Difference between revisions of "Team:William and Mary/Readjustment"

Line 37: Line 37:
  
 
<div style='padding-top: 0px;'></div>
 
<div style='padding-top: 0px;'></div>
−
<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >
+
<div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >While we have demonstrated a change in gene expression speed, recall that the steady state value for protein concentration is given as the production rate divided by the degradation rate. This means that as we increase the speed of gene expression, we are also decreasing the steady-state value. While some applications of genetic circuits may only be concerned with a gene’s expression as an on or off signal, we wanted our system to affect speed while maintaining the original steady state protein concentration. </div>
−
While we have demonstrated that we can see a real speed change, you might remember that in our math section we noted that the steady state value is given as the production rate divided by the degradation rate. This means that though we are increasing the speed of gene expression, we are also decreasing the steady state value. While some applications may only care about a gene’s expression an on or off signal, and not about the magnitude of expression, we wanted our system to be usable in any system. Since in our model gene expression speed is only regulated by degradation, it should be possible to readjust our steady state value back up to its original expression. Using pdt E as an example, we measured the time to steady state with and without mf-Lon at a given ATC induction level. We then showed that by increasing the ATC concentration (increasing production rate), we can return the steady state of the protease condition to the no protease condition while maintaining the same speed change, exactly as our model predicts. </div>
+
 
−
<div style='padding-top: 15px;'></div>
+
<div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >According to our model, gene expression speed is only regulated by degradation. This implies that it should be possible to readjust our steady-state value back up to its original expression level by manipulating protein production rate, without affecting the associated speed change. Using pdt E as an example, we measured the time to steady state with and without mf-Lon at a given ATC induction level. We then showed that by increasing the ATC concentration (increasing production rate), we can return the steady state of the with-protease condition to that of the without-protease condition while maintaining the same speed change, exactly as our model predicts.  
 +
</div>
  
 
<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;'>Modeling?  
 
<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;'>Modeling?  
Line 48: Line 49:
 
<div style='background: #808080; margin: 0px 190px 20px 190px; height:1px;></div>
 
<div style='background: #808080; margin: 0px 190px 20px 190px; height:1px;></div>
 
<div style='padding-top: 0px;'></div>
 
<div style='padding-top: 0px;'></div>
 +
 +
<center>
 +
<figure style='padding-left: px;'>
 +
<img src='https://2017.igem.org/File:T--William_and_Mary--mScarlet-I_Speed_MECY_Full.png' width = "65%"/>
 +
<figcaption><div style='padding-left: 20%;padding-right:20%; padding-top: 15px; color: #808080; font-size: 12px;'>Figure 1: Raw fluorescence values of mScarlet-I pdt constructs. Each data point represents the geometric mean of three biological replicates, and the shaded region represents one geometric standard deviation above and below the mean.
 +
</div></figcaption>
 +
</figure>
 +
</center>
  
 
<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >Figure 1 shows the raw fluorescence (MECY) of our ATC inducible mScarlet-I pdt constructs. Stronger pdts (earlier letters) show decreased fluorescence due to degradation. As a proof of concept, we show that our inducible pdt F construct can have it's raw fluorescence readjusted to previous values without any loss of gene expression speed (Figure 2)</div>
 
<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >Figure 1 shows the raw fluorescence (MECY) of our ATC inducible mScarlet-I pdt constructs. Stronger pdts (earlier letters) show decreased fluorescence due to degradation. As a proof of concept, we show that our inducible pdt F construct can have it's raw fluorescence readjusted to previous values without any loss of gene expression speed (Figure 2)</div>

Revision as of 16:50, 1 November 2017


Overview
While we have demonstrated a change in gene expression speed, recall that the steady state value for protein concentration is given as the production rate divided by the degradation rate. This means that as we increase the speed of gene expression, we are also decreasing the steady-state value. While some applications of genetic circuits may only be concerned with a gene’s expression as an on or off signal, we wanted our system to affect speed while maintaining the original steady state protein concentration.
According to our model, gene expression speed is only regulated by degradation. This implies that it should be possible to readjust our steady-state value back up to its original expression level by manipulating protein production rate, without affecting the associated speed change. Using pdt E as an example, we measured the time to steady state with and without mf-Lon at a given ATC induction level. We then showed that by increasing the ATC concentration (increasing production rate), we can return the steady state of the with-protease condition to that of the without-protease condition while maintaining the same speed change, exactly as our model predicts.
Modeling?
Results
Figure 1: Raw fluorescence values of mScarlet-I pdt constructs. Each data point represents the geometric mean of three biological replicates, and the shaded region represents one geometric standard deviation above and below the mean.
Figure 1 shows the raw fluorescence (MECY) of our ATC inducible mScarlet-I pdt constructs. Stronger pdts (earlier letters) show decreased fluorescence due to degradation. As a proof of concept, we show that our inducible pdt F construct can have it's raw fluorescence readjusted to previous values without any loss of gene expression speed (Figure 2)
Temp. Figures go to left (for #1, and below for #2). Link 1 https://2017.igem.org/File:T--William_and_Mary--mScarlet-I_Speed_MECY_Full.png Figure 1: Raw fluorescence values of mScarlet-I pdt constructs. Each data point represents the geometric mean of three biological replicates, and the shaded region represents one geometric standard deviation above and below the mean Figure 2: Raw fluorescence (A) and normalized fluorescence (B) for the original (50ng/mL) vs adjusted (85ng/mL) strength mScarlet-I pdt 3E construct. Each data point represents the geometric mean of three biological replicates, and the shaded region represents one geometric standard deviation above and below the mean