<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/2/2f/T--Exeter--RFPdark.jpg"> <!--RFP dark graph-->

<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/a/ae/T--Exeter--KRdark.jpg">  <!--KillerRed dark graph-->

<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/8/8e/T--Exeter--KRlight.jpg">  <!--KillerRed dark graph-->

<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/c/cb/T--Exeter--KOdark.jpg">  <!--KillerOrange dark graph-->

<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/b/be/T--Exeter--KOlight.jpg">  <!--KillerOrange light graph-->

<p id="pp"><b>Fig.1-6</b> The average percentage viable cells for induced and uninduced samples after 6 hrs of exposure to 12 W/m² of white light. Intensity was measured using an Ocean Optics USB2000+VIS-NIR-ES spectrometer, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre. 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 ℃

<p id="pp">To show the activity of lysozyme, a molecular probes EnzCheck lysozyme assay kit from Thermo fisher scientific was used. The CDS contains an OmpA signal peptide targeting it to the perisplasm therfore lysozyme will only be detectable if the cells have lysed. The kit uses a substrate containing <i>Micrococcus lysodeikticus</i> cell walls labelled with fluorescein to such as degree that fluorescence is quenched. The presence of lysozyme causes a sharp increase in fluorescence (AU) by easing the quenching. The increase in fluorescence is proportional to lysozyme activity in the sample. The fluorescence assay was used to measure the activity of the freshly transformed kill switch and that of the cultures grown in the ministat. CFUs were also used as a measure of efficiency by comparing number of colonies to a control. 5 ml ovenights of <i>E. coli</i> BL21 (DE3) transformed with pSB1C3 lysozyme were used to inoculate 250 ml Erlenmeyer flasks containing 50 ml of LB laced with 35 μg/ml chloramphenicol. Once an OD of 0.23 was reached IPTG was added to a final concentration of 0.2 nM. Protein production was allowed to proceed for 2 hrs. The sample was serially diluted (10^-2,10^-3,10^-4). 200 μl of each dilution factor was spread plated and incubated at 37 ˚C overnight. CFUs were then compared to a control treated in the same way.</p>  

<p id ="pp">The HGT experiment showed that DNA present in lysate can be successfully transformed into a different <i>E. coli</i> strain with an average of 4 colonies per transformation (stdev=3.38). The BL21 (DE3) competent cells all gained the antibiotic resistance and RFP marker from the plasmid present in the lysed DH5α. 2 colonies from each plate were cultured over night and showed a fluorescence value concordant with that of the original culture. The starting cultures of DH5α had an average starting OD of 1.11 and fluorescence value of 258 before lysis. The BL21 (DE3) cultures transformed with the lysate had an average OD of 0.75 and average fluorescence of 306. None of the spread plated lysate produced any colonies, showing that all cells were killed in the lysis reaction.</p>

<p id="pp">We have shown that KillerRed and KillerOrange can effectively kill cells under much lower light intensity than is used in the literature (reference). On investigation into the kind of light source that was needed to produce the 1 W/cm² of previous experiments (Bulina <i>et al</i>, 2005), it became clear that 1 W/cm² was impractically bright. We decided to use an LED array that produces 0.0012 W/cm² normally used for growing plants and expose our samples to light for a greater length of time. We showed that this was still effective with an average survival rate in the + IPTG condition of 2.2% for KillerRed and 12.7 % for KillerOrange. A wider range of exposure times and light intensities would greatly improve the characterisation of these parts, unfortunately time limitations prevented us from testing this. There was no (statistical) difference between the + IPTG condition and – IPTG condition. CFU counts for + IPTG conditions were within the standard error of – IPTG. For KillerRed the induced kill switch appears to be more effective whereas the uninduced switch is more effective in killer orange. The leakiness of the T7 promoter has likely lead to near equal expression both conditions, possibly exacerbated by the length of time that the cultures were left to grow in order for the protein to fully mature. The literature showed that cells had been kept in a cold room at 4 ℃ for 24 hrs before exposing the samples to light (reference), the reason given for this was to allow the protein to fully mature. We tested the validity of this as cultures were incubated at 37 ℃ 220 rpm overnight not 4 ℃ and the phototoxicity of KillerRed and KillerOrange was still evident. The light box itself had a negative effect on <i>E. coli</i> growth. Each sample was first diluted to 10^-3,10^-4 and 10^-5 before exposure to light. The control showed fewer colonies at each dilution factor as would be expected, with the CFU count at a 10^-3 dilution still being a lawn of bacteria. However in the dark condition, the control sample grew to a lawn of <i>E. coli</i> regardless of the starting dilution factor. (shown in graph)</p>

To resolve this we decided to carry out site directed mutagenesis to change one nucleotide base pair in each sequence of the restriction sites. Primers were designed for use with the Q5 site directed mutagenesis kit. The first attempt using this kit involved a 2 step PCR reaction, this was shown by gel electrophoresis of the product to have been unsuccessful. The protocol was changed to a 3 step PCR reaction and a successful product was produced. The PCR product underwent a KLD reaction and was transformed into <i>E. coli</i> DH5α.

             <li>Defrost one 100 μl (or 200 μl if available) aliquot of <i>E. coli</i> competent cells per transformation, plus one extra as a control (use whole tube and do not refreeze).

             <li>Add 1-5 μl of plasmid DNA (depending on concentration) to competent cells, mix by rolling or flicking the eppendorf.

             <li>Heat shock in eppendorf at 42 ˚C for 2 mins.</li>

             <li>Add 0.7 ml of pre-warmed LB medium, incubate at 37 ˚C, 200-220 rpm for 45-60 mins. </li>

             <li>Plate out 200 μl on agar laced with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 35 μg/ml chloramphenicol or 15 μg/ml tetracycline as appropriate.</li>

             <li>Incubate at 37 ˚C overnight.</li>

             <li>Prepare 5 ml overnight cultures of KillerRed, KillerOrange and RFP transformed into <i>E.coli</i> BL21 (DE3). This can be done from a glycerol stock or fresh transformation. Place in 37 ˚C 220 rpm incubator overnight.</li>

             <li>Prepare 250 ml erlenmeyer flasks with 50 ml of LB broth and add 50 μl of 35mg/ml chloramphenicol to a final concentration of 35 μg/ml. Cover the flasks in tin foil.

             <li>Once an OD of 0.23 has been reached, induce + IPTG samples with 100 μl of 0.1M IPTG to a final concentration of 0.2 nM. Incubate at 37˚C 220 rpm overnight.</li>

             <li>Perform a serial dilution of the samples by adding 5 μl of undiluted culture to 4995 μl of LB broth in a 10 ml falcon tube and invert 5-10 times, this is the 10^-3 culture. Take 500 μl of the 10^-3 and add it to 4500 μl of LB broth, invert the tube 5-10 times, this is the 10^-4. Finally take 500 μl of the 10^-4 and add it to 4500 μl of LB broth, invert the tube 5-10 times, this is the 10^-5 culture. Remove 500 μl from the 10^-5 culture, all falcon tubes will contain 4500 μl of sample.  Repeat this twice for all the samples (KillerRed induced, KillerRed not induced, KillerOrange induced, KillerOrange not induced, RFP and control: BL21 (DE3)). One set of samples should be covered in tin foil, the other set left uncovered.</li>

             <li>Spread plate 200 μl of each sample and incubate at 37˚C overnight.</li>

             <li>Add media to the containers. Add 100 μl of inoculum to the sterile culture chamber. Connect the media container to the culture chamber.</li>

             <li>Prepare a 5 ml overnight of pSB1C3 RFP transformed into <i>E. coli</i> DH5α in a 10 ml falcon tube.</li>

             <li>Incubate overnight at 55 ℃ (this step is critical to ensure all lysozyme is inactivated)</li>