Difference between revisions of "Team:Munich/Readouts"

• Cas13a
• Readouts
• Targets
• Detection Chip
• Amplification
• Biobrick

For our experimental design, we used different fluorescence and colorimetric readouts as stated below.

RNaseAlert Readout

To characterize our Cas13a, we first turned to the standard of the field, namely the RNase Alert detection kit. This was used by Gootenberg and Doudna to characterize the Cas13a and detect pathogen RNA sequences¹. In the absence of Cas13a activation, the physical proximity of the quencher dampens fluorescence from the Fluor and there is no fluorescence activity. When Cas13a is activated, the RNA substrate is cleaved, and the Fluor and quencher are spatially separated in solution, emitting a bright green signal when excited by light of the appropriate wavelength. We did most of our experiments using the RNaseAlert system and the corresponding results are in the Cas13a and target subsections.

The fluorescence signal quality of the RNaseAlert assay was very good, however one cannot use this readout system without the fluorescence detector. Also, since the RNaseAlert is a modified RNA, it is somehow expensive in comparison to other readouts.

Spinach Aptamer Readout

After the successful experimentation of the Cas13a with the RNaseAlert, we also tried out RNA aptamers for our readouts. For this, we used the Spinach aptamer which binds to the DFHBI changing its 3D structure². We activated the Cas13a by the specific target, which then cleaved the Spinach aptamer bound to DFHBI and were able to show that the fluorescence activity slowly decreases (Figure 1).

Although we could see a clear decrease in the fluorescence activity as soon as the Cas13a is activated, the original level of fluorescence is lower than in case of RNaseAlert. This could be due to the fact that as soon as the spinach aptamer binds to the DFHBI, the fluorescence is already released. And regarding the time factor needed to mix all the reaction components, we lose some fluorescence before the Cas13a cleavage activity starts.

Figure 1: The fluorescence activity of the Spinach aptamer decreased with the increase in the target concentration.

ssDNA Readout

One of the first colorimetric readout that we tried out was the ssDNA oligo based readout. The idea is based around the formation of a RNA/DNA dimer and the freeing of the DNA oligo by digestion of the RNA part which has the poly-U loops. The overall idea of this readout was to utilize the Cas13a freed small activator DNA-oligo strand in various signal amplifying chains:

1. Completion of an in vitro transcription target (in vitro Tx
2. Primer for an isothermal PCR
3. Completion of a DNA-target for a transcription/translation system (tx/tl)

We used parts of an already established synthetic circuit from our lab called Circuit 3 (C3). Circuit 3 already provided us with a dsDNA target (= in vitro transcription target ds-C3) with a single stranded overhang in the promotor region and the complementary short DNA oligo, which we used as our activator (= C3 activator, C3a)( Figure 2).