Biomolecular Breadboards:Protocols

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=== Cell-free circuit breadboard ===
=== Cell-free circuit breadboard ===
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The following protocols are available for the cell-free circuit breadboard:
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The main protocols for the cell-free circuit breadboard are described here:
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* [http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology] (JoVE, 2013)
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The following older protocols are also available for the cell-free circuit breadboard:
* [[Biomolecular Breadboards:Protocols:Reaction Preparation|Reaction Preparation]] - Cell-free expression of circuits from linear or plasmid DNA
* [[Biomolecular Breadboards:Protocols:Reaction Preparation|Reaction Preparation]] - Cell-free expression of circuits from linear or plasmid DNA
** [[Biomolecular Breadboards:Protocols:Crude Extract Prep|''E. Coli'' Crude Extract Preparation, 3X]]  
** [[Biomolecular Breadboards:Protocols:Crude Extract Prep|''E. Coli'' Crude Extract Preparation, 3X]]  
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** [[Biomolecular Breadboards:Protocols:3-PGA Buffer Prep|3-PGA Buffer Preparation, 14X]]
** [[Biomolecular Breadboards:Protocols:3-PGA Buffer Prep|3-PGA Buffer Preparation, 14X]]
* [[Media: TXTL_v2_3.xlsx | TXTL template, v2.3]] - TXTL spreadsheet for designing experiments
* [[Media: TXTL_v2_3.xlsx | TXTL template, v2.3]] - TXTL spreadsheet for designing experiments
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We are the in the process of publishing a paper in the ''Journal of Visualized Experiments'' ([http://www.jove.com JoVE]) that describes this protocol in much more detail.  The JoVE paper will be an open access paper.  If you would like a preprint, contact [[Biomolecular Breadboards:More Info|Richard Murray]].
 
===Vesicles===
===Vesicles===
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The cell-free circuit breadboard can be captured inside phospholipid vesicles, as described in [http://www.pnas.org/content/101/51/17669.full 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.
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The cell-free circuit breadboard can be captured inside phospholipid vesicles, as described in [http://www.pnas.org/content/101/51/17669.full 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 [http://openwetware.org/wiki/Biomolecular_Breadboards:Protocols:Vesicles here.]
=== Cost estimate ===
=== Cost estimate ===

Current revision

Home Protocols DNA parts Preliminary Data Models More Info

Contents

Cell-free circuit breadboard

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

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

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.

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