<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>
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<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>
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Revision as of 17:08, 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 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)
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 3: 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.
