T7 promoter + RBS + killerred
We add an T7 promoter in front of the killer red, and use IPTG to induce and then test the expression of killer red. In our project, we design this part to make it more easy to test.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 238
Killer red is a red fluorescent protein that could produce ROS (Reactive oxygen species) by light illumination. As shown in Fig.1, green light (585 nm) stimulation of the QYG chromophore of killer red produces ROS from water to kill cell</div>
Figure 1. The sketch map of killer red mechanism
Figure 2. Confocal images of killer red expression in E.coli. Then, We tried to cultivate the induced killer red under the light of oven, and the growth curve did not show significant difference between the experimental group and control group, illustrating that when under the illumination of white light, it would not produce ROS that could do harm to cells, confirming it’s safety in gut. Finally, to test the efficiency of killer red, we put it under the illumination of the specific light of fluorescent microscope for 4 hours, and then cultivate them in 37℃ drying oven overnight, and we could see it clearly that the culture medium which both under induction and illumination is significantly have less single clones, and the others(one illuminated by light and not induced, one induced without illumination of light, one neither induced nor illuminated) surface are rough, indicating the large amount of E.coli.
Figure 3. A: the experiment group which was induced and illuminated under the green light resource of fluorescent for 4 hours; B: the control group which was induced but without illumination; C: the group which was not induced but with illumination; D: the group without inducer and illumination