Biomolecular Breadboards:Protocols

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Cell-free circuit breadboard

The main protocols for the cell-free circuit breadboard are described here:

• Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology (JoVE, 2013)

The following older protocols are also available for the cell-free circuit breadboard:

• Reaction Preparation - Cell-free expression of circuits from linear or plasmid DNA
  • E. Coli Crude Extract Preparation, 3X
  • Amino Acid Preparation
  • 3-PGA Buffer Preparation, 14X
• TXTL template, v2.3 - TXTL spreadsheet for designing experiments

Vesicles

The cell-free circuit breadboard can be captured inside phospholipid vesicles, as described in Noireaux, V. & Libchaber, A. (2004) A vesicle bioreactor as a step toward an artificial cell. Proc. Natl. Acad. Sci. 101, 17669–17674. The protocol is availalble here.

Cost estimate

The cell-free circuit breadboard costs approximately 2.64 cents per uL, or 31.7 cents per 12 uL reaction. The cost distribution is shown in the table below.

Component	Material price per μL of reaction	Labor price per μL of reaction	Total price per μL of reaction	Total price per 12 μL reaction
E. coli crude extract	0.45 cents	0.62 cents	1.07 cents	12.84 cents
Amino acids	0.21 cents	0.03 cents	0.24 cents	2.93 cents
3-PGA	0.39 cents	0.94 cents	1.33 cents	15.96 cents
Total price	1.05 cents	1.6 cents	2.64 cents	31.7 cents

For a more detailed cost estimate, please see Breadboard Cost Estimate.

Design Cycle Time Analysis

Circuit	Assembly time
	In vitro testing (linear DNA)	In vitro testing (multiple plasmids)	In vitro testing (single plasmid)	In vivo testing (single plasmid)
Negatively autoregulated gene	6.5 hours	N/A	3 days	3 days*
Bistable switch	6.5 hours*	3 days	5 days*	5+ days*

* Estimated time based on similar constructions. All other times are measured.

For a more detailed description, please see Breadboard Cycle Time Measurements.