Difference between revisions of "Team:ETH Zurich/Detector Module"

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                 <div>
 
                 <div>
 
                     <h3>REACTIONS</h3>
 
                     <h3>REACTIONS</h3>
−
                     \begin{align*}
+
                     <p>
−
                    1) && P_{mNect} & \rightarrow P_{mNect} + mRNA_{mNect} \\
+
                        \begin{align*}
−
                    2) && P_{sfGFP} & \rightarrow P_{sfGFP} + mRNA_{sfGFP} \\
+
                        1) && P_{mNect} & \rightarrow P_{mNect} + mRNA_{mNect} \\
−
                    3) && mRNA_{mNect} & \rightarrow mRNA_{mNect} + mNect \\
+
                        2) && P_{sfGFP} & \rightarrow P_{sfGFP} + mRNA_{sfGFP} \\
−
                    4) && mRNA_{sfGFP} & \rightarrow mRNA_{sfGFP} + sfGFP \\
+
                        3) && mRNA_{mNect} & \rightarrow mRNA_{mNect} + mNect \\
−
                    5) && mRNA_{mNect} & \rightarrow \\
+
                        4) && mRNA_{sfGFP} & \rightarrow mRNA_{sfGFP} + sfGFP \\
−
                    6) && mRNA_{sfGFP} & \rightarrow \\
+
                        5) && mRNA_{mNect} & \rightarrow \\
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                    7) && mNect & \rightarrow \\
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                        6) && mRNA_{sfGFP} & \rightarrow \\
−
                    8) && sfGFP & \rightarrow \\
+
                        7) && mNect & \rightarrow \\
−
                    \end{align*}
+
                        8) && sfGFP & \rightarrow \\
 +
                        \end{align*}
 +
                    </p>
 
                 </div>
 
                 </div>
  
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                 <div>
 
                 <div>
 
                     <h3>STOCHASTIC REACTION RATES:</h3>
 
                     <h3>STOCHASTIC REACTION RATES:</h3>
−
                     \begin{align*}
+
                     <p>
−
                    1) \quad & k_{mRNAmnect} \cdot P_{mNect} \cdot P_{activity} \\
+
                        \begin{align*}
−
                    2) \quad & k_{mRNAsfgfp} \cdot P_{sfGFP} \cdot P_{activity} \\
+
                        1) \quad & k_{mRNAmnect} \cdot P_{mNect} \cdot P_{activity} \\
−
                    3) \quad & k_{mNect} \cdot mRNA_{mNect} \\
+
                        2) \quad & k_{mRNAsfgfp} \cdot P_{sfGFP} \cdot P_{activity} \\
−
                    4) \quad & k_{sfGFP} \cdot mRNA_{sfGFP} \\
+
                        3) \quad & k_{mNect} \cdot mRNA_{mNect} \\
−
                    5) \quad & d_{mRNAmnect} \cdot mRNA_{mNect} \\
+
                        4) \quad & k_{sfGFP} \cdot mRNA_{sfGFP} \\
−
                    6) \quad & d_{mRNAsfgfp} \cdot mRNA_{sfGFP} \\
+
                        5) \quad & d_{mRNAmnect} \cdot mRNA_{mNect} \\
−
                    7) \quad & d_{mNect} \cdot mNect \\
+
                        6) \quad & d_{mRNAsfgfp} \cdot mRNA_{sfGFP} \\
−
                    8) \quad & d_{sfGFP} \cdot sfGFP \\
+
                        7) \quad & d_{mNect} \cdot mNect \\
−
                    \end{align*}
+
                        8) \quad & d_{sfGFP} \cdot sfGFP \\
 +
                        \end{align*}
 +
                    </p>
 
                 </div>
 
                 </div>
 
                 <div>
 
                 <div>
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         <div class="sec white content_only">
 
         <div class="sec white content_only">
 
             <div>
 
             <div>
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                 <h2>CHARACTERIZATION</h2>
+
                 <h3>CHARACTERIZATION MODEL</h3>
 
                 <p>The reporter has been characterized by placing the fluorescent proteins under an aTc-inducible promoter. In this case the activity of the promoter is modeled as:</p>
 
                 <p>The reporter has been characterized by placing the fluorescent proteins under an aTc-inducible promoter. In this case the activity of the promoter is modeled as:</p>
−
                 \begin{align*}
+
                 <p>\begin{align*}
 
                     P_{activity}=l_{pTet}+(1-l_{pTet})\cdot\frac{[aTc]^{n}}{K_m^n+[aTc]^{n}}
 
                     P_{activity}=l_{pTet}+(1-l_{pTet})\cdot\frac{[aTc]^{n}}{K_m^n+[aTc]^{n}}
−
                 \end{align*}
+
                 \end{align*}</p>
 
                 <p>Where aTc is the tetracycline variant used for induction, $l_{pTet}$ is the leakiness of the promoter, $n$ the sensitivity to aTc and $K_m$ the affinity.</p>
 
                 <p>Where aTc is the tetracycline variant used for induction, $l_{pTet}$ is the leakiness of the promoter, $n$ the sensitivity to aTc and $K_m$ the affinity.</p>
 
             </div>
 
             </div>
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             <div>
 
             <div>
 
                 <h2>RESULTS</h2>
 
                 <h2>RESULTS</h2>
−
                 <h3></h3>
+
                 <h3>PARAMETERS</h3>
−
                 <p></p>
+
                 <p>We estimated the parameters for the reporter genes and the tet promoter stochastically using flow cytometry measurements. The simulated distribution was fitted to the measurements by <i>Approximate Bayesian computation (ABC)</i> usign the INSIGHT tool.</p>
 +
                <p>The figure below shows the distributions of the estimated parameters. The parameters page reports the <i>maximum a posteriori (MAP)</i> estimates, which are used in the simulation and analysis of our system.</p>
 +
 
 +
                <h3>EXPERIMENT DESIGN IMPROVEMENT</h3>
 +
                <p>Parameter estimation for the tet promoter revealed that the affinity $K_m$ of the tet promoter is in the order of 8000nM. This is almost double the maximum aTc concentration we were using for induction (2000ng/mL = 4320.6nM) in the experiments, meaning we were not using the full range of the promoter.</p>
 +
                <p>Since cells die at higher aTc concentrations, we need to reduce the concentration of the TetR repressor in the cells. We suggested to the experimentalists to use a low (~5) copy plasmid for TetR expression instead of the medium-low (~15-20) copy plasmid originally used.</p>
 
             </div>
 
             </div>
 
         </div>
 
         </div>
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     </body>
 
     </body>
 
</html>
 
</html>
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{{:Template:ETH_Zurich/footer}}
 
{{:Template:ETH_Zurich/footer}}

Revision as of 09:01, 16 October 2016

PAVLOV'S COLI

REPORTER MODULE

OVERVIEW

Figure 1: Schematic of the reporter of our circuit.

The reporter is the component of the circuit that enables readout of the stored information. The state of the switch is displayed by two different fluorescent proteins: sfGFP is expressed by DNA that has been switched, while DNA that didn't switch expresses mNectarine.

In order to implement multiplexing, the reporter proteins are expressed only when they are induced by the same candidate marker that triggered the switch.

GOALS

  • Assist the design of the reporter.
  • Characterize the reporter proteins.

MODEL

Our reporter system consist in two fluorenscent proteins that show the state of the switch. After induction, a switched plasmid expresses GFP, while a plasmid in it's original state expresses mNectarine.

Figure 2: Biological implementation of the integrase reporter. The figure shows both the switched and non switched state. Expression of the reporter proteins is repressed by default and induced in presence of the candidate marker.

The following section describes the species and reactions involved:

REACTIONS

\begin{align*} 1) && P_{mNect} & \rightarrow P_{mNect} + mRNA_{mNect} \\ 2) && P_{sfGFP} & \rightarrow P_{sfGFP} + mRNA_{sfGFP} \\ 3) && mRNA_{mNect} & \rightarrow mRNA_{mNect} + mNect \\ 4) && mRNA_{sfGFP} & \rightarrow mRNA_{sfGFP} + sfGFP \\ 5) && mRNA_{mNect} & \rightarrow \\ 6) && mRNA_{sfGFP} & \rightarrow \\ 7) && mNect & \rightarrow \\ 8) && sfGFP & \rightarrow \\ \end{align*}

SPECIES

Name Description
$P_{mNect}$ Non switched promoter, facing the mNectarine gene.
$P_{sfGFP}$ Switched promoter, facing the sfGFP gene.
$mRNA_{mNect}$ mRNA of the mNectarine protein.
$mRNA_{sfGFP}$ mRNA of the sfGFP protein.
$mNect$ mNectarine fluorescent protein.
$sfGFP$ Superfolder GFP protein.

STOCHASTIC REACTION RATES:

\begin{align*} 1) \quad & k_{mRNAmnect} \cdot P_{mNect} \cdot P_{activity} \\ 2) \quad & k_{mRNAsfgfp} \cdot P_{sfGFP} \cdot P_{activity} \\ 3) \quad & k_{mNect} \cdot mRNA_{mNect} \\ 4) \quad & k_{sfGFP} \cdot mRNA_{sfGFP} \\ 5) \quad & d_{mRNAmnect} \cdot mRNA_{mNect} \\ 6) \quad & d_{mRNAsfgfp} \cdot mRNA_{sfGFP} \\ 7) \quad & d_{mNect} \cdot mNect \\ 8) \quad & d_{sfGFP} \cdot sfGFP \\ \end{align*}

PARAMETERS

Name Description
$P_{activity}$ Fraction of the maximal activity of the promoter. This value is computed in the sensor module.
$k_{mRNAmnect}$ mNectarine mRNA transcription rate.
$k_{mRNAsfgfp}$ sfGFP mRNA transcription rate.
$k_{mNect}$ mNectarine translation rate.
$k_{sfGFP}$ sfGFP translation rate.
$d_{mRNAmnect}$ mNectarine mRNA degradation rate.
$d_{mRNAsfgfp}$ sfGFP mRNA degradation rate.
$d_{mNect}$ mNectarine degradation rate.
$d_{sfGFP}$ sfGFP degradation rate.

CHARACTERIZATION MODEL

The reporter has been characterized by placing the fluorescent proteins under an aTc-inducible promoter. In this case the activity of the promoter is modeled as:

\begin{align*} P_{activity}=l_{pTet}+(1-l_{pTet})\cdot\frac{[aTc]^{n}}{K_m^n+[aTc]^{n}} \end{align*}

Where aTc is the tetracycline variant used for induction, $l_{pTet}$ is the leakiness of the promoter, $n$ the sensitivity to aTc and $K_m$ the affinity.

RESULTS

PARAMETERS

We estimated the parameters for the reporter genes and the tet promoter stochastically using flow cytometry measurements. The simulated distribution was fitted to the measurements by Approximate Bayesian computation (ABC) usign the INSIGHT tool.

The figure below shows the distributions of the estimated parameters. The parameters page reports the maximum a posteriori (MAP) estimates, which are used in the simulation and analysis of our system.

EXPERIMENT DESIGN IMPROVEMENT

Parameter estimation for the tet promoter revealed that the affinity $K_m$ of the tet promoter is in the order of 8000nM. This is almost double the maximum aTc concentration we were using for induction (2000ng/mL = 4320.6nM) in the experiments, meaning we were not using the full range of the promoter.

Since cells die at higher aTc concentrations, we need to reduce the concentration of the TetR repressor in the cells. We suggested to the experimentalists to use a low (~5) copy plasmid for TetR expression instead of the medium-low (~15-20) copy plasmid originally used.

REFERENCES

  • [1] -

Thanks to the sponsors that supported our project: