Hardware and Cell-free systems

Week 05: Perform transcription and translation using cell-free systems. Design, 3D print, and test a microfluidic device.

Cell-free transcription You will set up a cell-free transcription reaction as shown below where the transcription mix produces fluorescent RNA aptamer called Broccoli, see this work by Filonov et al. The RNA is an aptamer that enhances the fluorescence of a small molecule ligand. Fluorescent RNA aptamers. In the transcription reaction, the Broccoli RNA should fold into a conformation which binds a ligand. The ligand--DFHBI--will fluoresce upon binding to the folded Broccoli. You will caluclate appropriate dilutions and individual volumes of reagents to be added using the table below to set up a reaction of a total volume of 500ul. You can then aliquot 40ul of the reaction into the wells of a 96 well plate. The energy and amino acid mix was prepared roughly according to the protocol based in this paper by Sun et al, while the extract was made by sonication according to protocol based on the this paper by Kwan et al. Once the 96-well plate is ready, incubate the reaction at 37C. You will use a plate reader to measure the fluorescence of aptamer for every 30mins and check if the mRNA is successfully expressed in the cell-free system. Explain in your webpage the importance of each of the reagents you used to transcribe mRNA using the cell-free system.