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<p> <font size="3.5"> Fluorometry experiments are performed using Jasco Spectrofluorometer FP-8500 machine. The solution necessary for transcription for our system consisted of nuclease-free water from Ambion(9914G), Ribonucleoside-5´-Triphosphate Solutions purchased from Epicentre(RN02825), RNA Polymerase Transcriptional Buffer from New England Biolabs(B9012S).
<p> <font size="3.5"> Fluorometry experiments are performed using Jasco Spectrofluorometer FP-8500 machine. The solution necessary for transcription for our system consisted of nuclease-free water from Ambion(9914G), Ribonucleoside-5´-Triphosphate Solutions purchased from Epicentre(RN02825), RNA Polymerase Transcriptional Buffer from New England Biolabs(B9012S).  Standard reactions contained 7.5 mM NTP, 24 mM MgCl2, and 1X RNA Polymerase Transcriptional Buffer, with a total volume of 60 uL.  Reactions were run at 30 °C in Starna Fluorimeter Cell Sub-Micro Quartz cuvettes (Starna, 16.45F/Q)?.
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Revision as of 10:16, 23 October 2014

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EDIT

Methods


DNA Strands, Fluorescent Dyes and Enzymes

All the strands were purchased from Integrated DNA Technologies, Coralville, IA. Strand K1 was labeled with Texas Red at the 5′ end and IOWA Black RQ-Sp quencher at the 3′ end. For transcription experiments, we used the SP6 RNA Polymerase (#EP0133), which was purchased from ThermoScientific, T7 RNA Polymerase (C-T7300K) purchased from CellScript, and E. coli RNase H from Ambion (#2292). DFHBI dye, used for Spinach genelet, was purchased from Lucerna technologies (#400-1). Malachite green dye was purchased from Sigma (#32745).


Fluorometry

Fluorometry experiments are performed using Jasco Spectrofluorometer FP-8500 machine. The solution necessary for transcription for our system consisted of nuclease-free water from Ambion(9914G), Ribonucleoside-5´-Triphosphate Solutions purchased from Epicentre(RN02825), RNA Polymerase Transcriptional Buffer from New England Biolabs(B9012S). Standard reactions contained 7.5 mM NTP, 24 mM MgCl2, and 1X RNA Polymerase Transcriptional Buffer, with a total volume of 60 uL. Reactions were run at 30 °C in Starna Fluorimeter Cell Sub-Micro Quartz cuvettes (Starna, 16.45F/Q)?.


Gel Electrophoresis

Denaturing Polyacrylamide Gels

Denaturing polyacrylamide gels (10% 19:1 acrylamide:bis and 6.93 M urea in 1x TBE buffer, 0.089M Tris, 0.089M boric acid, 0.002M EDTA) were run at 22 °C for 60-90 min with 10 V/cm in 1x TBE buffer. 10x TBE buffer was purchased from Ambion (AM9863). Samples were loaded with Gel Loading Buffer II from Ambion (AM8546G). A 10-base DNA ladder (Invitrogen, Carlsbad, CA; #1082- 015) was used as a reference. For imaging and quantifying, Denaturing gels were stained with SYBR Gold (Molecular Probes, Eugene, OR; #S-11494). Gels were scanned using the ChemiDoc XRS+Imager (Biorad, Hercules, CA) and analyzed using the Image Lab software (Biorad, Hercules, CA).


Non-Denaturing Polyacrylamide Gels

Non-Denaturing polyacrylamide gels (10% 19:1 acrylamide:bis and TAE buffer, 0.04M Tris, 0.004M Acetate, 0.001M EDTA) were run at 4 °C for 60-90 min with 15 V/cm in 1x TAE/12.5mM MgCl2 buffer. 10x TAE buffer was purchased from Ambion (AM9869). Samples were__________________. A 10-base DNA ladder (Invitrogen, Carlsbad, CA; #1082- 015) was used as a reference. For imaging and quantifying, Denaturing gels were stained with SYBR Gold (Molecular Probes, Eugene, OR; #S-11494). Gels were scanned using the ChemiDoc XRS+Imager (Biorad, Hercules, CA) and analyzed using the Image Lab software (Biorad, Hercules, CA)


RNA Extraction

AmpliScribe-T7-Flash Transcription Kit, from Epicentre (ASF3257), and MEGAscript SP6 Transcription Kit, from Ambion (AM1330), were used to perform rapid transcription of genelets for RNA Extractions. The samples are incubated at 37oC for 4 hours. Denaturing polyacrylamide gels (10% 19:1 acrylamide:bis and 6.93 M urea in 1x TBE buffer, 0.089M Tris, 0.089M boric acid, 0.002M EDTA) were run at 22 °C for 60-90 min with 10 V/cm in 1x TBE buffer. 10x TBE buffer was purchased from Ambion (AM9863). Samples were loaded with Gel Loading Buffer II from Ambion (AM8546G). Shortwave ultraviolet light was used to illuminate RNA strand of interest. The RNA strands were cut and submerged in 0.3M of sodium acetate (pH 5.3). The samples are incubated at 42oC for 20 hours. The supernatant is removed and 100% -20oC ethanol, from Sigma-Aldrich (E7023-500mL), and glycogen, from ThermoScientific (R0561) ), was added to the supernatant. The supernatant is incubated at -20oC for 20 hours. The sample is spun at 135000 rpm at 4oC for 15 minutes using Z 216 MK Refrigerated Microcentrifuge from Hermle Labnet. The supernatant was removed to keep the precipitate pellet. The precipitate pellet is washed twice with 70% ethanol and spun at 135000 rpm at 4oC for 5 minutes using Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35oC for 10 minutes using the Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938).


Simulations


Topology Design

Building on previous research, which suggests the possibility of complex, regulatory circuits, simulations were run to determine the existence of conditions necessary for oscillations and bistable systems using RNA aptamers. Stability analyses were performed using MATLAB, a computing software, to find stable equilibrium conditions for the bistable system and stable, yet dynamic conditions for the oscillator. A program was designed to search for parameters representing the conditions necessary for these systems to function.


Modelling and Tuning the Oscillator

MATLAB was used to model and simulate the oscillator. The chemical reactions involved in the schematic were translated into ODEs that represented the rate of synthesis and consumption of corresponding chemical components. The computing software was programmed to solve these equations and display a graphical representation of the behaviors of these ODES. This graph was designed to display the rates of change of the ODEs analogous to the reporters of the systems. A program searched for parameters and relationships suspected to promote oscillatory behavior. Further analysis compared the effects of changing different parameters on the simulations and provided more information on ideal, yet realistic conditions for strong and stable oscillations. Mathematical analysis of the ODES using matrices and phase plots served as compelling evidence for the existence of these conditions.


Modelling and Tuning the Bistable System

In addition to MATLAB, further extensive mathematical analysis was used to linearize the multidimensional and complex system. The ODEs were manipulated and analyzed to determine the two stable equilibrium points representing a toggle between two states. The third equilibrium point represented an unstable and unrealistic condition. Euler's formula, matrices, and phase plots all provided further evidence.

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