Difference between revisions of "Part:BBa K1914001"

Revision as of 12:39, 18 October 2016

pT7- E. coli optimised - KillerOrange (EOKO)

KillerOrange is a photoactiated kill switch with an IPTG inducible T7 promoter with E.coli optimised protein coding region of KillerRed mutant.

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
COMPATIBLE WITH RFC[1000]

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 ===Usage and Biology===
+KillerOrange is a mutant of the fluorescent protein KillerRed (BBa_K1141002, BBa_K1491015) activated by blue and green light, excitation maximum of 512 nm and emission maximum at 555 nm and absorbance spectrum has two peaks, at 455 and 514 nm.
+This device is BioBrick compatible and codon optimised for E. coli strain K12. All of our parts are under the pT7 inducible promoter (BBa_I712074) with an Elowitz ribosome binding site (BBa_B0034) before the protein coding region and a double terminator (BBa_B0015) after.We chose the parts we have used as they are either some of the most popular BioBricks used by other teams throughout the history of the iGEM competition or been awarded a registry star. One of the core aims of our project was to make it relatable and useful to as many future teams as possible. We believed that using the most popular parts on the registry reflects this intention.
+The phototoxicity of KillerOrange was compared to the commonly used Red fluorescence protein (RFP). Once we had established the efficiency ministat chambers were inoculated with samples of E.coli BL21 (DE3) with the plasmid coding for the protein to determine the robustness of the kill switches over time.
+The samples were tested for phototoxicity by exposing them to 12 W/m2 white light from a 4x8 LED array for 6 hrs. Samples were then spread plated and colony forming units (CFUs) were counted. The part was carried on the pSB1C3 plasmid and transformed into E. coli BL21 (DE3). Samples that were induced were done so with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 nM.
+We constructed a box around the LED array to prevent ambient light entering, and attached acetate colour filters to provide the desired excitation frequency. Access to the inside of the box was gained through an opening cut in the front. With help from Ryan Edginton, we used a portable spectrometer (Ocean Optics USB2000+VIS-NIR-ES, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre) to measure light spectra and absolute intensity in the visible range.
+The graphs below show the average percentage of viable cells for induced and uninduced samples after 6 hrs of exposure to 12 W/m2 of white light. CFU count for the control condition was treated as 100 % and viable cells calculated as a proportion of that value. CFUs were not counted above 300, any lawns were assigned the value of 300. Error bars represent the standard error of the mean. The average temperature in the light box was 38.63 °C.
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