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

Revision as of 20:41, 1 November 2017

Overview

While in previous sections we have demonstrated a change in gene expression speed, recall that in our model the steady state value concentration of a given protein is given as the protein's production rate divided by its degradation rate. This means that while we can use degradation to increase the speed of gene expression, at the same time we are inherently also decreasing the protein's steady-state value. While some applications of genetic circuits may only be concerned with a gene’s expression as a binary on or off signal, we wanted our system to provide a general solution, and thus we needed a way to change gene expression speed while still maintaining the original steady state protein concentration.

Recall that unlike steady-state concentration, in our simple kinetic model a given gene's expression speed is defined a function of degradation rate alone. This implies that it should be possible to readjust our steady-state value back up to its original expression level by increasing protein production rate, without affecting the associated speed change. After determining that we could successfully increase gene expression speed using our characterization parts, we next decided to test whether it was actually possible to adjust the steady-state fluorescence of one of our characterization constructs back to its previous (no degradation) condition. While we anticipate that a real-world implementation of a readjustment to steady-state would probably be implemented through a change in promoter or RBS strength, we decided to our protein production parameter by using a different concentration of inducer. This is analogous to either an RBS or promoter swap because we model protein production as an aggregate of transcription and translation, and so the only parameter we actually care about is how fast protein is being created.

Results

While we previously showed that we could increase the gene expression speed of our inducible mScarlet-I constructs (Figure 1A), it is important to note that the degradation needed to create this speed increase also causes a reduction in steady state protein concentration (Figure 1b).

Figure 1: Steady-state normalized (A) or 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.

To show that it is in fact possible to maintain our degradation mediated increase in gene expression speed while also maintaining existing steady state protein concentration, we determined the protein production parameter (ATC concentration) required to return our ATC inducible pTet mScarlet pdt E characterization construct to its previous steady state protein concentration (Figure 2A) while still maintaining a gene expression speed increase (Figure 2B).

Figure 2: Measurements of the fluorescence (A) or the steady state normalized fluorescence (B) over time of BBa_K2333432 (pTet mScarlet-I pdt E), induced at 50ng/mL ATC with and without mf-Lon, and readjusted at 85ng/mL ATC with mf-Lon. Each data point represents the geometric mean of three biological replicates, with at least 10,000 cells collected for each replicate. Shaded region represents +/- geometric standard deviation. Reporter constructs were used on pSB1C3 while a pSB3K3 version of BBa_K2333434 (pLac mf-Lon) was used.

@@ Line 34: / Line 34: @@
 <div style='padding-top: 0px;'></div>
-<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>
+<div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >While in <a href='https://2017.igem.org/Team:William_and_Mary/Speed_Control' style='text-decoration: underline;'>previous sections</a> we have demonstrated a change in gene expression speed, recall that in our model the steady state value concentration of a given protein is given as the protein's production rate divided by its degradation rate. This means that while we can use degradation to increase the speed of gene expression, at the same time we are inherently also decreasing the protein's steady-state value. While some applications of genetic circuits may only be concerned with a gene’s expression as a binary on or off signal, we wanted our system to provide a general solution, and thus we needed a way to change gene expression speed while still maintaining the original steady state protein concentration.</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 style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >Recall that unlike steady-state concentration, in our simple kinetic model a given gene's expression speed is defined a function of degradation rate alone.
+This implies that it should be possible to readjust our steady-state value back up to its original expression level by increasing protein production rate, without affecting the associated speed change. After determining that we could successfully increase gene expression speed using our characterization parts, we next decided to test whether it was actually possible to adjust the steady-state fluorescence of one of our characterization constructs back to its previous (no degradation) condition. While we anticipate that a real-world implementation of a readjustment to steady-state would probably be implemented through a change in promoter or RBS strength, we decided to our protein production parameter by using a different concentration of inducer. This is analogous to either an RBS or promoter swap because we model protein production as an aggregate of transcription and translation, and so the only parameter we actually care about is how fast protein is being created.
 </div>
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 <div style='padding-top: 0px;'></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 its 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;' >While we previously showed that we could increase the gene expression speed of our inducible mScarlet-I constructs (Figure 1A), it is important to note that the degradation needed to create this speed increase also causes a reduction in steady state protein concentration (Figure 1b).</div>
 <center>
 <figure style='padding-left: px;'>
 <img src='https://static.igem.org/mediawiki/2017/6/64/T--William_and_Mary--TruncAbsFluo.png' width = "50%"/>
-<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.
+<figcaption><div style='padding-left: 20%;padding-right:20%; padding-top: 15px; color: #808080; font-size: 12px;'>Figure 1: Steady-state normalized (A) or 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;' >To show that it is in fact possible to maintain our degradation mediated increase in gene expression speed while also maintaining existing steady state protein concentration, we determined the protein production parameter (ATC concentration) required to return our  ATC inducible pTet mScarlet pdt E characterization construct to its previous steady state protein concentration (Figure 2A) while still maintaining a gene expression speed increase (Figure 2B).</div>