Part:BBa_K1914001
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.
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 [2].
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.
Percentage viable cells after 6 hrs in the light box. BL21 (DE3) transformed with KillerOrange (BBa_K1914001) is compared to a control with no plasmid. 10 ml falcon tubes containing 4.5 ml of sample were placed label down in the light box to allow maximum exposure to the light.
Percentage viable cells after 6 hrs in the light box. BL21 (DE3) transformed with KillerOrange (BBa_K1914001) is compared to a control with no plasmid. 10 ml falcon tubes containing 4.5 ml of sample were covered in tin foil before being placed in the light box.
We further characterised this kill switch by growing the culture in a ministat and carrying out the same testing procedure, illuminating induced cultures 24 hours after induction with100μM of 0.1M IPTG in the light box for 6 hours. CFU’s were counted to determine if the kill switch was successful in cultures grown in the ministat for 120 and 168 hours to test how long the kill switch remains functional.
Comparison of CFUs formed by KillerOrange exposed to light and kept in the dark. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUS. The effect is not as obvious in KillerOrange compared to KillerRed as the starting efficiency of KillerOrange is lower.
Reference [2]Sarkisyan, K.S., Zlobovskaya, O.A., Gorbachev, D.A., Bozhanova, N.G., Sharonov, G.V., Staroverov, D.B., Egorov, E.S., Ryabova, A.V., Solntsev, K.M., Mishin, A.S. and Lukyanov, K.A., 2015. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PloS one,10(12), p.e0145287.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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