Difference between revisions of "Team:Thessaloniki/Model/iffl"

 
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         <div class="row justify-center q-mb-md" :class="rM('text-dark', 'text-light')">
 
         <div class="row justify-center q-mb-md" :class="rM('text-dark', 'text-light')">
 
             <div class="col-xs-12 col-sm-10 col-md-9 col-lg-8">
 
             <div class="col-xs-12 col-sm-10 col-md-9 col-lg-8">
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                 <div class="flex flex-center">
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                 <div class="flex flex-center q-mb-md">
 
                     <h3>Attenuation iFFL</h3>
 
                     <h3>Attenuation iFFL</h3>
 
                 </div>
 
                 </div>
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+
                <div class="flex flex-center">
 
                 <h4>RNA - based regulation</h4>
 
                 <h4>RNA - based regulation</h4>
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                 <p>
+
                </div>
 +
               
 +
                 <p class="q-mt-md">
 
                         We studied whether the IFFL network can work at the RNA level with the transcriptional attenuator-activating sRNA regulators who’s function, can efficiently regulate the expression of proteins in different network motifs. Although at the experimental level we could not work on this circuit due to time limitations, we still think it is interesting, and decided to add the modeling structure of IFFL to RNA level for future analysis after the end of the competition.
 
                         We studied whether the IFFL network can work at the RNA level with the transcriptional attenuator-activating sRNA regulators who’s function, can efficiently regulate the expression of proteins in different network motifs. Although at the experimental level we could not work on this circuit due to time limitations, we still think it is interesting, and decided to add the modeling structure of IFFL to RNA level for future analysis after the end of the competition.
 
                 </p>
 
                 </p>
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                     <small class="text-grey-5">Figure 1: The blue line shows the concentration (nM)  of mature sfGFP  with copy number  at IFFL network.The red line shows change in concentration (nM) of mature sfGFP with copy number at NAR newtork.</small>
 
                     <small class="text-grey-5">Figure 1: The blue line shows the concentration (nM)  of mature sfGFP  with copy number  at IFFL network.The red line shows change in concentration (nM) of mature sfGFP with copy number at NAR newtork.</small>
 
                 </div>
 
                 </div>
−
 
+
                 <div class="q-mt-md q-mb-md">
−
                 <p>
+
 
                     The ODE for the negative autoregulation is described as follows
 
                     The ODE for the negative autoregulation is described as follows
−
                 </p>
+
                 </div>
 
                 <div id="ode-eq-1"></div>
 
                 <div id="ode-eq-1"></div>
  
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                 <div id="ode-eq-2"></div>
 
                 <div id="ode-eq-2"></div>
 +
               
  
−
                 <p>
+
                 <p class="q-mt-md">
−
                         The parameters used are all featured in literature [1]. The diagram above refers to the case where we use two tandem repressors as described in the design of the model (). Note that in order to change the number of the repressor, three system parameters change. Changes in parameter values refer to the repressor degradation, the repressor maturation step from R * to R, and the dissociation constant in the hill equation.
+
                         The parameters used are all featured in literature [1]. The diagram above refers to the case where we use two tandem repressors as described in the design of the model. Note that in order to change the number of the repressor, three system parameters change. Changes in parameter values refer to the repressor degradation, the repressor maturation step from R * to R, and the dissociation constant in the hill equation.
 
                 </p>
 
                 </p>
  
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                 <p>Parameters are differentiated for a single (first column values) and double repressor (second column values).</p>
 
                 <p>Parameters are differentiated for a single (first column values) and double repressor (second column values).</p>
  
−
 
+
               
−
                 <div style="overflow-x:auto;">
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                 <div class="flex flex-center">
−
                    <table class="simple-table text-center">
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                    <div style="overflow-x:auto;">
−
                        <tr>
+
                        <table class="simple-table text-center">
−
                            <td>
+
                            <tr>
−
                                <b>Parameters</b>
+
                                <td>
−
                            </td>
+
                                    <b>Parameters</b>
−
                            <td>
+
                                </td>
−
                                <b>Biological meaning</b>
+
                                <td>
−
                            </td>
+
                                    <b>Biological meaning</b>
−
                            <td>
+
                                </td>
−
                                <b>Values</b>
+
                                <td>
−
                            </td>
+
                                    <b>Values</b>
−
                            <td>
+
                                </td>
−
                                <b>Values</b>
+
                                <td>
−
                            </td>
+
                                    <b>Values</b>
−
                            <td>
+
                                </td>
−
                                <b>units</b>
+
                                <td>
−
                            </td>
+
                                    <b>units</b>
−
                        </tr>
+
                                </td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>bR</b>
+
                                <td>
−
                            </td>
+
                                    <b>bR</b>
−
                            <td>maximum transcription rate of Repressor</td>
+
                                </td>
−
                            <td>39.4</td>
+
                                <td>maximum transcription rate of Repressor</td>
−
                            <td>39.4</td>
+
                                <td>39.4</td>
−
                            <td>nM/sec</td>
+
                                <td>39.4</td>
−
                        </tr>
+
                                <td>nM/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>bM</b>
+
                                <td>
−
                            </td>
+
                                    <b>bM</b>
−
                            <td>maximum transcription rate of sfGFP</td>
+
                                </td>
−
                            <td>4.9</td>
+
                                <td>maximum transcription rate of sfGFP</td>
−
                            <td>4.9</td>
+
                                <td>4.9</td>
−
                            <td>nM/sec</td>
+
                                <td>4.9</td>
−
                        </tr>
+
                                <td>nM/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>dR</b>
+
                                <td>
−
                            </td>
+
                                    <b>dR</b>
−
                            <td>degradation rate of repressor</td>
+
                                </td>
−
                            <td>2.1 10-3</td>
+
                                <td>degradation rate of repressor</td>
−
                            <td>1.7 10 -3</td>
+
                                <td>2.1 10-3</td>
−
                            <td>1/sec</td>
+
                                <td>1.7 10 -3</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>rm</b>
+
                                <td>
−
                            </td>
+
                                    <b>rm</b>
−
                            <td>maturation rate of double repressor</td>
+
                                </td>
−
                            <td>6.9 10-5</td>
+
                                <td>maturation rate of double repressor</td>
−
                            <td>7.5 10-5</td>
+
                                <td>6.9 10-5</td>
−
                            <td>1/sec</td>
+
                                <td>7.5 10-5</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>K</b>
+
                                <td>
−
                            </td>
+
                                    <b>K</b>
−
                            <td>concentration of active Repressor R</td>
+
                                </td>
−
                            <td>289.5</td>
+
                                <td>concentration of active Repressor R</td>
−
                            <td>193.6</td>
+
                                <td>289.5</td>
−
                            <td>nM</td>
+
                                <td>193.6</td>
−
                        </tr>
+
                                <td>nM</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>a</b>
+
                                <td>
−
                            </td>
+
                                    <b>a</b>
−
                            <td>Maturation rate of sfGFP</td>
+
                                </td>
−
                            <td>1.7 10-2</td>
+
                                <td>Maturation rate of sfGFP</td>
−
                            <td>1.7 10-2</td>
+
                                <td>1.7 10-2</td>
−
                            <td>1/sec</td>
+
                                <td>1.7 10-2</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>kt</b>
+
                                <td>
−
                            </td>
+
                                    <b>kt</b>
−
                            <td>translation rate of sfGFP</td>
+
                                </td>
−
                            <td>2.6 10-4</td>
+
                                <td>translation rate of sfGFP</td>
−
                            <td>2.6 10-4</td>
+
                                <td>2.6 10-4</td>
−
                            <td>1/sec</td>
+
                                <td>2.6 10-4</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>dG</b>
+
                                <td>
−
                            </td>
+
                                    <b>dG</b>
−
                            <td>degradation of mature sfGFP</td>
+
                                </td>
−
                            <td>19 10-2</td>
+
                                <td>degradation of mature sfGFP</td>
−
                            <td>19 10-2</td>
+
                                <td>19 10-2</td>
−
                            <td>1/sec</td>
+
                                <td>19 10-2</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>p(t)</b>
+
                                <td>
−
                            </td>
+
                                    <b>p(t)</b>
−
                            <td>inefficiency in transcription caused by atennuator</td>
+
                                </td>
−
                            <td>0.18</td>
+
                                <td>inefficiency in transcription caused by atennuator</td>
−
                            <td>0.18</td>
+
                                <td>0.18</td>
−
                            <td>1/sec</td>
+
                                <td>0.18</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>dM</b>
+
                                <td>
−
                            </td>
+
                                    <b>dM</b>
−
                            <td>Degradation rate of mRNA sfGFP</td>
+
                                </td>
−
                            <td>5.9 10-4</td>
+
                                <td>Degradation rate of mRNA sfGFP</td>
−
                            <td>5.9 10-4</td>
+
                                <td>5.9 10-4</td>
−
                            <td>1/sec</td>
+
                                <td>5.9 10-4</td>
−
                        </tr>
+
                                <td>1/sec</td>
−
                        <tr>
+
                            </tr>
−
                            <td>
+
                            <tr>
−
                                <b>N </b>
+
                                <td>
−
                            </td>
+
                                    <b>N </b>
−
                            <td>number of tandem repressors(single or double)</td>
+
                                </td>
−
                            <td>1</td>
+
                                <td>number of tandem repressors(single or double)</td>
−
                            <td>2</td>
+
                                <td>1</td>
−
                            <td>-</td>
+
                                <td>2</td>
−
                        </tr>
+
                                <td>-</td>
−
                    </table>
+
                            </tr>
 +
                        </table>
 +
                    </div>
 
                 </div>
 
                 </div>
 
                 <p>
 
                 <p>
Line 185: Line 189:
 
                 <div>
 
                 <div>
 
                     <div id="0be8faf7-c08f-4520-b510-53f5f5c507e6" class="plotly-graph-div"></div>
 
                     <div id="0be8faf7-c08f-4520-b510-53f5f5c507e6" class="plotly-graph-div"></div>
−
                     <small class="text-grey-5">Figure 2. The blue line shows the concentration (nM)  of mature sfGFP over a time (sec)  with single-repressor . The red line shows change in concentration (nM) of mature sfGFP over a  time (sec) with double-repressor.</small>
+
                     <small class="text-grey-5">Figure 2. The blue line shows the concentration (nM)  of mature sfGFP over a time (sec)  with single-repressor . The red line shows change in concentration (nM) of mature sfGFP over a  time (sec) with double-repressor.<br><br><br><br><br></small>
 
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Latest revision as of 03:04, 18 October 2018

Attenuation iFFL

RNA - based regulation

We studied whether the IFFL network can work at the RNA level with the transcriptional attenuator-activating sRNA regulators who’s function, can efficiently regulate the expression of proteins in different network motifs. Although at the experimental level we could not work on this circuit due to time limitations, we still think it is interesting, and decided to add the modeling structure of IFFL to RNA level for future analysis after the end of the competition.

As mentioned in the design page and based on the literature [1], the Negative Autoregulation network motif was studied using sRNA regulators. This network includes an attenuator, which regulates the transcription of the sRNA repressor and the sfGFP gene reporter. Using differential equations to describe the system, it was proven that this particular network cannot maintain the production of sfGFP over different copy numbers. Nevertheless, the advantages it possesses such as reducing about half of the system response and protein derivation at a lower steady-state level can be used for the IFFL motif after rearrangement of the sequences as described below.

By analyzing the differential equations for the NAR network we would like to highlight the following features: NAR can include one or two tandem repressors in a series. Regarding the paper, it has been shown that a double repressor is one that displays the characteristics of a fast response and tighter repression. The sRNA requires time to mature, represented as a maturation step in the model, in order to be able to interact with the attenuator and repress its expression at the same time as repressing sfGFP. In the NAR network, due to the reason of repressing the production of the sRNA repressor, the production of the reporter gene cannot be dependent on the copy number. Using the same rates by changing the position of the attenuator so as not to affect the transcription of the sRNA, the network motif changes and is expressed as IFFL. Note that cooperativity for binding to sRNA is equal to 1 and the equations enter the probability limit where the RNA polymerase can continue transcription without disruption. The probability is raised in the N + 1 exponent for N number of sequences of the repressor sRNAs arranged.

The result of the differential equations simulations that express the NAR and the IFFL network by using two tandem repressors (N = 2) shows that under the same conditions expressed by the same rates for both networks, the concentration of sfGFP remains constant for our network.

Figure 1: The blue line shows the concentration (nM) of mature sfGFP with copy number at IFFL network.The red line shows change in concentration (nM) of mature sfGFP with copy number at NAR newtork.
The ODE for the negative autoregulation is described as follows
The ODE for IFFL is described as follows

The parameters used are all featured in literature [1]. The diagram above refers to the case where we use two tandem repressors as described in the design of the model. Note that in order to change the number of the repressor, three system parameters change. Changes in parameter values refer to the repressor degradation, the repressor maturation step from R * to R, and the dissociation constant in the hill equation.

Parameters

Parameters are differentiated for a single (first column values) and double repressor (second column values).

Parameters Biological meaning Values Values units
bR maximum transcription rate of Repressor 39.4 39.4 nM/sec
bM maximum transcription rate of sfGFP 4.9 4.9 nM/sec
dR degradation rate of repressor 2.1 10-3 1.7 10 -3 1/sec
rm maturation rate of double repressor 6.9 10-5 7.5 10-5 1/sec
K concentration of active Repressor R 289.5 193.6 nM
a Maturation rate of sfGFP 1.7 10-2 1.7 10-2 1/sec
kt translation rate of sfGFP 2.6 10-4 2.6 10-4 1/sec
dG degradation of mature sfGFP 19 10-2 19 10-2 1/sec
p(t) inefficiency in transcription caused by atennuator 0.18 0.18 1/sec
dM Degradation rate of mRNA sfGFP 5.9 10-4 5.9 10-4 1/sec
N number of tandem repressors(single or double) 1 2 -

In IFFL, the stabilization of the system is demonstrated for both cases with single or double repressor. The choice to use the double repressor is regardless of whether the result obtained in both is a stable sfGFP expression, is the fact that for N = 2 it reaches faster to steady-state and at a lower level of expression than N = 1.

As can be seen from the diagram

Figure 2. The blue line shows the concentration (nM) of mature sfGFP over a time (sec) with single-repressor . The red line shows change in concentration (nM) of mature sfGFP over a time (sec) with double-repressor.
REFERENCES
[1]Hu, C., Takahashi, M., Zhang, Y. and Lucks, J. (2018). Engineering a Functional Small RNA Negative Autoregulation Network with Model-Guided Design. ACS Synthetic Biology, 7(6), pp.1507-1518.