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).

The annealing of the two strands only worked to some extent, needing around 8 to 10x excess of inhibitor RNA to fully anneal with 500 nM of DNA activator oligo at room temperature. Room temperature was chosen, because it is extremely difficult to generate sharp temperature transitions on a small chip, which would be needed for good annealing results. Reasons for a bad annealing reaction of the two strands could be the mix of different length products of the inhibitor RNA in vitro transcription initially or the steady state of RNA digested with RNA in solution, resulting in a reannealing of RNA with the DNA activator.

The RNA background brought up major problems in combination with the transcription measurement and brought up disordered results with increasing RNA background concentration not directly resulting in lower transcriptional burst differentiation. Problems for the realization of a ssDNA activator readout lie for example in the various reaction environments needed for the dimerization, the RNA digestion and the final amplification step, may it be as a transcription, PCR or in a transcription/translation system.

Intein-Extein Readout

For the intein-extein system, we order two gblocks from IDT. These gblocks were then cloned into psB1C3 and psB4A5 respectively. The successful insertion was confirmed by Sequencing at GATC. The plasmids were then opened by BbsI and the coding sequence of beta-galactosidase was inserted via a Gibbson-Assembly. Once again, we confirmed the sequences at GATC and got full length reads for both constructs. However, the two biobricks could neither be submitted nor characterized, as the recloning to remove illegal cuts sites within the sequence was not finished prior to the deadline. The characterization was not possible, as the C-terminal fragment couldn’t be expressed in any of our E. coli expression strains, as it seemed to be toxic. The toxicity seemed to not affect E. Coli Turbo, which was used for the cloning. The N-terminal part alone couldn’t be characterized as the parts only work together.

We plan to express the C-terminal part with an in vitro expression system and are confident, that the purified proteins will work together as planned.

Gel pictures of the final steps in the purification of our four proteins