Biomolecular Breadboards:Preliminary Data: Difference between revisions
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==Plasmid Expression of GFP== | |||
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). | 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). | ||
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'''Figure 1. eGFP expression as a function of plasmid DNA template.''' Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-T500 is varied by concentration. | '''Figure 1. eGFP expression as a function of plasmid DNA template.''' Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-T500 is varied 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. These include: | 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. These include: | ||
# Protecting linear DNA using noncoding segments | # Protecting linear DNA using noncoding segments | ||
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# Adding thiosulfate bonds to 5' ends | # Adding thiosulfate bonds to 5' ends | ||
===Protection Sequences=== | |||
[[Image:linear.png|400px]] | [[Image:linear.png|400px]] | ||
<br>'''Figure 2: Exonuclease protection using non-coding DNA.''' Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 with varying amounts of noncoding DNA surrounding the coding sequence. | <br>'''Figure 2: Exonuclease protection using non-coding DNA.''' Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 with varying amounts of noncoding DNA surrounding the coding sequence. | ||
===GamS=== | |||
[[Image:gamSComp.png|400px]] | [[Image:gamSComp.png|400px]] | ||
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<br>'''Figure 4. eGFP expression as a function of linear DNA template, with gamS.''' Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 is varied by concentration. GamS supplied at 3uM concentration. | <br>'''Figure 4. eGFP expression as a function of linear DNA template, with gamS.''' Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 is varied by concentration. GamS supplied at 3uM concentration. | ||
===Thiosulfate Bonds=== | |||
[[Image:ts-1.png|400px]] | [[Image:ts-1.png|400px]] | ||
<br>'''Figure 5. Thiosulfate bonds protect against 5' exonuclease degradation.''' Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500. 5 thiosulfate bonds are present at each 5' end. GamS supplied at 3uM concentration. | <br>'''Figure 5. Thiosulfate bonds protect against 5' exonuclease degradation.''' Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500. 5 thiosulfate bonds are present at each 5' end. GamS supplied at 3uM concentration. | ||
== Protein Degradation == |
Revision as of 17:59, 18 November 2012
Home | Protocols | DNA parts | Preliminary Data | Models | More Info |
This page contains some data that we have taken with the TX-TL breadboard.
Plasmid Expression of GFP
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.
|
Figure 1. eGFP expression as a function of plasmid DNA template. Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-T500 is varied 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. These include:
- Protecting linear DNA using noncoding segments
- Inhibiting RecBCD exonuclease with gamS
- Adding thiosulfate bonds to 5' ends
Protection Sequences
Figure 2: Exonuclease protection using non-coding DNA. Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 with varying amounts of noncoding DNA surrounding the coding sequence.
GamS
Figure 3. (left) GamS from lambda phage, an inhibitor of RecBCD complex, inhibits template DNA degradation. Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500. GamS supplied at 3uM concentration. (right) Simulation results based on a simple ODE model.
Figure 4. eGFP expression as a function of linear DNA template, with gamS. Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 is varied by concentration. GamS supplied at 3uM concentration.
Thiosulfate Bonds
Figure 5. Thiosulfate bonds protect against 5' exonuclease degradation. Linear DNA templates, 2nM, are derived from plasmid DNA pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500. 5 thiosulfate bonds are present at each 5' end. GamS supplied at 3uM concentration.