Team:William and Mary/Results
Results
Construction of an Abstract IFFL Model:
Based on our understanding that incoherent feedforward loops (IFFL) could serve as temporal distinguishers, the first goal of our project was to replicate the model from Zhang, et al. (2016) [1]. We constructed an abstract kinetics based IFFL model, and found that IFFLs are indeed temporal classifiers. We found that when an IFFL was given an oscillatory input signal it will give qualitatively different outputs depending on the length input signals (Figure 1). In the presence of long input signals, an IFFL will give output pulses of a fixed height, whereas in the presence of shorter input signals it will produce a staircase like output where the output concentration increases with each new input pulse.
Figure 1: The results from our abstract IFFL model. When inputs are long (left), reporter concentration pulses at a fixed height in response to each input. When input lengths are shorter than the threshold time, each input pulse will generate a stepwise increase whose size is determined by pulse time.
The ultimate source of this behavior is the dynamics of the inhibitor; when inputs are short, the amount of inhibitor never crosses the threshold at which it efficiently degrades the output, leading to stepwise increases in the output. Conversely, when inputs are long the inhibitor crosses the threshold and reduces the concentration of the output. Importantly for our project, these results showed that IFFLs are capable of distinguishing the temporal properties of inputs, giving different outputs depending on the length of the input signal. That means that an IFFL can provide the basis for a system that is attempting to interpret dynamic, i.e. time encoded information.
Creating a Protease Based IFFL:
With the knowledge that an IFFL would provide a suitable architecture for the construction of our decoder in hand, we decided to utilize a previously characterized IFFL system that utilizes the mf-Lon protease system [2,3]. In this system the mf-Lon protease acts as an inhibitor by degrading a reporter with a protein degradation tag (pdt) (Figure 2).
Figure 2: Schematic of the dual inducible mScarlet-I-pdt mf-Lon IFFL system. Inducers act as independently tunable proxies for inputs and mf-Lon acts as an inhibitor of the reporter mScarlet-I pdt.
This is an extremely powerful and tunable system because the degradation rate constant can be tuned by using different strength pdts. By simultaneously activating the production of mf-Lon and mScarlet-I-pdt with two separate small molecule inducers, this system operates as an IFFL, with the induction of the circuit leading to the production of both the output (mScarlet-I-pdt) and its inhibitor (mf-Lon). However, by using two separate inducible systems, the production rate of the inhibitor and the output can be tuned independently of one another. Last year, work in iGEM confirmed that this system is capable of generating a pulsatile output from a continuous on signal. Our first experiment therefore was to test if we could replicate this behavior on the plate reader, which would serve the dual purpose of confirming one half of the theoretical basis of our model as well as allow us to comfortably use the plate reader for future experiments involving multiple conditions. Using the registry parts K2333434 (plLac mf-Lon) and K2333428 (ptet mScarlet-I pdt#3) we were able to successfully replicate this pulsatile behavior given a constant input (Figure 3).
Figure 3:
Testing Short Inputs:
We next wanted to confirm that this system would be capable of discerning between different length temporal inputs. However, in order to do this we would first need to be able to determine how we could dynamically change the concentration of inducer present in solution. While our first choice would have been to use a constantly flowing microfluidics chamber along with a fluorescence microscope, we were unable to due so because of the cost involved and lack of institutional experience with such devices. Instead, we developed our own protocol in which centrifugation was used to pellet cells, allowing us to wash them and replace them with fresh non inducer containing media. However, we had concerns with whether this protocol would be able to remove inducer without otherwise impacting the results of experiment. One obvious concern was that since spinning down cells takes a significant amount of time, the precision of our experiments could be compromised, given that they involve the precise timing of activation periods. Other potential issues included the impact that variable temperature and the forces of centrifugation could have on gene expression.
Because of these potential pitfalls, we recognized that our first step had to be confirming that this method is valid. In our first few trials, we had issues choosing the proper speed for the centrifuge and found that our cell density was too low to form substantial cell pellets. However, with time, we were able to improve upon our procedure, eventually coming to a protocol where we centrifuged 50 mL of cells at 2762 RCF for 3 minutes, washed with prewarmed media and then repeated and resuspended cells. When we tested if this method was effective at controlling the expression of a pTet inducible mScarlet by removing ATc, we found that it was effective. (Figure 4).
Figure 4: Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa_K2333427 and BBa_K2333434 when grown at 37C and then chemically induced with 100ng/mL ATC and 0.1mM IPTG from times 0-120. At timepoint 120, each replicate were split into two tubes and spun down. Half of the cells were washed with media without ATC, and the other half were washed with media containing ATC. Dots represent the geometric mean of 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean.
With a way to dynamically alter the input of our circuits, we next wanted to confirm the prediction that short inputs would lead to a stepwise increase in output concentration. We tested this prediction in the small molecule induced IFFL by taking single cell fluorescence measurements of an input paradigm of 40 minutes on, 40 minutes off, 40 minutes on. Using this paradigm we were able to validate the prediction of the model, showing that IFFLs do indeed function as temporal distinguishers (Figure 5).
Figure 5: Normalized (% max) fluorescence measurements of the chemically induced circuits BBa_K2333429 and BBa_K2333434 when grown at 37C and then chemically induced with 100ng/mL ATC and 0.1mM IPTG from times 0-40 and 80-120 (represented by shaded region). Dots represent the geometric mean of at least calibrated 10,000 single cell measurements of 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean.
However, when we tried to extend our results we had trouble testing more cycles of inputs and shorter input pulse durations. Often times after the second centrifugation step we would obtain a small or nonexistent size pellet of cells, resulting in us have to abort the experiment. While we considered that it might be possible to scale up the size of our culture volumes, we reasoned that for this reason as well as others, the use of the chemically inducible system would limit our ability to characterize interesting dynamic patterns of input. For more information on our tests with this system, see the chemically inducible system page.