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  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA'><span>Home</span></a></li>
  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Motivation_&_Objectives'><span>Motivation & Objectives</span></a></li>
  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Results'><span>Results</span></a></li>
  <li class='active'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Methods'><span>Methods</span></a></li>
  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Supplement'><span>Supplement</span></a></li>
  <li class='last'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Team'><span>Team</span></a></li>

</ul> </div> </body> </html>

Methods

DNA strands, enzymes, dyes and buffers

Modeling and Simulations

DNA strands, enzymes, dyes and buffers

All the strands were purchased from Integrated DNA Technologies, Coralville, IA. Strand D1 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, E. coli RNase H from Ambion (#2292) and Pyrophosphatase from New England Biolabs (#M0296L). DFHBI dye, used for Spinach genelet, was purchased from Lucerna technologies (#400-1). Malachite green dye was purchased from Sigma (#32745). Transcription buffer was purchased from Epicentre Biotechnologies (#E7525), and transcription buffer was purchased from New England Biolabs (#E8590.)

Transcription

The genes were first annealed with 10% (v/v) 10× transcription buffer from 90°C to 37°C for 1 h 30 min at a target concentration of 10 μM. Then genes are mixed to a transcription mix composed of 10% (v/v), 10× transcription buffer, 7.5 mM each NTP, 24 mM MgCl₂ 4% (v/v) T7 RNA polymerase, 4-6% (v/v) SP6 RNA Polymerase, 1.2%(v/v) Pyrophosphatase (Ppase). When needed, we used 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl, unless noted otherwise.

Fluorometry

General Fluorometry Protocol

Fluorometry experiments are performed using a Jasco Spectrofluorometer FP-8500. Reactions were run at 30 °C in 50 μL chamber cuvettes manufactured by Starna (model 16.50F-Q-10/Z15). Samples were covered with 40 μL hexadecane oil to prevent evaporation.

Fluorometry Protocol for Specific Experiments

A. Transcription of Spinach gene by SP6 RNA polymerase

The following transcription solution was prepared with the following components, along with DHFBI Dye as the fluorescent reporter:

Component	Concentration
DHFBI Dye	5 μM
Spinach Gene	300 nM
MgCl₂	0.024 M
NTPs	7.5 mM

Solution also contained nuclease-free water and RNA polymerase reaction buffer. 2 μL of SP6 RNA polymerase was added to initiate transcription. Reaction took place at 30°C, with a total reaction volume of 60 μL.

B. Enzyme Inhibition with RNA Aptamers

For experiments testing T7 RNA Polymerase inhibition, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:

Component	Concentration
Malachite Green Dye	25 μM
Malachite Green Gene	50 nM
Gene G2	150 nM
MgCl₂	0.024 M
NTPs	7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 μL of the T7 RNAP enzyme and 3 μL of SP6 RNAP enzyme was added. The transcription reactions took place at 30°C.

For experiments testing SP6 RNA Polymerase inhibition, DHFBI was employed as the reporter dye. The transcriptional solution contained the following components:

Component	Concentration
DHFBI Dye	25 μM
Spinach Gene	50 nM
Gene G1	150 nM
MgCl₂	0.024 M
NTPs	7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 μL of the T7 RNAP enzyme and 3 μL of SP6 RNAP enzyme was added. The transcription reactions took place at 30°C.

C. Bound Aptamer-Kleptamer Interactions

For experiments testing T7 RNA Polymerase reactivation, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:

Component	Concentration
Malachite Green Dye	16 μM
Malachite Green Gene	100 nM
MgCl₂	0.024 M
NTPs	7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 1.3 μL of the T7 RNAP enzyme was added to solution along with 0.7 μL of Ppase to prevent inhibition due to diphosphate waste. The transcription reactions took place at 30°C. For the experimental cuvette, 800 nM of R3 was added as well as 1.2 μM of R2. The total volume used in each cuvette was 60 μL SP6 RNA Polymerase

D. Re-activation of inhibited SP6 RNA polymerase using a kleptamer

For experiments testing SP6 RNA Polymerase reactivation, DHFBI was the reporter dye. The transcriptional solution contained the following components:

Component	Concentration
DHFBI Dye	25 μM
Spinach Gene	100 nM
MgCl₂	0.024 M
NTPs	7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 μL of the SP6 RNAP enzyme was added to solution. The transcription reactions took place at 30°C. For the experimental cuvette, 1.77μM of R1 was added as well as 2.67μM D1. The total volume used in the cuvette was 67.421 μL.

E. Molecular Beacons as Reporters

For the experiments on the molecular beacon D1 (which is the main reporter in our systems), the transcriptional solution contained the following components:

Component	Concentration
D1	100nM
R1 aptamer	200 nM
MgCl₂	0.014 M
NTPs	5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 1 μL of the T7 RNAP enzyme was added to solution along with 0.7 μL of Ppase to prevent inhibition due to diphosphate waste and 2μL of RNase H to degrade the RNA bound to D1. The transcription reactions took place at 30°C. The total volume used in each cuvette was 60 μL.

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). 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 37^oC 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 42^oC for 20 hours. The supernatant is removed and 100% -20^oC ethanol, from Sigma-Aldrich (E7023-500mL), and glycogen, from ThermoScientific (R0561), was added to the supernatant. The supernatant is incubated at -20^oC for 20 hours. The sample is spun at 135000 rpm at 4^oC for 15 minutes using a 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 4^oC for 5 minutes using a Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35^oC for 10 minutes using a Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938).

Oscillator Reactions

Oscillator Experiment 1

The experiment shown in Fig. 13 in the results section was done as follows. Fluorometry was performed as described in 'General Fluorometry Protocols' section above. The reaction temperature was, 30°C. The reaction conditions are listed below:

Oscillator Experiment 2: RNaseH titration

The experiment shown in Fig. 14 in the results section was done as follows. Fluorometry was performed as described in 'General Fluorometry Protocols' section above. The reaction temperature was, 30°C. The reaction conditions are listed below:

Modeling and Simulations

To build our models, we first wrote all the reactions designed to occur in each system. Then, we used the law of mass action to derive ordinary differential equations (ODEs). The complete set of reactions and the ODEs are shown in the Supplement. ODE models were numerically integrated using MATLAB's ode23s solver. The nucleic acid binding rates and enzyme transcription and degradation rates were chosen to be in a physically plausible range based on the literature [1, 2]. All the simulation parameters are reported in Tables 1 and 2 in the Supplement.

Because both models are nonlinear, to gain insights into their behavior we performed a local linear analysis. Local linear analysis means that we approximated each nonlinear model with its linearized version around each equilibrium point. In practice, if our initial system is written as [math]\displaystyle{ \dot x = f(x) }[/math], and its equilibrium is [math]\displaystyle{ \bar x }[/math] such that [math]\displaystyle{ f(\bar x)=0 }[/math], then nearby point [math]\displaystyle{ \bar x }[/math] we can approximate the system as:

[math]\displaystyle{ \dot \xi = \frac{\partial f}{\partial x}(\bar x) \xi, }[/math]

where [math]\displaystyle{ \xi = x-\bar x }[/math]. The partial derivative [math]\displaystyle{ \frac{\partial f}{\partial x}(\bar x) }[/math] is evaluated at the equilibrium [math]\displaystyle{ \bar x }[/math], so in our case it is a constant matrix, known as the Jacobian of the system. The formula above is a direct application of Taylor series expansion stopped at the first order. First, for both models we found closed form expressions for their equilibria, by setting the ODEs to zero. These expressions, which are shown in the Supplement, are complex and had to be solved numerically to find the actual value of the equilibria. This was done again writing MATLAB scripts. Then, we found expressions for the Jacobian matrices [math]\displaystyle{ J= \frac{\partial f}{\partial x}(\bar x) }[/math] at each equilibrium point. To derive the plots shown in Figures 2 and 4 of the Results section, we wrote a script to find the eigenvalues of the Jacobian. Based on the nature of the eigenvalues, we classified the equilibrium as stable (eigenvalues have negative real part) or unstable (eigenvalues have positive real part). If there is a pair of complex conjugate eigenvalues with positive real part, then the point presents local oscillations. We also manipulated the models by re-ordering their variables, so that their Jacobians would clearly reveal the overall feedback interconnection of the systems. The matrices are shown in the Supplement.

References

All Medline abstracts: PubMed | HubMed

  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA'><span>Home</span></a></li>
  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Motivation_&_Objectives'><span>Motivation & Objectives</span></a></li>
  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Results'><span>Results</span></a></li>
  <li class='active'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Methods'><span>Methods</span></a></li>
  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Supplement'><span>Supplement</span></a></li>
  <li class='last'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Team'><span>Team</span></a></li>

</ul> </div> </body> </html>

Biomod/2014/UCR/Breaking RNA/Methods: Difference between revisions

Latest revision as of 23:47, 25 October 2014

Contents

Methods

DNA strands, enzymes, dyes and buffers

Transcription

Fluorometry

General Fluorometry Protocol

Gel Electrophoresis

Denaturing Polyacrylamide Gels

Non-Denaturing Polyacrylamide Gels

RNA Extraction

Oscillator Reactions

Oscillator Experiment 1

Oscillator Experiment 2: RNaseH titration

Modeling and Simulations

References

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@@ Line 1: / Line 1: @@
-----
 {{UCR/Breaking-RNA/HEAD}}
+<html>
-[http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Methods&action=edit EDIT]
+<body>
+<div id='cssmenu'>
+<ul>
+   <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA'><span>Home</span></a></li>
+   <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Motivation_&_Objectives'><span>Motivation & Objectives</span></a></li>
+   <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Results'><span>Results</span></a></li>
+   <li class='active'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Methods'><span>Methods</span></a></li>
+   <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Supplement'><span>Supplement</span></a></li>
+   <li class='last'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Team'><span>Team</span></a></li>
+</ul>
+</div>
+</body>
+</html>
 <font size="5">
 <h1> Methods </h1>
 </font>
-<div id='submenu'>
+<table id='floatmenu'>
-     <ul>
+     <tr>
-      <li>[[#DNA Strands and Enzymes|DNA Strands, Fluorescent Dyes and Enzymes]]</li>
+        <td id='edge'>[[#DNA strands, enzymes, dyes and buffers|DNA strands, enzymes, dyes and buffers]]</td>
-      <li>[[#Fluorometry|Fluorometry]]</li>
+        <td id='button'>[[#Transcription|Transcription]]</td>
-      <li>[[#Gel Electrophoresis|Gel Electrophoresis]]</li>
+        <td id='button'>[[#Fluorometry|Fluorometry]]</td>
-      <li>[[#RNA Extraction|RNA Extraction]]</li>
+        <td id='button'>[[#Gel Electrophoresis|Gel Electrophoresis]]</td>
-      <li>[[#Simulations|Simulations]]</li>
+        <td id='button'>[[#RNA Extraction|RNA Extraction]]</td>
-    </ul>
+        <td id='button'>[[#Oscillator Reactions|Oscillator Reactions]]</td>
-</div>
+        <td id='button'>[[#Modeling and Simulations|Modeling and Simulations]]</td>
-<!--      <li>[[#Aptamer and Complement Design|Aptamer and Complement Design]]</li>-->
+    </tr>
+ </table>
 <br />
 <font size="4.5">
-<h1>DNA Strands, Fluorescent Dyes and Enzymes</h1>
+<h1>DNA strands, enzymes, dyes and buffers</h1>
 </font>
-<p> <font size="3.5">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).
+<p> <font size="3.5">All the strands were purchased from Integrated DNA Technologies, Coralville, IA. Strand D1 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, E. coli RNase H from Ambion (#2292) and Pyrophosphatase from New England Biolabs (#M0296L). DFHBI  dye, used for Spinach genelet, was purchased from Lucerna technologies (#400-1). Malachite green dye was purchased from Sigma (#32745). Transcription buffer was purchased from Epicentre Biotechnologies (#E7525), and transcription buffer was purchased from New England Biolabs (#E8590.)
 </font></p>
+<br />
+<font size="4.5">
+<h1>Transcription</h1>
+</font>
+<br>
+<p>The genes were first annealed with 10% (v/v) 10× transcription buffer from 90&deg;C to 37&deg;C for 1 h 30 min at a target concentration of 10 μM. Then genes are mixed to a transcription mix composed of 10% (v/v), 10× transcription buffer, 7.5 mM each NTP, 24 mM MgCl<sub>2</sub> 4% (v/v) T7 RNA polymerase, 4-6% (v/v) SP6 RNA Polymerase, 1.2%(v/v) Pyrophosphatase (Ppase). When needed, we used 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl, unless noted otherwise. </p>
 <br />
@@ Line 27: / Line 46: @@
 <h1>Fluorometry</h1>
 </font>
 <h3><u><font size="4.5">General Fluorometry Protocol</font></u></h3>
-<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 50 microliter-chamber Fluorimeter Cell cuvettes manufactured by Starna (model 16.50F-Q-10/Z15).</font></p>
+<p><font size="3.5"> Fluorometry experiments are performed using a Jasco Spectrofluorometer FP-8500. Reactions were run at 30 °C in 50 μL chamber cuvettes manufactured by Starna (model 16.50F-Q-10/Z15). Samples were covered with 40 &mu;L hexadecane oil to prevent evaporation.  </font></p>
-<p><u><font size="3.5">Specific Fluorometry Protocol</font></u></p>
+<p><u><font size="3.5">Fluorometry Protocol for Specific Experiments</font></u></p>
-<p><font size="3.5">A. Bound Aptamer-Kleptamer Interactions</font></p>
+<p><font size="3.5">A. Transcription of Spinach gene by SP6 RNA polymerase</font></p>
+The following transcription solution was prepared with the following components, along with DHFBI Dye as the fluorescent reporter:
-For the T7 RNA Polymerase reactivation, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:
 ::{| border = "1" class="wikitable" style="margin:auto;"
 ! scope="col" | Component
 ! scope="col" | Concentration
 |- align="center"
-| Malachite Green Dye|| 16 &mu;M
+| DHFBI Dye|| 5 &mu;M
 |- align ="center"
-| Malachite Green Gene|| 100 nM
+| Spinach Gene|| 300 nM
 |- align ="center"
 | MgCl<sub>2</sub>|| 0.024 M
@@ Line 50: / Line 67: @@
 |}
-The solution also contains RNA Polymerase transcription buffer and RNase free water. 1.3 &mu;L of the T7 RNAP enzyme was added to solution along with 0.7 &mu;L of phase to prevent inhibition due to diphosphate waste. The transcription reactions took place at 30&deg;C. For the experimental cuvette, 800 nM of R3 was added as well as 1.2 &mu;M of R2. The total volume used in each cuvette was 60 &mu;L SP6 RNA Polymerase
+Solution also contained nuclease-free water and RNA polymerase reaction buffer.  2 &mu;L of SP6 RNA polymerase was added to initiate transcription. Reaction took place at 30&deg;C, with a total reaction volume of 60 &mu;L.
-<br />
-<font size="4.5">
-<h1>Transcription Protocol</h1>
-</font>
-<br>
-<p>The DNA strands were annealed with 10% (v/v) 10× transcription buffer from 90◦C to 37◦C for 1 h 30 min at a target concentration of 10 μM. The genes are in a solution with 10% (v/v), 10× transcription buffer, 7.5 mM each NTP, 24 mM MgCl<sub>2</sub> 4% (v/v) T7 RNA polymerase, 4-6% (v/v) SP6 RNA Polymerase, 1.2%(v/v) Ppase, and 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl. </p>
-<br>
-<br />
-<font size="4.5">
-<h1>Gel Electrophoresis</h1>
-</font>
-<h3>Denaturing Polyacrylamide Gels</h3>
+<p><font size="3.5">B. Enzyme Inhibition with RNA Aptamers  </font></p>
-<p> <font size="3.5">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).</font></p>
-<br />
+For experiments testing T7 RNA Polymerase inhibition, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:
-<h3>Non-Denaturing Polyacrylamide Gels</h3>
-<p> <font size="3.5">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 MgCl<sub>2</sub> buffer. 10x TAE buffer was purchased from Ambion (AM9869).  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)</font></p>
-<p><u><font size="3.5">Specific Gel Protocols</font></u></p>
-<p><font size="3.5">A. Bound Aptamer-Kleptamer Interactions</font></p>
-For the first gel, the concentration of R1 and K1 used are 1 &mu;M and 2 &mu;M and the T7 RNA Polymerase volume used was 1 &mu;L. The total volume in each well was 7 &mu;L. R1 and SP6 RNAP were mixed together and incubated for 20 minutes at 30°C. Next, K1 was added to the mixture and the solution was incubated for another 10 minutes at 30°C. A list of the components in each well is given below:
 ::{| border = "1" class="wikitable" style="margin:auto;"
-! scope="col" | Lane
+! scope="col" | Component
-! scope="col" | Components
+! scope="col" | Concentration
 |- align="center"
-| 1|| 10 base pair ladder
+| Malachite Green Dye|| 25 &mu;M
 |- align ="center"
-| 2|| R1 aptamer
+| Malachite Green Gene|| 50 nM
 |- align ="center"
-| 3|| K1 Strand (23 base pair)
+| Gene G2|| 150 nM
 |- align ="center"
-|4|| K1 Strand (38 base pair)
+| MgCl<sub>2</sub>|| 0.024 M
-|-align ="center"
+|- align ="center"
-|5|| R1 aptamer + SP6 RNA Polymerase
+|NTPs|| 7.5 mM
-|-align ="center"
-|6|| R1 aptamer +SP6 RNA Polymerase +K1 (23 base pair)
-|-align ="center"
-|7|| R1 aptamer +SP6 RNA Polymerase +K1 (38 base pair)
 |}
-For the second gel, the concentrations of R1 and R4 used were both 0.7 &mu;L and the SP6 RNA Polymerase volume used was 1 &mu;L. The total volume used in each well was 7 &mu;L. R1 and SP6 RNA Polymerase were mixed together and incubated for 15 minutes at 30&deg;C. Next, R4 was added to the mixture and the solution was incubated for another 15 minutes. The composition of each lane is given below:
+The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 &mu;L of the T7 RNAP enzyme and 3 &mu;L of SP6 RNAP enzyme was added. The transcription reactions took place at 30&deg;C.
+For experiments testing SP6 RNA Polymerase inhibition, DHFBI was employed as the reporter dye. The transcriptional solution contained the following components:
 ::{| border = "1" class="wikitable" style="margin:auto;"
-! scope="col" | Lane
+! scope="col" | Component
-! scope="col" | Components
+! scope="col" | Concentration
 |- align="center"
-| 1|| 10 base pair ladder
+| DHFBI Dye|| 25 &mu;M
 |- align ="center"
-| 2|| R1 aptamer
+| Spinach Gene|| 50 nM
 |- align ="center"
-| 3|| R4 Strand
+| Gene G1|| 150 nM
+|- align ="center"
+| MgCl<sub>2</sub>|| 0.024 M
 |- align ="center"
-|4|| R1 aptamer + SP6 RNA Polymerase
+|NTPs|| 7.5 mM
-|-align ="center"
-|5|| R1 aptamer + SP6 RNA Polymerase + R4 aptamer
 |}
+The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 &mu;L of the T7 RNAP enzyme and 3 &mu;L of SP6 RNAP enzyme was added. The transcription reactions took place at 30&deg;C.
-<p><font size="3.5">B. Unbound Aptamer-Kleptamer Interactions</font></p>
+<p><font size="3.5">C. Bound Aptamer-Kleptamer Interactions</font></p>
-For this gel, the concentrations used for R1 aptamer and K1 strands are all 1 &mu;M. The R1 and K1 aptamers were mixed into solution together at the same concentration and incubated at 30°C for 10 minutes. The composition of each lane is given below:
+For experiments testing  T7 RNA Polymerase reactivation, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:
 ::{| border = "1" class="wikitable" style="margin:auto;"
-! scope="col" | Lane
+! scope="col" | Component
-! scope="col" | Components
+! scope="col" | Concentration
 |- align="center"
-| 1|| 10 base pair ladder
+| Malachite Green Dye|| 16 &mu;M
 |- align ="center"
-| 2|| R1 aptamer
+| Malachite Green Gene|| 100 nM
 |- align ="center"
-| 3|| K1 strand (38 base pair)
+| MgCl<sub>2</sub>|| 0.024 M
 |- align ="center"
-|4|| K1 strand (23 base pair)
+|NTPs|| 7.5 mM
-|-align ="center"
-|5|| R1 aptamer + K1 strand (38 base pair)
-|-align ="center"
-|6|| R1 aptamer + K1 strand (23 base pair)
 |}
-<h3><u><font size="4.5">2.5 Bistable Mechanisms</font></u></h3>
-<h3><u><font size="4.5">2.6 Oscillatory Mechanisms</font></u></h3>
+The solution also contains RNA Polymerase transcription buffer and RNase free water. 1.3 &mu;L of the T7 RNAP enzyme was added to solution along with 0.7 &mu;L of Ppase to prevent inhibition due to diphosphate waste. The transcription reactions took place at 30&deg;C. For the experimental cuvette, 800 nM of R3 was added as well as 1.2 &mu;M of R2. The total volume used in each cuvette was 60 &mu;L SP6 RNA Polymerase
-For the inhibition of T7 RNA polymerase with gene G3, 2 &mu;L of T7 ran Polymerase was used in this experiment. The transcriptional solution has Malachite Green as the reporter and the solution composition is listed below:
+<br>
+<p><font size="3.5">D. Re-activation of inhibited SP6 RNA polymerase using a kleptamer</font></p>
+For experiments testing  SP6 RNA Polymerase reactivation, DHFBI was the reporter dye. The transcriptional solution contained the following components:
 ::{| border = "1" class="wikitable" style="margin:auto;"
 ! scope="col" | Component
 ! scope="col" | Concentration
 |- align="center"
-| Malachite Green Dye|| 16 &mu;M
+| DHFBI Dye|| 25 &mu;M
 |- align ="center"
-| Malachite Green Gene|| 250 nM
+| Spinach Gene|| 100 nM
 |- align ="center"
 | MgCl<sub>2</sub>|| 0.024 M
@@ Line 147: / Line 140: @@
 |NTPs|| 7.5 mM
 |}
-The solution also contains RNA Polymerase transcription buffer and RN are free water. The gene G3 was added at 500 nM. Experiment was incubated at 30&deg;C.
+The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 &mu;L of the SP6 RNAP enzyme was added to solution. The transcription reactions took place at 30&deg;C. For the experimental cuvette, 1.77μM of R1 was added as well as 2.67μM D1. The total volume used in the cuvette was 67.421 &mu;L.
+<br>
+<p><font size="3.5">E. Molecular Beacons as Reporters</font></p>
-For the reactivation of T7 RNA Polymerase attempt with G3 and G2, 2 &mu;L of both, T7 and SP6 RNA Polymerase were used. The transcriptional solution has Malachite Green as the reporter and the solution composition is listed below:
+For the  experiments on the molecular beacon D1 (which is the main reporter in our systems), the transcriptional solution contained the following components:
 ::{| border = "1" class="wikitable" style="margin:auto;"
 ! scope="col" | Component
 ! scope="col" | Concentration
 |- align="center"
-| Malachite Green Dye|| 16 &mu;M
+| D1|| 100nM
 |- align ="center"
-| Malachite Green Gene|| 100 nM
+| R1 aptamer|| 200 nM
 |- align ="center"
-| MgCl<sub>2</sub>|| 0.024 M
+| MgCl<sub>2</sub>|| 0.014 M
 |- align ="center"
-|NTPs|| 7.5 mM
+|NTPs|| 5 mM
 |}
-The solution also contained RNA Polymerase transcription buffer along with RNase free water. An addition of 750 nM of G3 was added to the experimental cuvette along with 200 nM of G2 approximately 2 hours later. Experiment was incubated at 30&deg;C.
+The solution also contains RNA Polymerase transcription buffer and RNase free water. 1 &mu;L of the T7 RNAP enzyme was added to solution along with 0.7 &mu;L of Ppase to prevent inhibition due to diphosphate waste and 2μL of RNase H to degrade the RNA bound to D1. The transcription reactions took place at 30&deg;C. The total volume used in each cuvette was 60 &mu;L.
-[http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
+<br />
+<font size="4.5">
+<h1>Gel Electrophoresis</h1>
+</font>
+<h3>Denaturing Polyacrylamide Gels</h3>
+<p> <font size="3.5">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).</font></p>
+<br />
+<h3>Non-Denaturing Polyacrylamide Gels</h3>
+<p> <font size="3.5">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 MgCl<sub>2</sub> buffer. 10x TAE buffer was purchased from Ambion (AM9869).  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)</font></p>
 <br />
@@ Line 172: / Line 180: @@
 <h1>RNA Extraction</h1>
 </font>
-<p><font size="3">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 37<sup>o</sup>C 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 42<sup>o</sup>C for 20 hours. The supernatant is removed and 100% -20<sup>o</sup>C ethanol, from Sigma-Aldrich (E7023-500mL), and glycogen, from ThermoScientific (R0561) ), was added to the supernatant. The supernatant is incubated at -20<sup>o</sup>C for 20 hours. The sample is spun at 135000 rpm at 4<sup>o</sup>C 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 4<sup>o</sup>C for 5 minutes using Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35<sup>o</sup>C for 10 minutes using the Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938). </font></p>
+<p><font size="3">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 37<sup>o</sup>C 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 42<sup>o</sup>C for 20 hours. The supernatant is removed and 100% -20<sup>o</sup>C ethanol, from Sigma-Aldrich (E7023-500mL), and glycogen, from ThermoScientific (R0561), was added to the supernatant. The supernatant is incubated at -20<sup>o</sup>C for 20 hours. The sample is spun at 135000 rpm at 4<sup>o</sup>C for 15 minutes using a 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 4<sup>o</sup>C for 5 minutes using a Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35<sup>o</sup>C for 10 minutes using a Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938). </font></p>
 <br />
 <font size="4.5">
-<h1>Simulations</h1>
+<h1>Oscillator Reactions</h1>
+</font>
+<h3>Oscillator Experiment 1</h3>
+The experiment shown in Fig. 13 in the results section was done as follows. Fluorometry was performed as described in 'General Fluorometry Protocols' section above. The reaction temperature was, 30&deg;C. The reaction conditions are listed below:
+[[Image:Osc1Condition.jpg|thumb|550px|center|Oscillator conditions]]
+<br>
+<h3>Oscillator Experiment 2: RNaseH titration</h3>
+The experiment shown in Fig. 14 in the results section was done as follows. Fluorometry was performed as described in 'General Fluorometry Protocols' section above. The reaction temperature was, 30&deg;C. The reaction conditions are listed below:
+[[Image:Osc2Conditions.png|thumb|650px|center|Oscillator conditions]]
+<font size="4.5">
+<h1>Modeling and Simulations</h1>
 </font>
 <br />
-<h3>Topology Design</h3>
+To build our models, we first wrote all the reactions designed to occur in each system. Then, we used the  law of mass action to derive ordinary differential equations (ODEs). The complete set of reactions and the ODEs are shown in the Supplement. ODE models were numerically integrated using MATLAB's ode23s solver. The nucleic acid binding rates and enzyme transcription and degradation rates were chosen to be in a physically plausible range based on the literature <cite>p1,p2</cite>. All the simulation parameters are reported in Tables 1 and 2 in the Supplement.
-<p><font size="3">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.</font></p>
-<br />
+Because both models are nonlinear, to gain insights into their behavior we performed a local linear analysis. Local linear analysis means that we approximated each nonlinear model with its linearized version around each equilibrium point. In practice, if our initial system is written as <math> \dot x = f(x)</math>, and its equilibrium is <math>\bar x </math> such that <math> f(\bar x)=0 </math>, then nearby point <math>\bar x </math> we can approximate the system as:
-<h3>Modelling and Tuning the Oscillator</h3>
+<math> \dot \xi = \frac{\partial f}{\partial x}(\bar x) \xi, </math>
-<p><font size="3">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.</font></p>
+where <math> \xi = x-\bar x</math>. The partial derivative <math> \frac{\partial f}{\partial x}(\bar x) </math> is evaluated at the equilibrium <math> \bar x</math>, so in our case it is a constant matrix, known as the Jacobian of the system.  The formula above is a direct application of Taylor series expansion stopped at the first order. First, for both models we found closed form expressions for their equilibria, by setting the ODEs to zero. These expressions, which are shown in the Supplement, are complex and had to be solved numerically to find the actual value of the equilibria. This was done again writing MATLAB scripts. Then, we found expressions for the Jacobian matrices <math> J= \frac{\partial f}{\partial x}(\bar x) </math> at each equilibrium point. To derive the plots shown in Figures 2 and 4 of the Results section, we wrote a script to find the eigenvalues of the Jacobian. Based on the nature of the eigenvalues, we classified the equilibrium as stable (eigenvalues have negative real part) or unstable (eigenvalues have positive real part). If there is a pair of complex conjugate eigenvalues with positive real part, then the point presents local oscillations.
+We also manipulated the models by re-ordering their variables, so that their Jacobians would clearly reveal the overall feedback interconnection of the systems. The matrices are shown in the Supplement.
-<br />
+<font size="3.5">
+<h1>References</h1>
-<h3>Modelling and Tuning the Bistable System</h3>
+<biblio>
+ #p1 pmid=21921236
-<p><font size="3">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.</font></p>
+ #p2 pmid=18006742
+</biblio>
+</font>
 <html>
@@ Line 201: / Line 221: @@
 <ul>
     <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA'><span>Home</span></a></li>
-    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Project'><span>Motivation & Objectives</span></a></li>
+    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Motivation_&_Objectives'><span>Motivation & Objectives</span></a></li>
-    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Methods'><span>Results</span></a></li>
+    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Results'><span>Results</span></a></li>
-    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Results'><span>Methods</span></a></li>
+    <li class='active'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Methods'><span>Methods</span></a></li>
-    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Members'><span>Supplement</span></a></li>
+    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Supplement'><span>Supplement</span></a></li>
-    <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Achievements'><span>Team</span></a></li>
+    <li class='last'><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Team'><span>Team</span></a></li>
 </ul>
 </div>
 </body>
 </html>