*<p style=”text-align:justify;”>We confirmed our device yesterday, but we continued working with it. We proved that our device really works. To fulfill this aim, we transformed competent ''E. Coli'' C43. This ''E. coli'' strain can produce the T7 polimeraze by IPTG induction. Thus, when we put IPTG into the culture medium, we will have expression of T7 polimeraze, which will induce the T7 promoter of our device and then, the endolysin will be expressed. Our test consists in overexpression of the endolysin in order to break it's own peptidoglycan wall. We transformed cells with our device BBa_K284022 plasmid and with the BBa_K112806 plasmid (without promoter).</p>
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*<p style=”text-align:justify;”> After we confirmed the construction of our device we start planning our characterization tests to prove that our device really works. To fulfill this aim, our new device was transformed in ''E. coli'' C43 strain, an overexpression strain in which the T7 promoter can be induced by IPTG. We transformed cells with our device, BBa_K284022, and with the BBa_K112806(without promoter).</p>
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*<p style=”text-align:justify;”>So, after get the transformed cells, we put them to growth in 5 erlenmeyers (LB-AMP medium) until half log phase (OD-0.6 to 1). Then, we induced with IPTG (1mM) and measured the optical density during 4 hours after induction. We plated the 5 cultures in LB-AMP medium plates in order to confirm the diminished cellular growth. To have a definitive confirmation, we also performed SDS-PAGE to notice the protein overexpression. The results are shown in [https://2009.igem.org/Team:UNICAMP-Brazil/Coliguard/Results ColiGuard results].</p>
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*<p style=”text-align:justify;”>A transformed colony was incubated in LB medium with ampicillin at 37ºC overnight. The culture was diluted in new LB medium with ampicillin to an OD=0,2 and incubated at 37ºC until reach an OD=0,8. At this point, the cells were induced with IPTG 1 mM and incubated again for 4 hours. After the end of the induction period the cells were plated in LB agar with ampicillin in order to confirm the diminished cellular growth. To have a definitive confirmation, we also performed SDS-PAGE to notice the protein overexpression. The results are shown in [https://2009.igem.org/Team:UNICAMP-Brazil/Coliguard/Results ColiGuard results].</p>
After we confirmed the construction of our device we start planning our characterization tests to prove that our device really works. To fulfill this aim, our new device was transformed in E. coli C43 strain, an overexpression strain in which the T7 promoter can be induced by IPTG. We transformed cells with our device, BBa_K284022, and with the BBa_K112806(without promoter).
A transformed colony was incubated in LB medium with ampicillin at 37ºC overnight. The culture was diluted in new LB medium with ampicillin to an OD=0,2 and incubated at 37ºC until reach an OD=0,8. At this point, the cells were induced with IPTG 1 mM and incubated again for 4 hours. After the end of the induction period the cells were plated in LB agar with ampicillin in order to confirm the diminished cellular growth. To have a definitive confirmation, we also performed SDS-PAGE to notice the protein overexpression. The results are shown in ColiGuard results.
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