Part:BBa_K5096074

This sequence encodes the complete sgRNA9, which directs the dCas9 protein to a specific genomic site, specifically, the deGFP target region of the deGFP plasmid. The binding sequence is derived from P70a-deGFP, and both sgRNA9 and P70a-deGFP were originally developed by Marshall et al. (2018). The graph below illustrates the CRISPRi reaction using sgRNA9 to downregulate P70a-deGFP expression. The results were successful, with positive controls showing high fluorescence levels, while sgRNA9 effectively repressed deGFP production as expected. Quantification of repression showed that sgRNA9 achieved 59.8% downregulation. Additionally, sgRNA9 was characterized to determine the optimal concentration for use in TXTL lysates. A concentration curve was generated by varying the final concentration of sgRNA9 from 3nM to 10nM. Results indicated that a 5nM final concentration of sgRNA9 achieved approximately 44% repression, despite lower GFP repression. These findings, which can be seen from the graph below, suggest the CRISPRi reaction remained effective under these conditions. In order to optimize wetlab outcomes, Lambert iGEM conducted a series of simulations to assess the impact of varying sgRNA concentrations. The concentration tests include 3nm, 5nm, 8nm, and 10nm. After creating a set of models and observing peak areas, Lambert iGEM predicted the values of 125 for 3nm, 205 for 5nm, 140 for 8nm, and 175 for 10nm. This model suggested that the concentration of 5nm of sgRNA would yield the highest peak value, indicating optimal performance. The wetlab committee tested the concentrations using the model created, and the experimental concentrations were similar to the predictions. 5nm displayed the highest peak concentration, confirming it as the most optimized sgRNA concentration.