The experiments and protocols followed an organized sequence of steps that allowed us to validate the results obtained from our laboratory work. We focus on having several lines in our experiments that were carried out separately but still converge in the same goal:
- 3A Assembly of biosensor promoters
- 3A Assembly of Heavy metals
- Signal amplification cascade
- Mucolytic production in E. Coli
In order to develop our project, we looked for the appropriate protocols to success, however, it was necessary to modify some of them in order to adapt them for our needs and goals. The following list shows all the protocols used, please read the PDF document for the complete steps. We hope they could be easily repeated by future iGEM teams to ensure the repeatability of each experience.
- PCR amplification
- PCR clean-up
- Digestion
- Agarose gel electrophoresis
- DNA gel extraction
- Ligation
- 3A assembly
- Golden
- Competent Cells (E. Coli)
- Electro (stbl4)
- Calcium (dh5alpha and BL21)
- Transformation protocol (E. Coli)
- Heatshock
- Electroporation
- Competence and transformation (Bacillus subtilis)
- Glycerol stocks
- Plasmidic DNA extraction (Miniprep)
- Protein induction assay
- Protein purification assay
- Fluorescence kinetic curve
- Bradford