#  Cell-Free Protein Synthesis 

 



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[**CMI Nuclera resources and user guide**](/node/1876791#nuclera-resources)**⬇︎**

## Nuclera eProtein Discovery System

The eProtein Discovery system from [Nuclera](https://www.nuclera.com/) 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](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/2026-01/nuclera_crop.png?itok=ZHmjX6rI)
- 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



 



###    Standard Workflow  expand\_more  

 ![Nuclera cell-free protein synthesis ](/sites/g/files/omnuum4016/files/2025-07/Cell-free%20Protein%20Synthesis%20-%2016x9%20Standard%20Workflow.png)

 

 

 



###    Alternate Workflow - for transmembrane or difficult proteins  expand\_more  

 ![Nuclera cell-free protein synthesis - membrane workflow](/sites/g/files/omnuum4016/files/2025-07/Cell-free%20Protein%20Synthesis%20-%2016x9%20Membrane%20Workflow.png)

 

 

 



###    DNA Template Design  expand\_more  

 ![DNA template design](/sites/g/files/omnuum4016/files/2025-06/Nuclera%20DNA%20template%2016x9.png)

 

 

 



###    eProtein Discovery Video Playback  expand\_more  

Monitor data collection in real-time or review video after.

 

##  CMI Nuclera - Training Set 

 





 

 

 



 

 

 

 

 



 

 

 

 

 Nuclera Resources Required Supplies & Equipment Experimental Design Tips 

## Nuclera Resources

[CMI Nuclera eProtein Discovery Getting Started Guide](/sites/g/files/omnuum4016/files/2025-07/CMI%20Nuclera%20eProtein%20Discovery%20Getting%20Started%20Guide_4.pdf "CMI Nuclera eProtein Discovery Getting Started Guide") - New!

Resources from [Nuclera](https://www.nuclera.com/):

- Nuclera Quick Start Instructions:
    - [eGene Prep Kit Quick Start](https://info.nuclera.com/rs/208-WJR-240/images/Nuclera-Flyer-eGene_Prep_Kit_Quick_Start.pdf) - eGene Assembly simplified instructions
    - [Soluble Protein Workflow Quick Start](https://info.nuclera.com/guide-soluble-proteins-workflow-transfer-plate-and-beads-preparation-quick-start) - transfer plate preparation for standard workflow (192 conditions, 30 purifications)
    - [Membrane Protein Workflow Quick Start](https://info.nuclera.com/guide-membrane-proteins-workflow-transfer-plate-and-beads-preparation-quick-start) - transfer plate preparation for membrane protein workflow (88 conditions, 88 purifications)
- [Nuclera Knowledge Base](https://knowledge.nuclera.com/)
- [Nuclera - eGene Prep Kit User Guide](https://info.nuclera.com/rs/208-WJR-240/images/Nuclera-Guide-STS_eGene_Prep_Kit_NC3009_User_Guide.pdf) - eGene Assembly User Guide (full instructions)
- [Nuclera - Scale-up Expression and Purification Guide](https://knowledge.nuclera.com/docs/scale-up-expression-and-purification/) - for scale-up
- [Nuclera - Custom Additives - chemical compatibility](https://info.nuclera.com/manual-custom-additives-chemical-compatibility-list) - guidelines for working with custom additives
- [Nuclera - Circular eGene assembly guidelines](https://urldefense.proofpoint.com/v2/url?u=https-3A__info.nuclera.com_manual-2Degenes-2Dto-2Dcircular-2Degenes-2Dassembly-2Dguidelines.html&d=DwMFaQ&c=WO-RGvefibhHBZq3fL85hQ&r=Nb1w1TbDaWUGG3MwCc3q6BGgXDXIFk_zHi_eprmxr1U&m=qIsDqMtPPpBmefUseCra17nWLMa69qHlNuxn_bCXncV-dfxSOdvcWRvVpjGU4MJv&s=lTqqWYLEgxev9LWqs5znRjbtpBc2PLqcoCicN7hJM2I&e=) - guidelines to convert Linear eGene constructs into plasmids

 

 

 

## 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.