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.