HTGAA: Hardware and Cell-free systems

Class Material

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

Judee Sharon, Nathaniel Gaut, Brock Cash, Suryateja Jammalamadaka, Sarah Vitak, Nathaniel Borders, Pat Pataranutaporn, and Berenice Estrada

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.

Reagents Intermediate stock concentration Working stock concentration
Nuclease free water
Transcription buffer 10x 1x
NTP mix 20mM 4mM
Template 50uM 0.14uM
Pyrrophosphatase 10x 1x
Homemade T7 125x 1x
RNAse inhibitor 50 1
DFHBI 100mM 0.1mM

Cell-free transcription and translation

You will set up cell-free protein expression experiment as shown below. The template (plasmid) is mCherry and its spectrum. Thaw everything on ice for 15-20mins. Set up a reaction of a total volume of 525ul. You can then aliquot 40ul of the reaction into the wells of a 96 well plate. You will use a plate reader to measure the fluorescence of mCherry for every 30mins and check if the protein is successfully expressed in the cell-free system. One thing to note, the amino acids form a precipitate, so vortex then immediately and add it to the master mix while preparing. Add the enzymes at the very end. Gently flick after you have combined all of the ingredients.
Note: The cell extract is in a series of 4 200 uL PCR tubes. Combine the volumes of cell extract and then take the required aliquot. This reaction doesn't produce fluorescent RNA aptamer but the expressed protein has fluoroscence.
Once the reaction is setup, incubate the reaction at 30C. Explain in your webpage the importance of each of the reagents you used to transcribe mRNA using the cell-free system.

Tx-Tl Master Mix Formulation (Component) Example TxTl 15ul Reaction (uL) 35 rxn's of MM (uL)
Mg-glu 1 M 0.18 6.30
K-glu 3 M 0.65 22.75
DTT 0.1 M 0.15 5.25
Energy Mix 1.5 52.50
Amino Acids 1.5 52.50
RNAse Inhinitor A (murine) 40k U/mL 0.3 10.50
T7 homemade (1.5 uM Final) e.g. @ 35 uM Stock 0.643 22.50
Water 3.58 125.30
Cell Extract 5.00 175.00
MM/reaction 13.50 N/A
Template 1.50 N/A


Design, build, and test a microfluidic device

  1. Design and print your own microfluidic chip or choose and print a microfluidic chip from Metafluidics, an open source, community-driven repository.
  2. Upload your files (.stl only) here. We will print your chip and notify you when it is ready.
  3. In the wetlab, test your microfluidic chips by playing with food coloring to 1) make a gradient; 2) mix colors; or 3) produce droplets.


Pro Challenge

Pro challenge is to perform cell free transcription and translation in a microfluidic chip.


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