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<p> <center>Figure 6: Effect of different wash steps on overall transformation efficiency (TrE). All competent cell preparation followed the standard MgCl2-CaCl2 protocol, with only the wash steps altered. 0 Wash - initial culture pellet followed by immediate aliquot of 100 uL storage/transformation buffer. 1 wash – a combined 100 mM MgCl2 and 100 mM CaCl2 buffer with 1 wash step. MgCl2 + CaCl2 – a combined 100 mM MgCl2 and 100 mM CaCl2 buffer with original two wash steps. MgCl2/CaCl2 – a 100 mM MgCl2 wash step, followed by a separate 100 mM CaCl2 wash step as per standard protocol. No significant impact on TrE (Kruskal-Wallis, H = 1.34, d.f. = 3, p = 0.720) was shown. Removing the wash step was the most effective (mean TrE = 2.30 x 106), with the more time consuming MgCl2-CaCl2 protocol being the second most effective (mean = 2.18 x 106). The least effective were the combined MgCl2/CaCl2 two wash (mean TrE = 1.79 x 106) and one wash (mean TrE = 1.66 x 106).</p> | <p> <center>Figure 6: Effect of different wash steps on overall transformation efficiency (TrE). All competent cell preparation followed the standard MgCl2-CaCl2 protocol, with only the wash steps altered. 0 Wash - initial culture pellet followed by immediate aliquot of 100 uL storage/transformation buffer. 1 wash – a combined 100 mM MgCl2 and 100 mM CaCl2 buffer with 1 wash step. MgCl2 + CaCl2 – a combined 100 mM MgCl2 and 100 mM CaCl2 buffer with original two wash steps. MgCl2/CaCl2 – a 100 mM MgCl2 wash step, followed by a separate 100 mM CaCl2 wash step as per standard protocol. No significant impact on TrE (Kruskal-Wallis, H = 1.34, d.f. = 3, p = 0.720) was shown. Removing the wash step was the most effective (mean TrE = 2.30 x 106), with the more time consuming MgCl2-CaCl2 protocol being the second most effective (mean = 2.18 x 106). The least effective were the combined MgCl2/CaCl2 two wash (mean TrE = 1.79 x 106) and one wash (mean TrE = 1.66 x 106).</p> | ||
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<p><center>Figure 11: Prediction profiler modelling the effect that varying reagent concentration has on overall transformation efficiency (TrE). Overall prediction profile set to most desirable transformation buffer composition. Black line indicates concentration relative to predictive TrE. Vertical dashed red line indicates the concentration of reagent at most desirable composition while horizontal dashed red line indicates predicted average TrE at desirable composition. Top panels show calculated effects of reagents after TrE was calculated after a 37℃ overnight 16 hour incubation post transformation recovery step. Bottom panels show calculated effects of reagents after TrE was recalculated after 96 hour incubation at room temperature (22-25℃) post transformation recovery step. Highlighted green panel indicate that a higher MgCl2.6H2O concentration had a statistically significant (p=0.0096) positive increase in overall TrE after a 96 hour incubation. Its effect after 16 hours incubation was shown to be positive, however not statistically significant. Highlighted blue panels indicate a statistically significant decrease in overall TrE when concentration is increased. Blue panel (A) indicates that the presence NiCl2 is significantly inhibitory to overall TrE (p = 0.0058) after 16 hours post recovery. Blue panel (B) suggests that CaCl2.6H2O has a negative impact with increasing concentration (p = 0.0490) and blue panel (C) indicates that kOAc has a significant inhibitory effect (p = 0.0153) on overall TrE after 96 hours post incubation. [Co(NH3)6]Cl3 at both post transformation time points is shown to have negligible effect on TrE. </p></center> | <p><center>Figure 11: Prediction profiler modelling the effect that varying reagent concentration has on overall transformation efficiency (TrE). Overall prediction profile set to most desirable transformation buffer composition. Black line indicates concentration relative to predictive TrE. Vertical dashed red line indicates the concentration of reagent at most desirable composition while horizontal dashed red line indicates predicted average TrE at desirable composition. Top panels show calculated effects of reagents after TrE was calculated after a 37℃ overnight 16 hour incubation post transformation recovery step. Bottom panels show calculated effects of reagents after TrE was recalculated after 96 hour incubation at room temperature (22-25℃) post transformation recovery step. Highlighted green panel indicate that a higher MgCl2.6H2O concentration had a statistically significant (p=0.0096) positive increase in overall TrE after a 96 hour incubation. Its effect after 16 hours incubation was shown to be positive, however not statistically significant. Highlighted blue panels indicate a statistically significant decrease in overall TrE when concentration is increased. Blue panel (A) indicates that the presence NiCl2 is significantly inhibitory to overall TrE (p = 0.0058) after 16 hours post recovery. Blue panel (B) suggests that CaCl2.6H2O has a negative impact with increasing concentration (p = 0.0490) and blue panel (C) indicates that kOAc has a significant inhibitory effect (p = 0.0153) on overall TrE after 96 hours post incubation. [Co(NH3)6]Cl3 at both post transformation time points is shown to have negligible effect on TrE. </p></center> | ||
− | <img src="https://static.igem.org/mediawiki/2018/e/ec/T--Newcastle--MeasurementFigure12.jpg"> | + | <img src="https://static.igem.org/mediawiki/2018/e/ec/T--Newcastle--MeasurementFigure12.jpg">{width}5px |
<p><center>Figure 12: Prediction profile modelling the interactions of transformation buffer (TB) constituents deemed to significantly affect overall transformation efficiency (TrE) after 16 hours of post-recovery incubation. Reagents were selected based on their significance, with only reagents with individual P values < 0.1 being selected for modelling. (A) Prediction profile set to most desirable TB composition. Black line indicates concentration relative to predictive TrE. Vertical dashed red line indicates the concentration of reagent at most desirable composition while horizontal dashed red line indicates predicted average TrE at desirable composition. Highlighted blue panels indicate a significant inhibitory effect on TrE. Highlighted green panels indicate significantly positive increase in TrE. Both MgCl2.6H2O and RbCl have a positive interaction with increasing concentration (P = 0.0095). NiCl2 and DMSO have a significant inhibitory interaction with individual increasing concentration (P = 0.0002 and P = 0.0479), however when both concentrations are increased they have a positive interaction with each other (P = 0.0054). (B) Interaction profile describing notable interactions between all significant reagents. Each panel represents an interaction between two reagents, with one reagent set at maximum concentration and the other at either lowest concentration (red line) or highest concentration (blue line). Panels positioned above or below the reagents in the graph are the reagents at maximum concentration, whereas panels positioned left or right are the reagents at either low or high concentrations. All other reagents included in the model but are not being examined in the panel are set to maximum. </p></center> | <p><center>Figure 12: Prediction profile modelling the interactions of transformation buffer (TB) constituents deemed to significantly affect overall transformation efficiency (TrE) after 16 hours of post-recovery incubation. Reagents were selected based on their significance, with only reagents with individual P values < 0.1 being selected for modelling. (A) Prediction profile set to most desirable TB composition. Black line indicates concentration relative to predictive TrE. Vertical dashed red line indicates the concentration of reagent at most desirable composition while horizontal dashed red line indicates predicted average TrE at desirable composition. Highlighted blue panels indicate a significant inhibitory effect on TrE. Highlighted green panels indicate significantly positive increase in TrE. Both MgCl2.6H2O and RbCl have a positive interaction with increasing concentration (P = 0.0095). NiCl2 and DMSO have a significant inhibitory interaction with individual increasing concentration (P = 0.0002 and P = 0.0479), however when both concentrations are increased they have a positive interaction with each other (P = 0.0054). (B) Interaction profile describing notable interactions between all significant reagents. Each panel represents an interaction between two reagents, with one reagent set at maximum concentration and the other at either lowest concentration (red line) or highest concentration (blue line). Panels positioned above or below the reagents in the graph are the reagents at maximum concentration, whereas panels positioned left or right are the reagents at either low or high concentrations. All other reagents included in the model but are not being examined in the panel are set to maximum. </p></center> |
Revision as of 10:19, 17 October 2018