Overview

We demonstrated that each of the modules of our platform (extraction, amplification and detection of pathogenic RNA) is functional, although we did not yet fully integrate all the modules into a final product.

What worked

• Demonstrated functionality of Lbu and Lwa Cas13a.
• Modelled the detection limit of our circuit and confirmed it experimentally (~10 nM RNA).
• Detected pathogen RNA sequence from in vitro and in vivo sources.
• Differentiated viral sequences from bacterial sequences.
• Used RNase Alert and Spinach aptamer read-out circuits.
• Used gold nanoparticles to detect general RNase activity.
• Detected RNA in bulk, on paper, and from lyophilized Cas13a.
• Constructed a functional fluorescence detector with high sensitivity and low production cost.
• Detected RNA in bulk, on paper, and from lyophilized Cas13a.
• Amplified target with RPA and transcription on paper.
• Improved the biobrick BBa_K1319008 by adding a 6x His-tag and provided Cas13a Lwa as three different composite biobricks.
• Characterized the GFP degradation tags and sent them as a part collection.

What presented issues

• Optimizing the purification protocol for Cas13a.
• Demonstrating functionality of Lsh Cas13a.
• Ruling out RNase contamination from heat-lysed in vivo samples.
• Detecting Q5 beta RNA.
• Reducing cross-talk between E.coli crRNA and B.subtilitis target RNA.
• Developing colorimetric read-outs.
• Optimizing the lyophilization and stability of Cas13a.
• Performing RPA and transcription on chip.