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.
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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.
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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)
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pTet-LacI (pBEST-pLTet01-UTR1-LacI-T500)
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| pLac-deCFP (pBEST-pLLac01-UTR1-deCFP-T500)
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pLac-TetR (pBEST-pLLac01-UTR1-TetR-T500)
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Negatively autoregulated gene
pTet-TetR-deGFP (fusion) (pBEST-pLTet01-UTR1-TetR-linker-deGFP-T500)
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