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$("#Content11").html("<h2>Monday 20/08/2018</h2><p>Cells were tested in a plate reader for induction of sfGFP by anhydrotetracycline (ATC). This was conducted overnight at 37C with shaking. The data shows 50ng/ml worked most effectively at generating high fluorescence/OD600 (0.5uL in 1mL from a 100ug/ml stock)</p>");

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$("#Content12").html("<h2>Tuesday 21/08/2018</h2><p>Plate reader was used to test which of our plasmid constructs interacted most effectively with ATC at 100ng/ml overnight at 37C as well as determine if the basal fluorescence of our non-induced constructs (when centrifuged they formed green pellets, indicating sfGFP expression) was in fact satisfactory when comparing them with the induced cultures. Double-transformed cells containing plasmid constructs tested were PAJ379+PAJ390 (3A igRNA switch and 3A sRNA respectively) and PAJ426+PAJ471 (HIV3 igRNA switch and HIV VIF sRNA respectively).<br><br><table id='smoltable' style='width: 75%;'><tr><td></td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td></tr><tr><td>A</td><td></td><td>426 1</td><td>426 ATC 1</td><td>HIV 1</td><td>HIV ATC 1~~/td>~~<td>3A 1</td><td>3A ATC 1</td></tr><tr><td>B</td><td>LB ATC blank</td><td>426 2</td><td>426 ATC 2</td><td>HIV 2</td><td>HIV ATC 2</td><td>3A 2</td><td>3A ATC 2</td></tr><tr><td>C</td><td>LB blank</td><td>426 3</td><td>426 ATC 3</td><td>HIV 3</td><td>HIV ATC 3</td><td>3A 3</td><td>3A ATC 3</td></tr></p>");

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$("#Content12").html("<h2>Tuesday 21/08/2018</h2><p>Plate reader was used to test which of our plasmid constructs interacted most effectively with ATC at 100ng/ml overnight at 37C as well as determine if the basal fluorescence of our non-induced constructs (when centrifuged they formed green pellets, indicating sfGFP expression) was in fact satisfactory when comparing them with the induced cultures. Double-transformed cells containing plasmid constructs tested were PAJ379+PAJ390 (3A igRNA switch and 3A sRNA respectively) and PAJ426+PAJ471 (HIV3 igRNA switch and HIV VIF sRNA respectively).<br><br><table id='smoltable' style='width: 75%;'><tr><td></td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td></tr><tr><td>A</td><td></td><td>426 1</td><td>426 ATC 1</td><td>HIV 1</td><td>HIV ATC 1<td>3A 1</td><td>3A ATC 1</td></tr><tr><td>B</td><td>LB ATC blank</td><td>426 2</td><td>426 ATC 2</td><td>HIV 2</td><td>HIV ATC 2</td><td>3A 2</td><td>3A ATC 2</td></tr><tr><td>C</td><td>LB blank</td><td>426 3</td><td>426 ATC 3</td><td>HIV 3</td><td>HIV ATC 3</td><td>3A 3</td><td>3A ATC 3</td></tr></p>");

$("#Content13").html("<h2>Wednesday 22/08/2018</h2><p>Transformed cells showed no correlation between ATC and no ATC in the culture. Single plasmids (PAJ426 with sfGFP inhibited by LacI) also showed no response to ATC but had high basal fluorescence, implying sfGFP was being expressed in large amounts.<br>This is highly indicative of loss of LacI; GFP is constitutively expressed due to loss of its Z1 cassette and, therefore, loss of TetR, SpR and LacI. To test this we incubated the cells with spectinomycin for 6 hours. The cells did not grow. Therefore our hypothesis is correct.<br>To fix this, we decided to use a different batch of Z1 cells and ensure spectinomycin was present at all times to prevent loss of the Z1 cassette. Transformed negative control cells (pSB1AK8) and both HIV3 (PAJ426+PAJ471) and 3A (PAJ379+PAJ390) plasmids into new batch of competent Z1 cells.<br>To make the best of our cells that lost the Z1 cassette, we will use them as a positive control as we no longer need to insert IPTG to make them fluoresce, due to the loss of LacI from their chromosome.</p>");

Difference between revisions of "Team:Warwick/Notebook"

Revision as of 12:14, 16 October 2018

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@@ Line 155: / Line 155: @@
          $("#Content11").html("<h2>Monday 20/08/2018</h2><p>Cells were tested in a plate reader for induction of sfGFP by anhydrotetracycline (ATC). This was conducted overnight at 37C with shaking. The data shows 50ng/ml worked most effectively at generating high fluorescence/OD600 (0.5uL in 1mL from a 100ug/ml stock)</p>");
-         $("#Content12").html("<h2>Tuesday 21/08/2018</h2><p>Plate reader was used to test which of our plasmid constructs interacted most effectively with ATC at 100ng/ml overnight at 37C as well as determine if the basal fluorescence of our non-induced constructs (when centrifuged they formed green pellets, indicating sfGFP expression) was in fact satisfactory when comparing them with the induced cultures. Double-transformed cells containing plasmid constructs tested were PAJ379+PAJ390 (3A igRNA switch and 3A sRNA respectively) and PAJ426+PAJ471 (HIV3 igRNA switch and HIV VIF sRNA respectively).<br><br><table id='smoltable' style='width: 75%;'><tr><td></td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td></tr><tr><td>A</td><td></td><td>426 1</td><td>426 ATC 1</td><td>HIV 1</td><td>HIV ATC 1/td><td>3A 1</td><td>3A ATC 1</td></tr><tr><td>B</td><td>LB ATC blank</td><td>426 2</td><td>426 ATC 2</td><td>HIV 2</td><td>HIV ATC 2</td><td>3A 2</td><td>3A ATC 2</td></tr><tr><td>C</td><td>LB blank</td><td>426 3</td><td>426 ATC 3</td><td>HIV 3</td><td>HIV ATC 3</td><td>3A 3</td><td>3A ATC 3</td></tr></p>");
+         $("#Content12").html("<h2>Tuesday 21/08/2018</h2><p>Plate reader was used to test which of our plasmid constructs interacted most effectively with ATC at 100ng/ml overnight at 37C as well as determine if the basal fluorescence of our non-induced constructs (when centrifuged they formed green pellets, indicating sfGFP expression) was in fact satisfactory when comparing them with the induced cultures. Double-transformed cells containing plasmid constructs tested were PAJ379+PAJ390 (3A igRNA switch and 3A sRNA respectively) and PAJ426+PAJ471 (HIV3 igRNA switch and HIV VIF sRNA respectively).<br><br><table id='smoltable' style='width: 75%;'><tr><td></td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td></tr><tr><td>A</td><td></td><td>426 1</td><td>426 ATC 1</td><td>HIV 1</td><td>HIV ATC 1<td>3A 1</td><td>3A ATC 1</td></tr><tr><td>B</td><td>LB ATC blank</td><td>426 2</td><td>426 ATC 2</td><td>HIV 2</td><td>HIV ATC 2</td><td>3A 2</td><td>3A ATC 2</td></tr><tr><td>C</td><td>LB blank</td><td>426 3</td><td>426 ATC 3</td><td>HIV 3</td><td>HIV ATC 3</td><td>3A 3</td><td>3A ATC 3</td></tr></p>");
          $("#Content13").html("<h2>Wednesday 22/08/2018</h2><p>Transformed cells showed no correlation between ATC and no ATC in the culture. Single plasmids (PAJ426 with sfGFP inhibited by LacI) also showed no response to ATC but had high basal fluorescence, implying sfGFP was being expressed in large amounts.<br>This is highly indicative of loss of LacI; GFP is constitutively expressed due to loss of its Z1 cassette and, therefore, loss of TetR, SpR and LacI. To test this we incubated the cells with spectinomycin for 6 hours. The cells did not grow. Therefore our hypothesis is correct.<br>To fix this, we decided to use a different batch of Z1 cells and ensure spectinomycin was present at all times to prevent loss of the Z1 cassette. Transformed negative control cells (pSB1AK8) and both HIV3 (PAJ426+PAJ471) and 3A (PAJ379+PAJ390) plasmids into new batch of competent Z1 cells.<br>To make the best of our cells that lost the Z1 cassette, we will use them as a positive control as we no longer need to insert IPTG to make them fluoresce, due to the loss of LacI from their chromosome.</p>");