Description
The aim of our project was to extensively test the viability of different of kill switches across an array of variables. We focused on the ones that have already been used by previous teams (Pekin 2014, Oxford 2015) as well as a KillerRed homologue KillerOrange. This will allow future teams to more accurately chose a kill switch that suits their project the best as we have providing better understanding on the durability of the different kill switches as well as their efficiency.
We submitted a total of X new parts all of which are BioBrick compatible and codon optimised for E. coli strain K12. See list below for more details.
KillerRed
Name
pT7- E. coli optimised - KillerRed (EOKR)
Description
KillerRed is a red fluorescent protein that generates reactive oxygen species after illumination with light between the wavelengths of 540-580nm[1].
We further characterised this kill switch by illuminating induced cultures 24 hours after induction with IPTG as well as uninduced cultures. After 6 hours in the light box CFU’s were counted to determine if the kill switch was successful. This was also reproduced on cultures grown in a ministat for 120 and 168 hours to test how long the kill switch remains functional. The full protocol can be found here and the results can be found here.
The mechanism by which KillerRed kills cells isn’t fully understood yet
Here we are submitting KillerRed as a composite part under a T7 promoter (code), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).
The sequence for KillerRed protein coding region can be found here (BBa_K1141002)
Biobrick Code
BBa_K1914003
KillerOrange
Name:
pT7- E. coli optimised - KillerOrange (EOKO)
Description:
KillerOrange is a mutant of the fluorescent protein KillerRed (BBa_K1141002, BBa_K1491015) activated by blue and green light. It carries a tryptophan-based chromophore that is novel for photosensitizers [2].
KillerOrange has an excitation maximum of 512 nm and emission maximum at 555 nm and its absorbance spectrum has two peaks, at 455 and 514 nm.
It is believed that was confers KillerOrange's ability to generate reactive oxygen species depends on a water-filler channel reaching the chromophore area from the end cap of the β-barrel [3].
The mechanism by which KillerOrange kills cells isn’t fully understood yet[3].
We characterised this part in the same way as KillerRed (link to protocol), and the results of which can be found here (link to results)
Here we are submitting KillerOrange as a composite part under a T7 promoter (code), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).
The sequence for KillerOrange protein coding region can be found here (BBa_K1914000)
Biobrick Code
BBa_K1914001
LysozymeC
Name:
LysozymeC
Description:
Lysozyme C from chicken egg white, attacks the cell wall in bacteria by hydrolysing the β-1,4 linkages between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan [4].
We are submitting this Lysozyme C as a composite part under a T7 promoter, an Elowitz ribosome binding site (biobrick code) and a double terminator (BBa_B0015). We have codon optimised the protein coding region, added a FLAG tag and signal peptide directing Lysozyme to the periplasm. The sequence for just Lysozyme C can be found here (biobrick code).
Characterisation of this part involved using the Enzcheck assay kit which detects lysozyme activity as a measure of fluorescence as well as using the same protocol we used to characterise KillerRed (link to protocol)and the results of which can be found here (link to results)
Here we are submitting lysozymeC as a composite part under a T7 promoter (code), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).
The sequence for LysozymeC protein coding region can be found here (BBa_K1914004)
Biobrick Code
BBa_K1914005
