznanrhamnoselbgraph.png
+https://parts.igem.org/File:Teamuampoznanrhamnosem9graph.png
+The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.
+All three rhamnose promoters are induced by rhamnose and repressed by glucose.
+Turns out that single point mutation in EcoRI restriction site in Rha1 significantly increased the activity of the promoter as comapred to the wild type construct.
+Rha2 is a weak promoter, perfect for people who would like to manufacture small amount of the protein of interest.
+We also checked the tightness of our promoters.
+All of our promoters are induced only by their respective sugars with a small exception of xylose promoters being slightly induced by arabinose and arabinose promoters being slightly induced by xylose. All of our promoters are repressed by glucose.
+https://static.igem.org/mediawiki/parts/4/43/Teamuampoznanpromoterscomparisongrpah.png