Our subject will be used to diagnose inflammation and serve as a probiotic to facilitate the generation of hesperetin in response to inflammation risk in the human intestine.
Although the E. coli strain we have chosen has little or no risk of pathogenicity, accidentally ingesting might cause health hazards to our team membrane. Moreover, sharp equipment in our lab also potentially causes harm to our team membrane. Therefore, we conduct lab training for every single person in our team. During the training, team members learned how to conduct experiments safely and to follow biosafety rules. We comply with the "Waste Disposal Act" by Taiwan's Environmental Protection Administration and laboratory safe hygiene precautions of our institution.
Engineered bacteria might be released into the environment when users of our diagnostic device do not dispose of waste correctly, or anti-inflammatory bacteria leave the intestine and survive. Escaption of our engineered bacteria might cause horizontal gene transfer and affect the gene expression of other bacterial strains. In the lab, our biohazard waste is sterilized with the hypochlorous acid solution or steam. To prevent bacteria from escaping in the situation outside of our lab, we adapted the kill-switch design in our project. We use BBa_K115002, a thermo-switch activated at 37°C, and LacI, a plac promoter to control BBa_K145151, a ccdB gene that leads to bacteria dying. The thermo-switch ribosomal binding mechanism will be used in our project to terminate the engineered bacteria once it is released into the environment.
We constructed a kill switch genetic circuit by combining the BBa_K115002, lacI, and BBa_K145151.
Firstly, we designed primers P-R-r ( 5’ atACTAGTTTTCTCCTCTTTCTCTAGcctcctgaatcactattcaaaag 3’) and ccdB-his-r (5’ aTACTAGTAttaGTGATGGTGATGGTGATGtattccccagaacatcaggt3’).
Then, we performed the PCR reactions with VF2 + P-R-r and psB1C3- BBa_K115002 as template to obtain the BBa_K115002-B0034 fragment (~200 bp). Besides, we also used the primers VF2, ccdB-his-r, and pSB1C3- BBa_K115002 to generate the BBa_K115002 with his tag (~460 bp).
The electrophoresis gel results are shown in Figure 1.
We also confirmed our ligation results of the kill switch circuit, the BBa-K115002-B0034-Bba-K145151 in the pSB1C3.After the ligation experiment, we should get the plasmid as large as 2470 bp.
After the plasmid extraction experiments, we used the EcoRI and EcoRI + SpeI to do the RE digestion assay to confirm the ligation results. We anticipated that we should get the single linear product, 2470 bp in length, after EcoRI digestion. As for EcoRI + SpeI digestion, we should see the fragments as long as 420 bp and 2050 bp.
Figure 2. shows that two colonies, A and B, were selected in these experiments. A-1 and B-1 meant the digestion with EcoRI, as for A-2 and B-2, the EcoRI + SpeI were used.
Here we can see the clear enzyme digestion results as we anticipated, except a light one around 1.5 kb. Currently, we are trying to figure out what this is.
+886-2-2826-7000
nycu.taipei.igem@gmail.com
© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at gitlab.igem.org/2023/nycu-taipei.