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.
Buffer regions of non-coding DNA protect coding regions from exonuclease degradation.
One main 3’ exonuclease present is the RecBCD complex, which can be inhibited by gamS protein produced by lambda phage.