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[[Image:Approach.png|center|800px]]
[[Image:Approach.png|center|800px]]
<p></p>
<p></p>
<h2><div style= "text-align: center;">Motivation</div></h2>
<h1>Motivation</h1>


<u>Overview</u>
<h3><u>Overview</u></h3>
<br />
<p>Biological circuitry plays a pivotal role in proper cell functioning. Since the discovery of the Lac Operon in 1961, a myriad of circuit-like operons have been shown to exist in a wide variety of organisms. Many synthetic biologists have taken advantage of the concept of an operon to manipulate cellular activity. However, most mechanisms involve thousands of unknown regulatory pathways. This ambiguity severely impedes the ability to reliably predict and program cellular function. Our project focuses on alleviating this dilemma by designing biological circuits in-vitro. Using basic biological components (RNA Polymerase & Oligonucleotide sequences) we are attempting to design and implement programmable and reliable bistable and oscillatory circuits.</p>
<p>Biological circuitry plays a pivotal role in proper cell functioning. Since the discovery of the Lac Operon in 1961, a myriad of circuit-like operons have been shown to exist in a wide variety of organisms. Many synthetic biologists have taken advantage of the concept of an operon to manipulate cellular activity. However, most mechanisms involve thousands of unknown regulatory pathways. This ambiguity severely impedes the ability to reliably predict and program cellular function. Our project focuses on alleviating this dilemma by designing biological circuits in-vitro. Using basic biological components (RNA Polymerase & Oligonucleotide sequences) we are attempting to design and implement programmable and reliable bistable and oscillatory circuits.</p>


<u>Approach</u>
<h3><u>Approach</u></h3>
<br />
<p>In order to develop complex circuitry, there must first be a wide set of tools to choose from. Thus, it's imperative that more ways to control biological components are developed. One approach is to use specific strands of oligonucleotides. RNA is unique in that it has the ability to be used as an informative polymer, such as mRNA, or as a catalyst, such as rRNA. This catalytic behavior is a result of its ability to readily undergo a unique secondary structure. Using literature results, we can utilize this property of RNA to cause inhibition of specific RNA Polymerases. RNA structures with this ability are known as aptamers. Likewise, this can be reversed with the use of DNA or RNA strands to bind and remove the aptamer from the enzyme. Strands with this novel property are called kleptamers. These tools can be integrated together for the creation of a novel biological circuits such as bistable or oscillatory systems. Our project helps to expand the toolbox of in-vitro synthetic biology. </p>
<p>In order to develop complex circuitry, there must first be a wide set of tools to choose from. Thus, it's imperative that more ways to control biological components are developed. One approach is to use specific strands of oligonucleotides. RNA is unique in that it has the ability to be used as an informative polymer, such as mRNA, or as a catalyst, such as rRNA. This catalytic behavior is a result of its ability to readily undergo a unique secondary structure. Using literature results, we can utilize this property of RNA to cause inhibition of specific RNA Polymerases. RNA structures with this ability are known as aptamers. Likewise, this can be reversed with the use of DNA or RNA strands to bind and remove the aptamer from the enzyme. Strands with this novel property are called kleptamers. These tools can be integrated together for the creation of a novel biological circuits such as bistable or oscillatory systems. Our project helps to expand the toolbox of in-vitro synthetic biology. </p>
   
  <h1>Objectives</h1>
<h2> <div style= "text-align: center;">Objectives</div></h2>
   <div id='objectives'>  
   <div id='objectives'>  
<h3>Engineering biological circuits necessitates the integration of three crucial components:</h3>
<ul>
<ul>
      <li> Engineering biological circuits necessitates the integration of three crucial components:</li>
     <li> 1)&nbsp;&nbsp;&nbsp;Modeling the candidate clock and switch architectures. </li>
     <li> &nbsp;&nbsp;i)&nbsp;&nbsp;&nbsp;Modeling the candidate clock and switch architectures. </li>
     <li> 2)&nbsp;&nbsp;&nbsp;Aptamer and Kleptamer Design </li>
     <li> &nbsp;ii)&nbsp;&nbsp;&nbsp;Aptamer and Kleptamer Design </li>
     <li> 3)&nbsp;&nbsp;&nbsp;Assembly of the circuits. </li>
     <li> iii)&nbsp;&nbsp;&nbsp;Assembly of the circuits. </li>
   </ul>
   </ul>
 
    </div>
<h3>Modeling</h3>
<p></p>
<li>Approach: We build ODEs from the list of reactions and sample the behavior of the circuit for realistic parameter values.</li>
<h1>1. Modeling</h1>
<h3><font size="4.5">Approach: We build ODEs from the list of reactions and sample the behavior of the circuit for realistic parameter values.</font></h3>
<p>Creating and designing simulations using computing programs, such as MATLAB, are important for testing the various mechanisms involved in the systems. This is particularly useful for coming up with preliminary conditions for in vitro experiments, as well as determining the robustness of the schematic.</p>
<p>Creating and designing simulations using computing programs, such as MATLAB, are important for testing the various mechanisms involved in the systems. This is particularly useful for coming up with preliminary conditions for in vitro experiments, as well as determining the robustness of the schematic.</p>


<h2><div style= "text-align: center;">The Topology</div></h2>
<h3><u>The Topology</u></h3>
E1 and E2 refer to the RNA polymerase enzymes involved in the regulation of RNA species. g1,g2,g3,g4 refer to the genes that are involved. In the topology, E1 inhibits E2 via g1, while E2 activates E1 via g4. There is a self-inhibiting loop on E1 involving g3 and a self-activating loop on E2 involving g4. This represents the general idea for how the oscillator would function.
<p>E1 and E2 refer to the RNA polymerase enzymes involved in the regulation of RNA species. g1,g2,g3,g4 refer to the genes that are involved. In the topology, E1 inhibits E2 via g1, while E2 activates E1 via g4. There is a self-inhibiting loop on E1 involving g3 and a self-activating loop on E2 involving g4. This represents the general idea for how the oscillator would function.</p>


<h3><div id = "Aptamer and Kleptamer Design" style = "text-align:center;"><u>Aptamer Design</u></div></h3>
<h1><div id = "Aptamer and Kleptamer Design">2. Aptamer and Kleptamer Design</div></h1>
Specific inhibtion of enzymatic activity is of major importance for our system. The RNA aptamers used to inhibit SP6 and T7 RNA Polymerases was found in the literature. Mori, et. al. determined the proper sequence of RNA that could be used to inhibit SP6 RNA Polymerase. Likewise, Ohuchi, et. al. determined the aptamer sequence and structure for T7 RNAP inhibition. The method used to design these sequences is known as Systems Evolution of Ligand by Exponential Enrichment (SELEX), which employs evolutionary principles to optimize chemical properties of a ligand. In this case, a large pool of randomly generated RNA sequences and those that bound the best to the enzyme were extracted and amplified using PCR. Then the process was repeated at least 10 times over to yield a strand that has the best binding ability. Next, these strands were tested on their ability to inhibit the enzyme. Our DNA strands used as a template for RNAP are designed to produce a slightly modified version of these aptamers which still retains the desired activity.  
<h3><u>Aptamer Design</u></h3>
<p>Specific inhibition of enzymatic activity is of major importance for our system. The RNA aptamers used to inhibit SP6 and T7 RNA Polymerases was found in the literature. Mori, et. al. determined the proper sequence of RNA that could be used to inhibit SP6 RNA Polymerase. Likewise, Ohuchi, et. al. determined the aptamer sequence and structure for T7 RNAP inhibition. The method used to design these sequences is known as Systems Evolution of Ligand by Exponential Enrichment (SELEX), which employs evolutionary principles to optimize chemical properties of a ligand. In this case, a large pool of randomly generated RNA sequences and those that bound the best to the enzyme were extracted and amplified using PCR. Then the process was repeated at least 10 times over to yield a strand that has the best binding ability. Next, these strands were tested on their ability to inhibit the enzyme. Our DNA strands used as a template for RNAP are designed to produce a slightly modified version of these aptamers which still retains the desired activity. </p>


<h3><div id = "Aptamer and Kleptamer Design" style = "text-align:center;"><u>Kleptamer Design</u></div></h3>
<h3><u>Kleptamer Design</u></h3>
<p>[[Image: complement_design_scheme.png|center|700px]]</p>
<p>[[Image: complement_design_scheme.png|center|700px]]</p>
<p>Similar to inhibition, it is also crucial to allow the reactivation of the enzymes for oscillations. In order to reactivate the RNA polymerases, the RNA aptamers must be removed. We can do this by using a DNA strand that is complementary to the aptamer. The DNA complement will have a higher affinity to the RNA aptamer than does the enzyme. Thus, allowing the inhibitor to be "peeled" off from the enzyme and restoring transcription. It's important to ensure that the complement strand does not bind to the RNA aptamer not bound to the enzyme. If it does, there will be no chance for the aptamer to inhibit the RNA polymerase! </p>
<p>Similar to inhibition, it is also crucial to allow the reactivation of the enzymes for oscillations. In order to reactivate the RNA polymerases, the RNA aptamers must be removed. We can do this by using a DNA strand that is complementary to the aptamer. The DNA complement will have a higher affinity to the RNA aptamer than does the enzyme, allowing the inhibitor to be "peeled" off from the enzyme and restoring transcription. It's important to ensure that the complement strand does not bind to the RNA aptamer not bound to the enzyme. If it does, there will be no chance for the aptamer to inhibit the RNA polymerase! </p>
[[Image: Oruchi.png|right|300px|thumb| <font size="1.5">Secondary structure of RNA aptamer when bound to SP6 RNA Polymerase.</font>]] [[Image: aptamer_solution.png|right|300px|thumb|<font size="1.5">Secondary structure of RNA aptamer when free in solution. ]]  
[[Image: Oruchi.png|right|250px|thumb| <font size="1.5">Secondary structure of RNA aptamer when bound to SP6 RNA Polymerase.</font>]] [[Image: aptamer_solution.png|right|240px|thumb|<font size="1.5">Secondary structure of RNA aptamer when free in solution.</font> ]]  
<p>Thus, it is important to take advantage of the differences in the secondary structure of the unbound and bound forms of the RNA aptamer. </p>
<p>Thus, it is important to take advantage of the differences in the secondary structure of the unbound and bound forms of the RNA aptamer. </p>
      
      
Line 42: Line 41:
<br>
<br>
<br>
<br>
Mori, ''et. al.'' were able to determine the secondary structure when attached to SP6 RNA polymerase and NUPACK was used to determine the structure of the unbound aptamer. Strands that are complement to the areas boxed in red are likely to have little interactions occurring, whereas, complements to nucleotides boxed in green will likely have high interactions. It is ideal that a strand is complement to most or all of the nucleotides that are boxed in order to get the most favorable interactions.
Mori, ''et. al.'' were able to determine the secondary structure when attached to SP6 RNA polymerase and NUPACK was used to determine the structure of the unbound aptamer. Strands that are complement to the areas boxed in red are likely to have little interactions occurring, whereas, complements to nucleotides boxed in green will likely have high interactions. It is ideal that a strand is complement to most or all of the nucleotides that are boxed in order to get the most favorable interactions.</p>
 
<br>
<h3><div id = "Aptamer and Kleptamer Design" style = "text-align:center;"><u>Assembly of Circuits</u></div></h3>
<h1><div id="Assembly of Circuits">3. Assembly of Circuits</div></h1>
Now that the tools are designed, the next step is to begin piecing the system together to ensure each component is working properly. There are two main methods used to show evidence for our system, gel electrophoresis and fluorometry. Gel electrophoresis provides an excellent means of qualitatively determined the desired interactions are taking place. Our experiments pertain mainly to the use of 10% Polyacrylamide native gels. Since our system involves a dynamical aspect, this method is not sufficient. Fluorometry can be used in this case to determine the kinetics and the dynamics.  
Now that the tools are designed, the next step is to begin piecing the system together to ensure each component is working properly. There are two main methods used to show evidence for our system, gel electrophoresis and fluorometry. Gel electrophoresis provides an excellent means of qualitatively determined the desired interactions are taking place. Our experiments pertain mainly to the use of 10% Polyacrylamide native gels. Since our system involves a dynamical aspect, this method is not sufficient. Fluorometry can be used in this case to determine the kinetics and the dynamics.  


Line 58: Line 57:
[[Image:T7inhibitor.png|left|300x175px|thumb|Inhibition of T7 RNAP with R3]]
[[Image:T7inhibitor.png|left|300x175px|thumb|Inhibition of T7 RNAP with R3]]
[[Image:R3 T7 R2.png|right|300x175px|thumb|Reactivation of T7 RNAP with R2]]
[[Image:R3 T7 R2.png|right|300x175px|thumb|Reactivation of T7 RNAP with R2]]
 
<br><br><br><br><br><br><br>
<div style= "align-center;"><font size="3.5">The aptamer encoded by T7 RNA Polymerase must be able to bind and inhibit to SP6 RNA Polymerase. This can be ascertained throughout the use of glee electrophoresis and fluorometry. Once this is determined, it's important to ensure that K1 can properly remove the aptamer from SP6 RNA Polymerase. This also was done through the use of gel electrophoresis and fluorometry. Another kleptamer involved in SP6 reactivation is strand R4 and was determined by gel electrophoresis only. are two mechanism that need to be verified for T7 RNAP. First, T7 needs to be able to self-inhibit itself via the transcription of G3. Finally, T7 must also be shown to be reactivated by the kleptamer R2. Both of these were verified using fluorometry and gel electrophoresis.Once all of the parts have been verified, the full oscillator can then begin to be implemented. Even at this point, however, it still can be very challenging since proper functioning can often times be very sensitive to initial conditions. Thus, models created using MATLAB will help to guide these experiments in order to help fine tune the initial concentrations of genes and enzymes to induce the proper parameters. Finally, the system is ready to begin full assembly. The model plays a crucial role at this point for giving approximations on the concentration of each component. However, the model will not be perfect and it will be necessary to perform tuning experiments. In this case, each concentration of each component will be varied one at a time. This will ensure that each concentration is optimized for the system to perform most efficiently. </font></div>
<p><font size="3.5">The aptamer encoded by T7 RNA Polymerase must be able to bind and inhibit to SP6 RNA Polymerase. This can be ascertained throughout the use of glee electrophoresis and fluorometry. Once this is determined, it's important to ensure that K1 can properly remove the aptamer from SP6 RNA Polymerase. This also was done through the use of gel electrophoresis and fluorometry. Another kleptamer involved in SP6 reactivation is strand R4 and was determined by gel electrophoresis only. are two mechanism that need to be verified for T7 RNAP. First, T7 needs to be able to self-inhibit itself via the transcription of G3. Finally, T7 must also be shown to be reactivated by the kleptamer R2. Both of these were verified using fluorometry and gel electrophoresis.Once all of the parts have been verified, the full oscillator can then begin to be implemented. Even at this point, however, it still can be very challenging since proper functioning can often times be very sensitive to initial conditions. Thus, models created using MATLAB will help to guide these experiments in order to help fine tune the initial concentrations of genes and enzymes to induce the proper parameters. Finally, the system is ready to begin full assembly. The model plays a crucial role at this point for giving approximations on the concentration of each component. However, the model will not be perfect and it will be necessary to perform tuning experiments. In this case, each concentration of each component will be varied one at a time. This will ensure that each concentration is optimized for the system to perform most efficiently. </font></p>

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Motivation

Overview

Biological circuitry plays a pivotal role in proper cell functioning. Since the discovery of the Lac Operon in 1961, a myriad of circuit-like operons have been shown to exist in a wide variety of organisms. Many synthetic biologists have taken advantage of the concept of an operon to manipulate cellular activity. However, most mechanisms involve thousands of unknown regulatory pathways. This ambiguity severely impedes the ability to reliably predict and program cellular function. Our project focuses on alleviating this dilemma by designing biological circuits in-vitro. Using basic biological components (RNA Polymerase & Oligonucleotide sequences) we are attempting to design and implement programmable and reliable bistable and oscillatory circuits.

Approach

In order to develop complex circuitry, there must first be a wide set of tools to choose from. Thus, it's imperative that more ways to control biological components are developed. One approach is to use specific strands of oligonucleotides. RNA is unique in that it has the ability to be used as an informative polymer, such as mRNA, or as a catalyst, such as rRNA. This catalytic behavior is a result of its ability to readily undergo a unique secondary structure. Using literature results, we can utilize this property of RNA to cause inhibition of specific RNA Polymerases. RNA structures with this ability are known as aptamers. Likewise, this can be reversed with the use of DNA or RNA strands to bind and remove the aptamer from the enzyme. Strands with this novel property are called kleptamers. These tools can be integrated together for the creation of a novel biological circuits such as bistable or oscillatory systems. Our project helps to expand the toolbox of in-vitro synthetic biology.

Objectives

Engineering biological circuits necessitates the integration of three crucial components:

  • 1)   Modeling the candidate clock and switch architectures.
  • 2)   Aptamer and Kleptamer Design
  • 3)   Assembly of the circuits.

1. Modeling

Approach: We build ODEs from the list of reactions and sample the behavior of the circuit for realistic parameter values.

Creating and designing simulations using computing programs, such as MATLAB, are important for testing the various mechanisms involved in the systems. This is particularly useful for coming up with preliminary conditions for in vitro experiments, as well as determining the robustness of the schematic.

The Topology

E1 and E2 refer to the RNA polymerase enzymes involved in the regulation of RNA species. g1,g2,g3,g4 refer to the genes that are involved. In the topology, E1 inhibits E2 via g1, while E2 activates E1 via g4. There is a self-inhibiting loop on E1 involving g3 and a self-activating loop on E2 involving g4. This represents the general idea for how the oscillator would function.

2. Aptamer and Kleptamer Design

Aptamer Design

Specific inhibition of enzymatic activity is of major importance for our system. The RNA aptamers used to inhibit SP6 and T7 RNA Polymerases was found in the literature. Mori, et. al. determined the proper sequence of RNA that could be used to inhibit SP6 RNA Polymerase. Likewise, Ohuchi, et. al. determined the aptamer sequence and structure for T7 RNAP inhibition. The method used to design these sequences is known as Systems Evolution of Ligand by Exponential Enrichment (SELEX), which employs evolutionary principles to optimize chemical properties of a ligand. In this case, a large pool of randomly generated RNA sequences and those that bound the best to the enzyme were extracted and amplified using PCR. Then the process was repeated at least 10 times over to yield a strand that has the best binding ability. Next, these strands were tested on their ability to inhibit the enzyme. Our DNA strands used as a template for RNAP are designed to produce a slightly modified version of these aptamers which still retains the desired activity.

Kleptamer Design

Similar to inhibition, it is also crucial to allow the reactivation of the enzymes for oscillations. In order to reactivate the RNA polymerases, the RNA aptamers must be removed. We can do this by using a DNA strand that is complementary to the aptamer. The DNA complement will have a higher affinity to the RNA aptamer than does the enzyme, allowing the inhibitor to be "peeled" off from the enzyme and restoring transcription. It's important to ensure that the complement strand does not bind to the RNA aptamer not bound to the enzyme. If it does, there will be no chance for the aptamer to inhibit the RNA polymerase!

Secondary structure of RNA aptamer when bound to SP6 RNA Polymerase.
Secondary structure of RNA aptamer when free in solution.

Thus, it is important to take advantage of the differences in the secondary structure of the unbound and bound forms of the RNA aptamer.

The binding properties of the RNA aptamers rely on their secondary structure. For our strands, secondary structure is different when bound to the RNA polymerase than when unbound. Using NUPACK and Mori, et. al. we can make an estimation of how the DNA complement will interact with either of the two secondary structures.

Mori, et. al. were able to determine the secondary structure when attached to SP6 RNA polymerase and NUPACK was used to determine the structure of the unbound aptamer. Strands that are complement to the areas boxed in red are likely to have little interactions occurring, whereas, complements to nucleotides boxed in green will likely have high interactions. It is ideal that a strand is complement to most or all of the nucleotides that are boxed in order to get the most favorable interactions.


3. Assembly of Circuits

Now that the tools are designed, the next step is to begin piecing the system together to ensure each component is working properly. There are two main methods used to show evidence for our system, gel electrophoresis and fluorometry. Gel electrophoresis provides an excellent means of qualitatively determined the desired interactions are taking place. Our experiments pertain mainly to the use of 10% Polyacrylamide native gels. Since our system involves a dynamical aspect, this method is not sufficient. Fluorometry can be used in this case to determine the kinetics and the dynamics.

Unfortunately, we can't measure directly the activity of the enzymes. However, we can determine activity indirectly with the use of fluorescent dyes. Tsien, et. al. report the observation that specific aptamers can be used to switch on fluorescence of Triphenylmethane dyes, more specifically, Malachite green. Likewise, Jaffrey, et. al. have used SELEX to find an aptamer sequence that will bind to, and greatly enhance the fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolidine (DFHBI). These molecules are incorporated into the design as the reporter system. This is done by using genes that code for an aptamer that will bind to the fluorophore. In that way the RNAPs will code the aptamer which will bind to the fluorophore and increase fluorescence intensity! We have designated that DFHBI aptamer gene has an SP6 RNAP promoter, whereas Malachite green aptamer gene has a T7 RNAP promoter. Thus, it can be determined which enzyme is activated or inhibited by viewing the rate of fluorescence intensity increase for each respective fluorophore.

A cartoon representation of the reporter system. The aptamer will interact with the dye, resulting in enhanced fluorescence of the molecule.

Before the circuits can be assembled together to make a circuit, the individual components should be tested. This has two advantages. First, the circuit cannot function if even one portion does not work. By performing experiments on the components it ensures the system is plausible. Second, by performing individual reactions one at a time, the parameters used in the model can be better estimated to give more accurate approximations. There are five major portions that need to be tested before running the circuit. These include the following:

Inhibition of SP6 RNAP with R1
Reactivation of SP6 RNAP with K1
Reactivation of SP6 RNAP with R4
Inhibition of T7 RNAP with R3
Reactivation of T7 RNAP with R2








The aptamer encoded by T7 RNA Polymerase must be able to bind and inhibit to SP6 RNA Polymerase. This can be ascertained throughout the use of glee electrophoresis and fluorometry. Once this is determined, it's important to ensure that K1 can properly remove the aptamer from SP6 RNA Polymerase. This also was done through the use of gel electrophoresis and fluorometry. Another kleptamer involved in SP6 reactivation is strand R4 and was determined by gel electrophoresis only. are two mechanism that need to be verified for T7 RNAP. First, T7 needs to be able to self-inhibit itself via the transcription of G3. Finally, T7 must also be shown to be reactivated by the kleptamer R2. Both of these were verified using fluorometry and gel electrophoresis.Once all of the parts have been verified, the full oscillator can then begin to be implemented. Even at this point, however, it still can be very challenging since proper functioning can often times be very sensitive to initial conditions. Thus, models created using MATLAB will help to guide these experiments in order to help fine tune the initial concentrations of genes and enzymes to induce the proper parameters. Finally, the system is ready to begin full assembly. The model plays a crucial role at this point for giving approximations on the concentration of each component. However, the model will not be perfect and it will be necessary to perform tuning experiments. In this case, each concentration of each component will be varied one at a time. This will ensure that each concentration is optimized for the system to perform most efficiently.

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