Difference between revisions of "Part:BBa K1741006"

Latest revision as of 07:46, 27 September 2015

sfGFP under short rhamnose promoter (very weak)

Both CRP sites were removed from the Rha1 promoter. It seems likely that RhaS binding site has been affected as well, so even though the resulting promoter is still induced by rhamnose, the induction is very weak.

Design

Legend:

RhaWT - rhamnose wild type

Rha1 - [BBa_K1741005]

Rha2 - [BBa_K1741006]

Our wild type rhamnose promoter is commercially available. However, we had to modify it to remove EcoRI restriction site to fit BioBrick standards. We achieved it by a single point mutation of cytosine to thymine - that is how Rha1 was created. Afterwards, we decided to further minimize the promoter sequence by removing the CRP site - we called this construct 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. 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 148

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1741006 short</partinfo>
-From rhaBAD1 promoter we removed both CRP binding sites. It seems likely thatt RhaS binding site has been also affected, so however the resulting promoter is still induced by rhamnose, the induction is very weak.
+Both CRP sites were removed from the Rha1 promoter. It seems likely that RhaS binding site has been affected as well, so even though the resulting promoter is still induced by rhamnose, the induction is very weak.
+===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 promoter is commercially available. However, we had to modify it to remove EcoRI restriction site to fit BioBrick standards. We achieved it by a single point mutation of cytosine to thymine - that is how Rha1 was created. Afterwards, we decided to further minimize the promoter sequence by removing the CRP site - we called this construct 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.
+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
 <!-- Add more about the biology of this part here