Difference between revisions of "Part:BBa K1741005"
 
(8 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K1741005 short</partinfo>
 
<partinfo>BBa_K1741005 short</partinfo>
  
−
RhamnoseWT 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===
  
−
===Characteristic and Results===
+
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:
 
Legend:
  
Line 18: Line 37:
  
 
https://static.igem.org/mediawiki/parts/9/92/Teamuampoznanrhamnoselbgraph.png
 
https://static.igem.org/mediawiki/parts/9/92/Teamuampoznanrhamnoselbgraph.png
−
https://static.igem.org/mediawiki/parts/9/97/Teamuampoznanmelibiosem9graph.png
+
https://static.igem.org/mediawiki/parts/0/0d/Teamuampoznanrhamnosem9graph.png
 +
 
  
 
The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.
 
The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.
  
 
All three rhamnose promoters are induced by rhamnose and repressed by glucose.
 
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.
+
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.
 
Rha2 is a weak promoter, perfect for people who would like to manufacture small amount of the protein of interest.
  

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

TeamUAMpoznanRhaWTseq.jpg


Rha1 - [BBa_K1741005]

TeamuampoznanRha1seq.jpg


Rha2 - [BBa_K1741006]

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 - [BBa_K1741005]

Rha2 - [BBa_K1741006]

Teamuampoznanrhamnoselbgraph.png 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. Teamuampoznanpromoterscomparisongrpah.png


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