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

Once we confirmed that degradation was working reliably, and that our tags had a variety of different strength tags, we next tested whether we had control over gene expression speed. Using the ATC inducible mScarlet-I constructs from the previous section, we confirmed that we could change the gene expression speed of our constructs (Figure 3a). Further, if you plot the gene expression speed of each construct against its degradation rate then the shape of the curve resembles that of a constant/degradation, just as we would expect from our model (Figure 3B). Together, this data represents the first experimental confirmation of the relationship between gene expression speed and degradation rate.

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.

Our measurements have demonstrated that the pdt system can be used to predictably control the speed of a given gene’s expression. We then wanted to use this control over speed to obtain control over the temporal dynamics of a circuit. One of the simplest examples of a dynamical circuit is the incoherent feed forward loop (IFFL), which consists of three proteins X, Y, and Z which regulate each other such that X activates Y and Z, and Y represses Z. This circuit architecture can generate a pulsatile response upon activation of X (Figure 6).

An important dynamical property of this circuit is the pulse sharpness. There are two ways to tune this property: (1) By increasing the strength of Y -| Z, you make the damping to the lower steady state occur faster, which means the pulse is narrower. (2) By increasing the speed of X->Z, you make the rise-time to the peak faster, which makes the pulse taller. Both properties lead to sharper pulses. We realized that we would be able to construct a minimal IFFL circuit by using Lon’s proteolytic degradation of a tagged protein as the inhibition step in the circuit.

While we can schematize the Lon-based circuit to classify as an IFFL, the fact that one of the nodes in the circuit is a small-molecule inducer and that the inhibition of mScarlet by Lon is a post-translational degradation process rather than an actual inhibition of transcription, it is not immediately intuitive that our Lon-based circuit would indeed function as a canonical IFFL and generate a pulse. In order to address this issue, we simulated an ODE model of the Lon-based circuit over a wide range of parameters, and determined that the system can indeed exhibit pulsatile behavior representative of a canonical IFFL circuit (Figure 7).

We next moved to experimentally validate whether our Lon-based IFFL can generate measurable pulses in experimental conditions. We measured mScarlet-I pdtA for a 200-minute timecourse, with the Lon either induced simultaneously with the mScarlet-I pdtA or induced beforehand, allowing its concentration to reach steady state before mScarlet-I pdtA becomes activated. We should expect to see that the simultaneous induction condition will exhibit a pulse-like response, while the pre-induced Lon condition will not. Indeed, we observe that this is the case (Figure 8).

After demonstrating the functionality of our system using basic reporter constructs, we wanted to exhibit an example of a practical application for speed control. To that end, we collaborated with the University of Maryland iGEM team by creating and characterizing the speed of pdt-tagged versions of their copper detecting circuit. We then sent the tagged constructs back to them for future use. Though we were unable to perform the full range of characterization that we wanted due to time concerns, we found that we were able to achieve an increase in speed similar to that of our simple reporter circuits. This serves as a practical proof of concept, as we were successfully able to show that our ready-cloning parts could be used to increase the speed of an arbitrary genetic circuit.

Once we felt that we could understand and control gene expression speed, we next wanted to make our system more accessible to future iGEM teams. While our system is inherently easy to clone and implement, as it only consists of only a 27 amino acid residue pdt and the associated mf-Lon protease, we wanted to make it even easier to implement. With this in mind, we created a suite of ready-to-clone pdt constructs and added them to the registry. Each part contains one of our six different strength E. coli-optimized protein degradation tags with a double stop codon and a double terminator. Combining all these parts together into one construct prevents extra cloning steps, saving time, money and aggravation. In addition to the functional elements above, each construct also contains two BsaI restriction sites for Golden Gate Assembly, two Universal Nucleotide Sequences for Gibson Assembly, as well as a number of well-tested primer sequences that can be used for any other type of cloning. We also made it easy to swap and design large libraries of constructs with different speeds, by making sure that the only difference between each ready-cloning construct was a small unique region in the pdt. That means there is no need to switch primers to use a different strength pdt. Alongside our well-characterized construct Bba_K2333434 (pLac mf-Lon), these ready-to-clone parts should make it cheap and easy for future teams to test their constructs with a wide variety of different gene expression speeds, either by changing the pdt or the concentration of mf-Lon.