Difference between revisions of "Team:Munich/Results"

Bacterial targets used for the experiments

Escherichia coli

We took 16s rRNA of the E. coli as our target RNA. Since 16s rRNA is highly conserved in all bacterial species and can used as a well characterized site for our cleavage assays. It can also be easily extracted from bacterial cultures. For our experiments, we used only a part of the 16s rRNA since the whole 16s rRNA is too large to be transcribed (1500 bp). For this particular target RNA sequence we took, we designed the crRNA and in vitro transcribed the crRNA and the target RNA in our lab. We also performed RNA extraction using chemical lysis and heat lysis for the E. coli samples. Although the chemical lysis gave us good quality and detectable concentration of the RNA, the heat lysis didn’t work so well. There was always some cellular residues, RNases present in the sample due to which the fluorescence activity in the cleavage assay was way higher than the positive controls.

16s rRNA part used for the experiment

Figure 1: Gel picture showing the our 16s rRNA partial sequence used for our experiments

Figure 2: Urea gel picture of the different crRNAs

Bacillus subtilis

We also focused on trying out our experiment with other target RNAs and for this we chose the gram positive Bacillus subtilis since it is widely used in microbiological research. Plus we wanted to see if one can detect the difference between the 16s rRNAs of B. subtilis and E. coli . For B. subtilis , we did not perform any in vitro transcription, rather we directly used the bacterial culture for the RNA extraction. However, we did encounter some problems due to the spore forming nature of the Bacillus subtilis . Also, the quality of the extracted RNA was not so good and there were some cellular residues apart from the RNA which caused some problems during the assay.

crRNA designed for the Bacillus subtilis 16s RNA

Viral targets used for the experiments

Noro virus

Noro virus originally called Norwalk virus, of the family Caliciviridae, is one of the major cause of viral gastroenteritis in humans and it affects patients of all age groups. It is also the cause of high rate of deaths and is associated with hospital infections. For our experiments, we took the 5’ UTR of the Noro virus and also did in vitro transcription to get the target RNA and the crRNA. The 5’ UTR of the viruses are very specific to each individual virus so one can use this part to design the crRNA and detect different viral RNAs using the Cas13a system.

crRNA designed for the Noro virus

Hepatitis C virus

HCV is a small single stranded RNA virus of family Flaviviridae which is the major cause of the Hepatitis C and liver cancer. Common setting for transmission of HCV is also intra-hospital (nosocomial) transmission, when practices of hygiene and sterilization are not correctly followed in the clinic. There are no vaccines for HCV virus. For our experiments, we took the 5’ UTR of the HCV virus and also did in vitro transcription to get the target RNA and the crRNA.

crRNA designed for the HCV virus

Gel picture

Cas13a strains used for the experiments

The genus Leptotrichia was one of the first microorganisms to be drawn and described by the Antoni van Leeuwenhoek. The generic name was first used in 1879 for filamentous organisms found in the human mouth. We used the following strains of Cas13a for our experiments.

• Leptotrichia buccalis (referred as Lbu in our experiments)
• Leptotrichia wadei (referred as Lwa in our experiments)
• Leptotrichia shahii (referred as Lsh in our experiments)

We expressed our His-tagged proteins in E. coli strains and purified them using a Äkta purification system or Ni-NTA agarose. To cleave off the His-SUMO or His-MBP tags from Cas13a proteins, we incubated them with the SUMO or TEV protease (BBa_K2323002) during dialysis overnight, respectively. In some cases, we reloaded the cleaved protein solution again on Ni-NTA agarose to get rid of the thereby binding His-tag. For higher purity, we loaded the proteins on a size exclusion column. Protein purity was always checked by SDS PAGE.

Both the Cas13a Lbu and Lwa are the central component of our diagnostic platform. The TEV Protease is part of our idea to the Intein-Extein readout, but apart from that, served as molecular tool for cleaving off the protein tags. So far, we managed to express and purify all three mentioned Cas13a proteins and the TEV protease as you can see in following chromatograms and SDS gels. 

Äkta purification

His purification Äkta graph Lbu plus gel

Nickel NTA purification of Lwa

HCV is a small single stranded RNA virus of family Flaviviridae which is the major cause of the Hepatitis C and liver cancer. Common setting for transmission of HCV is also intra-hospital (nosocomial) transmission, when practices of hygiene and sterilization are not correctly followed in the clinic. There are no vaccines for HCV virus. For our experiments, we took the 5’ UTR of the HCV virus and also did in vitro transcription to get the target RNA and the crRNA.

Lwa gel from ni nta

Size exclusion purification

SEC purification Lbu plus gel

Affinity purification and Size exclusion purification of TEV protease

His purification TEV

Assays used for the experiments

For our experimental design, we used different fluorescence assays as stated below:

RNaseAlert Assay

This is a commercial kit readily available in the markets, which can be used for the detection of the RNase activity and sensitivity in real time. The RNaseAlert® QC System uses a novel RNA substrate tagged with a fluorescent reporter molecule (fluor) on one end and a quencher on the other. In the absence of RNases, the physical proximity of the quencher dampens fluorescence from the Fluor to extremely low levels. When RNases are present, however, the RNA substrate is cleaved, and the Fluor and quencher are spatially separated in solution. This causes the Fluor to emit a bright green signal when excited by light of the appropriate wavelength. Since the fluorescence of the RNaseAlert substrate increases over time when RNase activity is present, results can be easily monitored. For the detection and monitoring of the kinetics of the fluorescence, we used the plate readers in lab and our self-made fluorescence detector.

Spinach Aptamer Assay

The spinach aptamer assay is based on a fluorophore DMHBI which was the first molecule against which a SELEX experiment was run. However, DFHBI was extracted from eGFP and it exhibits a higher extinction coefficient and lead to a brightness increase of eGFP. In 2012, Paige et al. developed the 24-2 aptamer, mostly known as Spinach due its green fluorescence when bound to DFHBI. The Spinach aptamer exclusively binds the deprotonated variant of eGFP (DFHBI) with a dissociation constant of Kd = 390nM. It increases the quantum yield of DFHBI from 0.0007 when free to 0.72 when bound to the aptamer. Figure (a) Structure of the Spinach aptamer in absence (yellow) and in presence (green) of DFHBI. (b) G-quadruplex motif of the Spinach aptamer in absence (yellow) and in presence (green) of DFHBI.

Aptamer

The aptamer structure is elongated and it folds with two helical stems adjacent to the binding region, which exhibits a G-quadruplex pattern. The Spinach aptamer binds the DFHBI in a planar conformation. Hydrogen bonds are formed between the G-nucleotides and the fluorophore, and the aptamer changes its 3d-structure when bound to the DFHBI. In the absence of the fluorophore, the base triplet formed by the nucleotides A53-U29-A58 collapses on the G-quadruplex site. Spinach shifts the absorbance maximum of the DFHBI by approximately 60 nm comparing with the unbound form, from 405 nm to 469 nm. Spinach has been used for imaging protein and gene expression, and it has been also modified in order to be used as a sensor of biological reactions.

Proof of principle

To characterize key protein of our diagnostic device we conducted several experiments.

Firstly, we confirmed that Cas13a activity is target dependent. Despite the fact that Cas13a exhibits RNase activity in absence of target RNA, its activity in presence of target RNA is up to 8 times higher. However, this is true at low protein concentrations. At high concentrations of Cas13a presence of target RNA does not have significant effect on enzyme activity as depicted in the Figure 3. Secondly, we verified that enzyme is activated by crRNA. As Figure 4 (this is the only figure with old enzyme, so concentrations are completely off the values of enzyme purified and used later on) shows, enzyme is active only in the presence of crRNA. It can be seen the higher is the concentration of crRNA, the more of enzyme gets activated, which is in accordance with the first step of reaction --link to overall reaction equation--. Besides that, crRNA when forming a complex with Cas13a defines specificity of ribonuclease. This was confirmed by cross-reactivity experiment.

please place results of cross-reactivity experiment here

And most importantly we determined detection limit of Cas13a-crRNA complex by varying target RNA concentration. Figure 2 shows that target concentrations above two-digits in nanomolar range can be detected.

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Crosstalk experiments

To show that the Cas13a is highly specific for a particular target, we tested the CRISPR RNA designed for Noro virus with different targets, namely E. coli , HCV and Noro virus. As observed in the bar graph on the right, the Cas13a activity is visible only there is the presence of the target as Noro virus itself. Whereas in presence of other targets there is very low background fluorescence visible, which is also measurable when no target is present. The results observed showed that there is no crosstalk between the targets and that a particular crRNA is specific for one type of target RNA only. With this we can confirm that our system CascAID can be efficiently used to differentiate different viral and bacterial target RNAs.

In vivo (chemical lysis)

After the successful experiments with the in vitro transcribed target RNA from E.coli, we decided to extract the 16s RNA from the E.coli culture in the lab and to perform the same RNaseAlert assays with the extracted RNA. At first, we used the chemical lysis using SDS plus heat followed by phenol chloroform purification. However, there was presence of some RNases in the extracted RNA which led to higher fluorescence activity of the RNaseAlert in presence of Cas13a. Therefore, we took the extracted RNA and then conducted a serial dilution to use them for our experimental setup. The extracted target RNA was 40 x concentrated.

Graph

As shown in the graph above, the level of fluorescence activity increases with the increasing target concentration which again verifies the activity and the sensitivity of the Cas13a. Since the RNA was extracted directly from the E. coli culture, we can say that the Cas13a can be easily manipulated to be used not only for in vitro samples but also for in vivo samples, which makes it even more suitable for practical uses.

Paperstrips

After the implementation of the working principle of the Cas13a to detect different targets in the plate reader, we decided to go further and do the same experiment by adding the samples to the glass fiber filter paper. We used overnight BSA treated glass fiber filter paper, which was then dried for 20-30 mins at 70C before pipetting the reaction mix. For this, we 3D printed a 96 well plate with 2 upper and lower parts which was suited for placing the paper in between the parts.

The first figure shown above shows that even in the paper, we can observe the increasing amount of fluorescence with the increasing target concentration. The second linear graph which is time plot showing the amount of fluorescence released, also shows that we can the Cas13a system on the paper to detect the fluorescence activity. Both graphs above thus prove that the Cas13a can be used as an efficient system that can be integrated into the paper for the simple lab on chip detection of viral and bacterial infections.

Hardware

We also designed a simple fluorescence reader with an exchangeable paper-based chip during our project, targeting the areas which do not access to advanced and space consuming machines. We basically tried it out with the Fluorescein in the beginning to see how far could we go with our detection limit. Then we worked on optimizing the device conditions and we could measure concentrations down to 100 nM. Finally, we tried our experimental setting of plate reader on to the fluorescence detector that we assembled along with the positive control containing RNase A and negative control containing RNase inhibitor only. From the given graph, it is clearly visible that we can measure the fluorescence activity of the RNaseAlert substrate over time in the presence of Cas13a using our simple fluorescence detector with a paper based chip. This also shows that our CascAID system has can be developed further for a wide scale production.

Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The positive control contains RNaseA and RNaseAlert. The negative control contains only RNaseAlert. Data points are connected with lines for the convenience of the eye. Error bars represent the measurement uncertainties of the detector.

Variety targets

We tested different targets apart from E.coli 16s RNA. Our other targets were gram positive B. subtilis, Noro virus and the Hepatitis C virus.

E. coli

In case of E.coli , we took the 16s RNA as our target RNA. We first worked with a part of the 16s RNA sequence ( 161 bp) which was then in vitro transcribed and was used for our assays. Later, we also did the extraction of the RNA from the E.coli overnight culture and performed the same experiments. The results are visible under section in vivo and proof of principle.

B. subtilis

For B. subtilis , we did not perform any in vitro transcription. We basically extracted the RNA from the B. subtilis culture and then did the RNaseAlert assay. We had a low-quality RNA after the extraction and the reason could be that since it is a gram-positive spore forming bacteria, the RNA extraction didn’t work so well. However, the results from the RNaseAlert assay are as convincing as in case of E. coli . As seen in the results on the right, although the quality of the extracted RNA is not so good, one can use the Cas13a to detect the RNA present in the extracted sample.

Noro virus