Difference between revisions of "Part:BBa K1741005"

Latest revision as of 08:54, 27 September 2015

sfGFP under rhamnose promoter with removed EcoRI site

RhaWT promoter has been copied from E. coli genome. Then EcoRI site was removed by PCR in order to obtain Rha1 in the BioBrick standard. The one point mutation makes the promoter slightly stronger and more sensitive to rhamnose.

Design

Legend:

RhaWT - rhamnose wild type

Rha1 - [BBa_K1741005]

Rha2 - [BBa_K1741006]

Our wild type rhamnose-induced promoter is commercially available. However, we had to modify it to remove EcoRI restriction site to comply with BioBrick standards. We achieved it by a single point mutation of cytosine to thymine - that is how Rha1 was created. Next, we decided to further minimize the promoter sequence by removing the CRP site - we called this contstruct Rha2.

Results

Legend:

RhaWT - rhamnose wild type

Rha1 - [BBa_K1741005]

Rha2 - [BBa_K1741006]

The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.

All three rhamnose promoters are induced by rhamnose and repressed by glucose. It turns out that single point mutation in EcoRI restriction site in Rha1 significantly increased the activity of the promoter as compared 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
+RhaWT promoter has been copied from ''E. coli'' genome.
-then Eco RI site was removed by PCR
+Then EcoRI site was removed by PCR in order to obtain Rha1 in the BioBrick standard.
-to obtain Rha1 in a biobrick standard:
+The one point mutation makes the promoter slightly stronger and more sensitive to rhamnose.
-ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTTaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt
-The only one point mutation makes the promoter slightly stronger and more sensitive to rhamnose.
+===Design===
+Legend:
+RhaWT - rhamnose wild type
+https://static.igem.org/mediawiki/parts/d/d5/TeamUAMpoznanRhaWTseq.jpg
+Rha1 - [[https://parts.igem.org/Part:BBa_K1741005 BBa_K1741005]]
+https://static.igem.org/mediawiki/parts/3/3a/TeamuampoznanRha1seq.jpg
+Rha2 - [[https://parts.igem.org/Part:BBa_K1741006 BBa_K1741006]]
+https://static.igem.org/mediawiki/parts/3/34/TeamuampoznanRhaS1.jpg
+Our wild type rhamnose-induced promoter is commercially available. However, we had to modify it to remove EcoRI restriction site to comply with BioBrick standards. We achieved it by a single point mutation of cytosine to thymine - that is how Rha1 was created. Next, we decided to further minimize the promoter sequence by removing the CRP site - we called this contstruct Rha2.
+===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://static.igem.org/mediawiki/parts/0/0d/Teamuampoznanrhamnosem9graph.png
+The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.
+All three rhamnose promoters are induced by rhamnose and repressed by glucose.
+It turns out that single point mutation in EcoRI restriction site in Rha1 significantly increased the activity of the promoter as compared 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