Team:William and Mary/Readjustment
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.
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 (B) of mScarlet-I pdt constructs. Data was collected using standard time course protocol, and normalization was performed relative to steady state as described previously. For comparison, raw fluorescence data has identical time points displayed. Full data can be found on parts pages or in the other results sections. 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). After restoring the steady state protein concentration to it's no-degradation control, we still see the same increase in gene expression speed (Figure 2B). This demonstrates that we can tune gene expression speed while maintaining desired steady-state concentrations of our proteins.
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. Time courses were taken for 180 minutes without dilution. 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.