The central goal of our project this year was to develop a better way for teams to control temporal dynamics by creating an easy to use modular gene expression speed control system. Using a protein degradation tag (pdt) based degradation system, we successfully showed that we can change the speed (time to steady state) for our characterization circuits. While the relationship between degradation and time to steady state had previously been mathematically formulated, we believe that our project represents the first reported biological confirmation of this relationship.
We then expanded on our system by successfully collaborating with University of Maryland iGEM to create a proof of concept version of their copper sensor with increased gene expression speed, and by using our system to create a dynamic circuit, the incoherent feed forward loop. We believe that we have fulfilled this medal requirement because we have successfully demonstrated that our gene expression speed control system functions both at the basal level of controlling the speed of a single gene and at the level of controlling a circuit's dynamics. Please see our other pages for more background and results. Additionally, see our medal requirements for information on how we fufilled our medal requirements.
Gene Expression Speed Control
Using a series of reporter constructs (see parts for more details), we successfully demonstrated that we could change the speed of gene expression by tuning the degradation rate (Figure 1A). The relationship between speed and degradation rate was a scaling of 1/(degradation rate), consistent with our theoretical understanding of the system (Figure 1B). We then expanded on this result and showed that, as predicted by mathematical models, we could successfully alter the gene expression speed of a given circuit while maintaining the steady state value (Figure 2).
Figure 1: A. Measurements of gene expression normalized to steady state using our ATC inducible mScarlet-I constructs. Data is shown for each construct until steady state is reached (at least two consecutive subsequent data points do not increase fluorescence). Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.
B. Comparison of calculated t1/2 vs degradation rate. Degradation rate was obtained as above, and t1/2 was defined as time at which each biological replicate's regression line reached half of steady state. The blue line is a guide to the eye that scales as to 1 / degradation rate.
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.
Collaboration
Using our pdt system, we were successfully able to show that we could in fact change the gene expression speed of University of Marylands copper sensing circuit (Figure 3). Since University of Marylands circuit uses different proteins, reporter, and different (and more toxic) inducers, we view these initial results as evidence that our system can be used to change the gene expression speed of arbitrary circuits. For more see our collaboration page.
Figure 3: Time course assay of Copper Sensor pdt parts, values are steady state normalized geometric means of of three biological replicates taken on the FL3 channel. Shaded region represents one geometric std above and below mean
Dynamic Circuit Control
Guided by math modeling, we determined that it would be possible to make an incoherant feed forward loop, using our existing circuit architecture. We determined that by using inducing our Lon and reporter simultaneously, we could generate pulse behavior from our circuit, further we demonstrated that this effect is dependent on the degradation rate, and that it does not occur in weaker tags or when the concentration of mf-Lon has already reached steady state (Figure 4).
Figure 4: The increased speed from the pdt system is preserved in the simultaneous induction case. (A) Pre-inducing Lon to steady state before the activation of mScarlet allows one to observe a faster speed to steady state, as expected. (B) The pdt-tagged circuit is faster than the untagged circuit even in the case of simultaneous Lon induction. Experimental conditions and data processing procedures are as in Figure 5.
