Biomolecular Breadboards:Preliminary Data: Difference between revisions
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=== Temperature === | === Temperature === | ||
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(B) Red blue color map presents preliminary data showing the transient response as a function of temperature. Evaporation dominates at 37 °C. Measurements were acquired over approximately 8 hour duration with indiviudal measurements every 3 minutes. | (B) Red blue color map presents preliminary data showing the transient response as a function of temperature. Evaporation dominates at 37 °C. Measurements were acquired over approximately 8 hour duration with indiviudal measurements every 3 minutes. | ||
(C) Red dots represent the dependence of temperature of parameter estimates obtained from this data. These estimates are for the constitutive promoter activity β. | (C) Red dots represent the dependence of temperature of parameter estimates obtained from this data. These estimates are for the constitutive promoter activity β. | ||
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[[Image:TXTL-negative feedback Figure9.png|600px]] | [[Image:TXTL-negative feedback Figure9.png|600px]] | ||
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(B) Red blue color map presents preliminary data showing the transient response as a function of temperature. Evaporation dominates at 37 °C. Measurements were acquired over approximately 8 hour duration with indiviudal measurements every 3 minutes. | (B) Red blue color map presents preliminary data showing the transient response as a function of temperature. Evaporation dominates at 37 °C. Measurements were acquired over approximately 8 hour duration with indiviudal measurements every 3 minutes. | ||
(C) and (D) Red dots represent temperature dependence of parameter estimates obtained from this data. These estimates are for promoter constants β and k. | (C) and (D) Red dots represent temperature dependence of parameter estimates obtained from this data. These estimates are for promoter constants β and k. | ||
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Revision as of 10:26, 11 May 2013
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
GamS
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Thiosulfate Bonds
Protein Degradation
Implementing protein degradation is integral to enabling the function of many dynamical circuits, such as oscillators or feed-forward loops. We have been exploring the overexpression of AAA ATPases ClpXP to selectively target proteins with degradation tags. Initial experiments indicate that ClpXP, when overexpressed, can accelerate degradation of purified eGFP-ssrA (Fig. 1). We will further characterize this AAA ATPase family, as well as try to demonstrate its use through an incoherent feed-forward loop. We have not been able to replicate the results by adding purified ClpXP protein. However, we intend to express ClpXP off of plasmids or to create custom extract with ClpXP already overexpressed to enable rapid protein degradation.
Environmental Context
The TX-TL system can be used to study the operation of circuits in different environmental contexts, including salts, energy levels and temperature.
Magnesium and Potassium concentration
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Temperature
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