Biomolecular Breadboards:DNA parts

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This page contains a description of the DNA parts used for the biomolecular breadboards. These parts will be submitted to Addgene (eventually).

Control Plasmid: pBEST-OR2-OR1-Pr-UTR1-deGFP-T500

This section contains a description of the deGFP control plasmid for the cell-free expression breadboard.

Control sequence: pBEST_OR2_OR1_Pr_UTR-deGFP-T500.gb (GenBank file)

Plasmid description

P70	Lambda phage promoter OR2-OR1-Pr specific to E. coli σ70	GenBank: J02459.1
UTR1	The untranslated region containing the T7 g10 leader sequence for highly efficient translation initiation	GenBank: M35614.1
deGFP	The enhanced green fluorescent protein truncated and modified in N- and C- terminal	Shin, J., and Noireaux, V. (2010) Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70, J Biol Eng 4, 8.
T500	Transcription terminator for E. coli RNA polymerase	Larson, M. H., Greenleaf, W. J., Landick, R., and Block, S. M. (2008) Applied force reveals mechanistic and energetic details of transcription termination, Cell 132, 971-982.

Preliminary data

Using pBEST-OR2-OR1-Pr-UTR1-deGFP-T500, a plasmid enhanced for GFP expression, the biomolecular breadboard is able to express mass at equal concentrations to comparable bacteriophage in-vitro systems (J. Shin and V. Noireaux, 2010).

Expression of plasmids can be optimized by concentration.

Raw data file: plasmid_deGFP-070812.xls (Excel spreadsheet) Information about protocol on "Outline" sheet; plotted data is on the sheet "plotted-data" Control plasmid file: pBEST_OR2_OR1_Pr_UTR-deGFP-T500.gb (GenBank format)

Figure 1. eGFP expression as a function of plasmid DNA template. Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is varied by concentration. File:PTet-TetR-deGFP (fusion).png

Bistable toggle switch: 4 plasmid circuit

pTet-deGFP (pBEST-pLTet01-UTR1-deGFP-T500)

pTet-LacI (pBEST-pLTet01-UTR1-LacI-T500)

pLac-deCFP (pBEST-pLLac01-UTR1-deCFP-T500)

pLac-TetR (pBEST-pLLac01-UTR1-TetR-T500)

Negatively Autoregulated Gene

pTet-TetR-deGFP (fusion) (pBEST-pLTet01-UTR1-TetR-linker-deGFP-T500)