Cell-Free Protein Synthesis

CMI Nuclera resources and user guide⬇︎

Nuclera eProtein Discovery System

The eProtein Discovery system from Nuclera is a platform for Cell-Free Protein Synthesis (CFPS), also known as in vitro transcription/translation, that enables rapid protein prototyping. The system uses a digital microfluidic chip and custom reagents to screen protein expression and solubility of up to 192 conditions, including up to 24 constructs in 8 cell-free expression conditions. 

Experiments begin by designing and assembling Nuclera-compatible DNA templates, using gene blocks and Nuclera megaprimers. Each DNA construct incorporates a split-GFP peptide (GFP11) for detecting expression level, a Strep tag II for purification, and optional solubility tags. The eProtein Discovery system dispenses user-generated DNA template, cell-free core reagent and additives for gene expression onto the digital microfluidic cartridge for transcription and translation, then detection using split-GFP (green-fluorescent protein) complementation.  Nuclera scale-up reagents allow expression and purification in the μg-mg/ml range, that are reproducibly predicted from the expression and purification screen yield estimates. Alternatively, selected constructs may be adapted for expression in bacteria or eukaryotic cells. 

Applications

  • Rapid Protein prototyping

  • Protein expression and purification screening

    Nuclera eProtein Discovery
  • Solubility tag screening

Key Features 

  • Screen and Scale-up protein production in 48 hours
  • Standard workflow: 192 conditions screened
    • 24 constructs in 8 cell-free conditions
    • 30 purification tests
  • Membrane workflow: 88 conditions screened
    • 11 constructs in 8 cell-free conditions
    • 88 purification tests
  • Reagent kits for 200 μl to 1 ml Scale-up

Nuclera Resources

CMI Nuclera eProtein Discovery Getting Started Guide - New!

 

Resources from Nuclera:

Required Supplies & Equipment

Gene Assembly (at User's Lab)

  • Nuclera-compatible gene fragment with 3C (5’ end) and TEV (3’ end)
  • Nuclera eGene Prep kit (choose one)
    • eGene Prep Kit: Solubility Tag Screen (NC3009)
    • eGene Prep Kit: FlexiVariant Screen (NC3008)
  • High fidelity PCR mastermix
    • Platinum SuperFi II PCR MasterMix by Thermo (PN 12368010) highly recommended
  • 0.2 mL thin-walled PCR tubes or 96-well PCR plate
  • PCR purification kit (column- or bead-based methods, do NOT use gel extraction)
  • DNA gel electrophoresis: 1% (w/v) agarose gel, DNA gel stain, Loading buffer, Electrophoresis running buffer, DNA ladder
  • User-supplied equipment: Thermocycler, Electrophoresis apparatus and Gel doc

eProtein Discovery Load and Run (at CMI)

  • Nuclera eProtein Discovery Cartridge (NC3006)
  • Nuclera Cartridge Reagent Kit (choose one)
    • Cartridge Reagent Kit (NC3010)
    • Cartridge Reagent Kit: Membrane Protein (NC3013)
  • 96-well Transfer plate (PCR plate or V-bottom)
  • Required equipment: 30C incubator, magnetic tube rack, centrifuge (available at CMI)

Scale-up  (at User’s Lab or CMI)

  • Scale-up kit Strep Beads (NC3011)
  • Scale-up additives (NC3005)
  • DNA construct (Nuclera eGene prep kit)
  • Required equipment: 29C incubator, magnetic tube rack, centrifuge

Experimental Design Tips

Gene Design

  • Remove initial methionine and stop codons from the sequence(s).
  • Avoid internal 3C and TEV sites.
  • Codon-optimize for E.coli.
  • For genes longer than 3Kb use “confidential sequence” feature to enter bp and protein mass.

Gene Assembly

  • Check DNA quality by gel electrophoresis (make sure you see a single PCR product band and that the positive and negative controls look as expected)
  • Purified PCR products must be eluted using eGene prep kit Elution Buffer since it contains a surfactant that is essential for downstream experimental setup
  • When measuring DNA concentration using a Nanodrop
    • blank with Elution Buffer (the surfactant in the buffer causes a high background absorbance)
    • measure at multiple dilutions for better accuracy

Cartridge load and run

  • Cartridge fluid must be degassed at 30C for at least 1 hr before run time.
  • Plate setup for cartridge loading should be done right before experimental startup.
    • Do not let a filled plate sit idle for more than an hour.
  • Take care to avoid air bubbles during loading of the cartridge.

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.