Biomolecular Breadboards:Preliminary Data
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Plasmid Expression of GFP
Using pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500, a plasmid enhanced for GFP expression, the biomolecular breadboard is able to express mass at equal concentration to comparable bacteriophage in-vitro systems (J. Shin and V. Noireaux, 2010).
Expression of plasmids can be optimized by concentration.
Protecting Linear DNA from Exonuclease-Mediated Degradation
Current standards for circuit design utilize plasmids for DNA template, which require time-consuming subcloning steps. However, circuits based on linear DNA require only PCR assembly or gene synthesis, which drastically decreases preparation time. As a purely extract-derived system, our biomolecular breadboard exhibits exonuclease activity which degrades linear DNA. We are developing multiple technologies to protect linear DNA from exonuclease degradation.
Figure 1: Exonuclease protection using non-coding DNA. Linear DNA templates, 2nM, are derived from pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 with varying amounts of noncoding DNA surrounding the coding sequence.
One main 3’ exonuclease present is the RecBCD complex, which can be inhibited by gamS protein produced by lambda phage.