Difference between revisions of "Team:Warwick/Notebook"

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         $("#Content15").html("<h2>Friday 24/08/2018</h2><p>Cells transformed successfully. To be sure the cells were transformed with the plasmids, IPTG was added at 1mM concentration and incubated at 37C for 1 hour. Centrifuged pellet showed much higher green fluorescence than that of the non-induced cells. Therefore, our cells have transformed successfully.<br>These cells were replated with chloramphenicol, kanamycin and spectinomycin on LBA. Meanwhile, liquid cultures of individual plasmid-containing cells (PAJ379 and PAJ390) were incubated with shaking overnight at 37C and appropriate antibiotics.</p>");
 
         $("#Content15").html("<h2>Friday 24/08/2018</h2><p>Cells transformed successfully. To be sure the cells were transformed with the plasmids, IPTG was added at 1mM concentration and incubated at 37C for 1 hour. Centrifuged pellet showed much higher green fluorescence than that of the non-induced cells. Therefore, our cells have transformed successfully.<br>These cells were replated with chloramphenicol, kanamycin and spectinomycin on LBA. Meanwhile, liquid cultures of individual plasmid-containing cells (PAJ379 and PAJ390) were incubated with shaking overnight at 37C and appropriate antibiotics.</p>");
  
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         $("#Content16").html("<h2>Monday 27/08/2018</h2><p>Cells were refreshed 1:200 in 2ml of M9 minimal media plus antibiotics plus anhydrotetracycline and grown at 37C with shaking at 220rpm for 2 hours. Cells were then added (1uL in 199uL ddH2O) to black-sided clear-bottom 96-well plates and incubated in a TECAN infinite F500 fluorescent plate reader at 37C with shaking. Optical density (OD600nm) and fluorescence (480/20nm excitation 530/25 emission) were measured in 15 minute intervals with 3 biological repeats and 3 technical repeats. All measurements were blanked against M9 media plus antibiotics plus ATC (100ng/ml), then against MG1655Z1 cells containing pSB1C3 and pSB1AK8 plasmid backbones so that they have no basal fluorescence.<br><br>Test plasmid constructs: Measure GFP levels in plate reader in M9 minimal media with spectinomycin (17μg/ml); Positive control - Cells with loss of the Z1 cassette to constitutively express sfGFP; Negative control - Cells, non-induced; Inducible - ATC (anhydrotetracycline) (moisture-, light-, and air-sensitive!) at 100ng/ml.<br>Fluorescence protocol<br><ul><ol>Grow cells overnight to early stationary phase at 37°C at 220rpm overnight in M9 minimal media with appropriate antibiotics (spec + chlor/kan) [and ATC for induced test cells at 100ng/ml]. Meanwhile, prepare negative control cells by transforming pSB1C3 and pSB1AK8 into MG1655Z1 cells.</ol><ol>Refresh cells 1:200 in 2ml M9 minimal media plus antibiotics [plus ATC for induced cells], and grow at 37°C at 220rpm for 2 hours. Add cells to black-sided clear-bottom 96-well plate (3 replicates of each; induced cells, non-induced cells, positive controls, negative controls, blanks)</ol><ol>Incubate plates in plate reader at 37°C with shaking (Standard Protocol - OD = 600nm, fluorescence = 480/20nm excitation, 530/25nm emission) in 15 minute intervals (3 repeats, and 3 replicates - 1 std deviation in error bars) blanked against M9 media with antibiotics and ATC as necessary, and then against MG1655Z1 cells containing empty plasmids (no igRNA/trigger/dCas9/sfGFP components to generate basal fluorescence levels).</ol></ul><br>Experiment: 3 replicates of each, 15 minute intervals, 37C, 80 cycles (20 hours)<br>OD = 600nm, Fluor for sfGFP = 480/20nm excitation, 530/25nm emission.<br><br>Induced (In) (E. coli MG1655Z1 cells with PAJ379 and PAJ390 + chlor + kan +spectinomycin + 100ng/ml ATC in M9)<br><br>Non-induced (NIn) (E. coli MG1655Z1 cells with PAJ379 and PAJ390 + chlor + kan +spectinomycin, no ATC in M9)<br>Positive control (Pos) (E. coli MG1655Z1 LacI-SpR cells with PAJ379 and PAJ390 + chlor + kan in M9)<br>Negative control (Neg) (E. coli MG1655Z1 cells with pSB1C3 and pSB1AK8 + chlor + kan in M9)<br>379 sfGFP (379) (E. coli MG1655Z1 cells with PAJ379 encoding 3A switch sgRNA and sfGFP + kan)<br>390 dCas9 (390) (E. coli MG1655Z1 cells with PAJ390 encoding the sRNA [3A trigger] and dCas9 + chlor)<br>ATC only (ATC) (0.1ug/ml ATC + chlor + kan + spectinomycin in M9)<br>Blank (Blnk) (M9 + chlor + kan + spectinomycin, no cells)</p><img src='https://static.igem.org/mediawiki/2018/5/51/T--Warwick--table0.png'>");
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         $("#Content16").html("<h2>Monday 27/08/2018</h2><p>Cells were refreshed 1:200 in 2ml of M9 minimal media plus antibiotics plus anhydrotetracycline and grown at 37C with shaking at 220rpm for 2 hours. Cells were then added (1uL in 199uL ddH2O) to black-sided clear-bottom 96-well plates and incubated in a TECAN infinite F500 fluorescent plate reader at 37C with shaking. Optical density (OD600nm) and fluorescence (480/20nm excitation 530/25 emission) were measured in 15 minute intervals with 3 biological repeats and 3 technical repeats. All measurements were blanked against M9 media plus antibiotics plus ATC (100ng/ml), then against MG1655Z1 cells containing pSB1C3 and pSB1AK8 plasmid backbones so that they have no basal fluorescence.<br><br>Test plasmid constructs: Measure GFP levels in plate reader in M9 minimal media with spectinomycin (17μg/ml); Positive control - Cells with loss of the Z1 cassette to constitutively express sfGFP; Negative control - Cells, non-induced; Inducible - ATC (anhydrotetracycline) (moisture-, light-, and air-sensitive!) at 100ng/ml.<br><br><br>Fluorescence protocol<br><ul><ol>Grow cells overnight to early stationary phase at 37°C at 220rpm overnight in M9 minimal media with appropriate antibiotics (spec + chlor/kan) [and ATC for induced test cells at 100ng/ml]. Meanwhile, prepare negative control cells by transforming pSB1C3 and pSB1AK8 into MG1655Z1 cells.</ol><ol>Refresh cells 1:200 in 2ml M9 minimal media plus antibiotics [plus ATC for induced cells], and grow at 37°C at 220rpm for 2 hours. Add cells to black-sided clear-bottom 96-well plate (3 replicates of each; induced cells, non-induced cells, positive controls, negative controls, blanks)</ol><ol>Incubate plates in plate reader at 37°C with shaking (Standard Protocol - OD = 600nm, fluorescence = 480/20nm excitation, 530/25nm emission) in 15 minute intervals (3 repeats, and 3 replicates - 1 std deviation in error bars) blanked against M9 media with antibiotics and ATC as necessary, and then against MG1655Z1 cells containing empty plasmids (no igRNA/trigger/dCas9/sfGFP components to generate basal fluorescence levels).</ol></ul><br>Experiment: 3 replicates of each, 15 minute intervals, 37C, 80 cycles (20 hours)<br>OD = 600nm, Fluor for sfGFP = 480/20nm excitation, 530/25nm emission.<br><br>Induced (In) (E. coli MG1655Z1 cells with PAJ379 and PAJ390 + chlor + kan +spectinomycin + 100ng/ml ATC in M9)<br><br>Non-induced (NIn) (E. coli MG1655Z1 cells with PAJ379 and PAJ390 + chlor + kan +spectinomycin, no ATC in M9)<br>Positive control (Pos) (E. coli MG1655Z1 LacI-SpR cells with PAJ379 and PAJ390 + chlor + kan in M9)<br>Negative control (Neg) (E. coli MG1655Z1 cells with pSB1C3 and pSB1AK8 + chlor + kan in M9)<br>379 sfGFP (379) (E. coli MG1655Z1 cells with PAJ379 encoding 3A switch sgRNA and sfGFP + kan)<br>390 dCas9 (390) (E. coli MG1655Z1 cells with PAJ390 encoding the sRNA [3A trigger] and dCas9 + chlor)<br>ATC only (ATC) (0.1ug/ml ATC + chlor + kan + spectinomycin in M9)<br>Blank (Blnk) (M9 + chlor + kan + spectinomycin, no cells)</p><img src='https://static.igem.org/mediawiki/2018/5/51/T--Warwick--table0.png'>");
  
 
         $("#Content17").html("<h2>Tuesday 28/08/2018</h2><p>Results obtained were subtracted by their respective blank measurements (ATC blank for induced cells, M9 blank for other measurements) then averaged across their 3 biological repeats. The final results show a 5.68-fold upregulation of sfGFP expression in induced cells compared with non-induced cells. Whilst the non-induced cells had significantly higher fluorescence than that of the individual plasmid constructs and the negative control (likely due to leakage in the circuit by dCas9-igRNA binding pLacIQ-1 without the sRNA, thereby inhibiting its expression), the positive control and induced cells had comparably higher fluorescence, indicating our circuit can be induced by the expression of the signalling RNA (sRNA) by ATC.</p><img src='https://static.igem.org/mediawiki/2018/a/a1/T--Warwick--FOD600.png'><br>Ordered forward and reverse primers for plasmid backbones and oligos to begin testing our own device.");
 
         $("#Content17").html("<h2>Tuesday 28/08/2018</h2><p>Results obtained were subtracted by their respective blank measurements (ATC blank for induced cells, M9 blank for other measurements) then averaged across their 3 biological repeats. The final results show a 5.68-fold upregulation of sfGFP expression in induced cells compared with non-induced cells. Whilst the non-induced cells had significantly higher fluorescence than that of the individual plasmid constructs and the negative control (likely due to leakage in the circuit by dCas9-igRNA binding pLacIQ-1 without the sRNA, thereby inhibiting its expression), the positive control and induced cells had comparably higher fluorescence, indicating our circuit can be induced by the expression of the signalling RNA (sRNA) by ATC.</p><img src='https://static.igem.org/mediawiki/2018/a/a1/T--Warwick--FOD600.png'><br>Ordered forward and reverse primers for plasmid backbones and oligos to begin testing our own device.");

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