Team:Kingsborough NY/LacZ
This page demonstrates the activity and function of our control constructs, K2268003 and K2268004, by measuring LacZ activity. Overall, our constructs appear to respond to light as intended - although the addition of an supplementary repressor in K2268004 does not seem to have lead to a greater degree of control on over our designed kill-switch.
Contents
- 1 Figure 1: LacZ expression of K2268004 grown in the Dark
- 2 Figure 2: Approach for ImageJ Analysis (Dark)
- 3 Figure 3: LacZ expression of K2268003 grown in the Light
- 4 Figure 4: Approach for ImageJ Analysis (Light)
- 5 Figure 5: Quantified Results comparing Light and Dark conditions
- 6 Figure 6: Optimizing X-gal concentrations
- 7 Figure 7: Direct Comparison of K2268003 and K2268004 with no IPTG added
- 8 Figure 8: Direct Comparison of K2268003 and K2268004 with 1mM IPTG added
Figure 1: LacZ expression of K2268004 grown in the Dark
(Figure 1)Shows the plating of K2268004 grown in the dark where the blue indicator shows the direction of plating. The center had the highest expression of LacZ suggesting that rate of cell growth is the highest at the center and rate of cell growth towards the edge of the plate is lower. The cell was induced with 1mM of IPTG in 20 ɥg/ɥl of X-gal.
Figure 2: Approach for ImageJ Analysis (Dark)
(Figure 2)Shows the ImageJ analysis for the average expression level. Each “cell” is a ratio of blue to gray and expression level was found by taking the ratio of the background to the cell.
Figure 3: LacZ expression of K2268003 grown in the Light
(Figure 3)Shows the plating of K2268003 grown in the light where the blue indicator shows the direction of plating. The center had the highest expression of LacZ suggesting that rate of cell growth is the highest at the center and rate of cell growth towards the edge of the plate is lower. The cell was induced with 1mM of IPTG in 20 ɥg/ɥl of X-gal.
Figure 4: Approach for ImageJ Analysis (Light)
(Figure 4)Shows the ImageJ analysis for the average expression level. Each “cell” is a ratio of blue to gray and expression level was found by taking the ratio of the background to the cell.
Figure 5: Quantified Results comparing Light and Dark conditions
(Figure5)Shows that there is a significant difference in expression level between K2268005 grown in light and K2268005 grown in dark with significantly higher expression in K2268005 grown in light, p<.001. Data analysis was done by comparing the mean expression level of figure 2 and figure 4 with GraphPad Software.
Figure 6: Optimizing X-gal concentrations
(Figure 6) shows that increasing x-gal from 20 ɥg/ɥl to 40 ɥg/ɥl showed a noticeable increase in LacZ expression for K2268004. Increased expression level shows that LacI inhibition is not strong and that LacZ expression is coming from the plasmid backbone (pSB1C3) or the bacteria itself.
Figure 7: Direct Comparison of K2268003 and K2268004 with no IPTG added
(Figure 7) Another demonstration of the expression of LacZ in both K2268003 (top right on each plate) and K2268004 (bottom right on each plate), together with a negative control plasmid (bottom left) that does not contain LacZ. These plates were grown either in complete darkness (right) or exposed to light (left) during the entire growth period - neither plate contains IPTG.
Figure 8: Direct Comparison of K2268003 and K2268004 with 1mM IPTG added
(Figure 8) Another demonstration of the expression of LacZ in both K2268003 (top right on each plate) and K2268004 (bottom right on each plate), together with a negative control plasmid (bottom left) that does not contain LacZ. These plates were grown either in complete darkness (left) or exposed to light (right) during the entire growth period - both plates contain 1mM IPTG, which should repress LacZ expression from K2268004.
