Difference between revisions of "Team:Munich/Protocols"

For our project we used different methods which are described in respective protocols. We also included a detailed list of all chemicals, materials and the aim of each experiment. This page is for everybody who wants to reproduce our work or wants to get an overview, but also to share our experiences with the iGEM community.

Cloning

We used different approaches to assemble genetic parts dependent on their design. Golden gate cloning was chosen for assembly of Cas13a-His-SUMO-Lwa into pSB1C3. We digested and ligated DNA parts of aeBlue to assemble this construct. For Intein-Extein we finally used a combination of different cloning methods to assembly the DNA fragments.

We amplified DNA fragments with standard PCR approaches. We used Q5, Phusion and Taq polymerases. Cells were transformed by chemical or electro transformation and outgrown at 37 °C for 1 h in SOC medium before plating on selective LB agar plates. We isolated plasmid DNA with the help of a commercial available kit and always eluted with nuclease-free water. With analytical restriction digest, colony PCR and agarose gels we checked the success of our cloning work. Final glycerol cryo stocks were always verified by sequencing.

RNA extraction

We tested different methods to lyse cells and purify RNA. We compared these methods with commercial available kits. RNA purity was checked in denaturating urea gels or agarose gels. To quantify RNA after FINA extraction we transcribed it into cDNA and then amplify using PCR and then tried to quantify the products on an agarose gel. To test our system, we first used defined transcribed RNAs which mimicked our targets. After in vitro transcription, RNA was further purified using a phenol chloroform based method.

Protein purification

We expressed our His-tagged proteins in E.coli strains and purified them using a Äkta purification system or Ni-NTA agarose. The latter had the advantage to incubate the cell lysate a certain time to get a better binding. To cleave off the His-SUMO or His-MBP tags from Cas13a proteins, we incubated them with the SUMO or TEV protease during dialysis over night, respecively. 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 in SDS PAGE gels.

Protein purification

We expressed our His-tagged proteins in E.coli strains and purified them using a Äkta purification system or Ni-NTA agarose. The latter had the advantage to incubate the cell lysate a certain time to get a better binding. To cleave off the His-SUMO or His-MBP tags from Cas13a proteins, we incubated them with the SUMO or TEV protease during dialysis over night, respecively. 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 in SDS PAGE gels.

Cas13a Lbu is 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.

Readout

For our experiments we chose Cas13a Lbu since it was reported to be the most active one. We first focused to get a running system and detect a fragment of E.coli 16S rRNA in plate reader experiments. We in vitro transcribed these target sequences, purified them using a phenol/chloroform based method and quantified resulting RNA in a denaturing SDS PAGE. As a reporter system we chose the RNase alert kit from IDT. Further testing included to detect different targets both bacteria and viruses. Next step was to detect RNA, which we isolated form actual bacterial sources. We then brought the system onto paper by lyophilization of our components.