Part:BBa_K784001
pT7_RBS_xyIE reporter gene
This part's purpose is to testify to the presence of the T7 RNA Polymerase by the xyIE reporter gene from iGEM2006_Edinburgh. It consists of an RNA T7 promoter, an RBS, a reporter gene- xyIE, and a T7 terminator. The assay for xyIE is very simple, refer to the xyIE basic part for instructions.
The xyIE+RBS part is between XhoI and XmaI restriction sites, therefore can be excised by these enzymes.
Expression of the gene XylE into CDO enzyme using the homemade cell free system OnePot PURE - EPFL,2019
Group: EPFL, 2019
Hypothesis:
Our goal was to express the gene XylE, into the enzyme Catechol 2,3-dioxygenase (CDO), using the cell free system OnePot PURE. CDO reacts with the substrate catechol to transform into another product, which causes a color change from transparent to yellow. For the purpose of our project we needed to induce this color change in order to create a signal.
Methods
To obtain this part, we started from the part BBa_J33204 to which we added a T7 promoter and T7 terminator.
To express this gene into the cell free system OnePot PURE, we followed the protocol we created (link to protocols.io). To assert the validity of the results, we performed two controls next to the reaction. The first control expressed XylE without catechol, and the second one had catechol but no XylE template. We incubated the reactions at 37°C and took pictures regularly to see how the color was changing throughout the reaction.
Results:
This picture shows the evolution of the reaction of CDO with catechol as well as the state of the controls. The first column are the pictures of the reaction. The second and third columns are controls, respectively, one with XylE template but without catechol, the other with catechol but without XylE template. A color change is observed in the reaction; after 30 minutes of incubation the color starts to become yellow and after 1 hour the color is bright and distinguishes clearly from the controls. Both controls remain transparent throughout the runtime.
The gene XylE with a T7 promoter was successfully expressed in the cell free system OnePot PURE into its corresponding enzyme, CDO. The reaction between CDO and catechol induces a color change from transparent to yellow which was clearly observed after 1 hour of incubation at 37°C.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 368
Illegal NgoMIV site found at 540
Illegal AgeI site found at 891 - 1000COMPATIBLE WITH RFC[1000]
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