As IFFLs require a reporter and its inhibitor to be expressed at the same time, we needed a way to have both of these products induced at the same time. Previously, iGEM teams have found that IPTG-induced mf-Lon and ATC-induced mscarlet-I can be used together to produce a functioning IFFL circuit. This system is ideal for creating an IFFL, as using two different chemicals for induction allows for tight control of the expression parameters of both molecules. Through experimentation, this allows for the creation of a circuit with strong IFFL properties.
Design
Initial testing for our chemically inducible system
The first experiment we perused was ensuring that our chemically induced parts function as we expected. To do this, we transformed bacteria with a 3K3 plasmid containing the IPTG induced mf-Lon and a 1C3 plasmid containing the ATC induced mscarlet-I. We then tested these cells on the plate reader with a variety of induction conditions as per Figure 1.
Figure 1: Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa_K2333427 and BBa_K2333434 when grown at 37C and then chemically induced. 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.
Development of a less time consuming protocol.
Now that we verified that our mf-Lon and mScarlet-I are compatible together, we now began to screen for pulses. However, due to the long time course of our experiment, this presents a challenge for using FACS, as measurements need to be taken in regular intervals within a short time frame. In order to make experiments less labor intensive, we decided to freeze the bacteria in liquid nitrogen when the time points were taken. As can be seen in figure 2, the absolute difference in fluorescence between frozen cells vs non-frozen cells is less than 0.5%. Thus demonstrating that cells can be frozen in liquid nitrogen, and run through a FACS machine at a later, and more convenient time. See our preserving cells for flow cytometry page
Figure 2: 3 colonies of cells transformed with a plasmid containing mScarletI and were grown in m9 medium + ATC in three separate falcon tubes, thus creating three bio replicates. Time points were take every 20 minutes for 2.5 hours. At each time point, some volume of culture was either immediately ran through the FACS and had their fluorescence measured, or was pipetted into glycerol and frozen in liquid nitrogen. The cells frozen in liquid nitrogen were then stored in the -80 and had their fluorescence measured at a later time. As can be seen in the graph, there was a less than 0.5% difference in fluorescence. This is a negligible difference, considering how this modified protocol will allow more teams to be able to conduct FACS experiments.
Screening for an IFFL Pulse.
With the new FACS protocol, we now had the ability to conduct longer, and more complex experiments, although running a FACS experiment for many different circuits and inductions combinations would still be very involved and time consuming. Due to the relative ease of running a plate reader experiment, a plate of different mf-lon and mscarletI circuit creations was made and tested with different induction conditions. As can be seen in figure 3, a system of mf-Lon and mscarletI is able to produce IFFL behavior.
Figure 3: 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. 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. As can be seen, the system exhibits an IFFL pulse.
Since we were using chemical inducers, turning the circuit off posed a bit of a challenge. As we did not possess advance microfluidic technology, we would need a way to remove the chemical inducers. In order to accomplish this, we decided to centrifuge the bacteria, then wash the pellets with media. To sufficiently wash out the inducer, this step would have to repeated at least three times. As can be seen in Figure 4. we were able to demonstrate success by doing this, However, this posed a problem, as each centrifugation step requires a significant amount of time to complete. Ultimately, it was determined that having to manually centrifuge the bacteria each time we wanted to turn the IFFL on/off would be infeasible. Thus an alternate induction system would be required in order for our project to proceed.
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, the bioreplicates were split into two tubes and spun down. Half of the cells were washed with media without ATC 3 times, and the other half of the cells were washed with media with 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. The result of this experiment is that the cells washed with media without ATC had significantly lower fluorescence than those with ATC. This demonstrates that we can remove inducer media with this method.
Figure 5: Fluorescence/OD600 (AU) 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 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean. The result of this experiment is a characteristic staircase increase in fluorescence due to the IFFL behavior of the system.
References
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[2] Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A., & Shapiro, M. G. (2016). Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nature Chemical Biology, 13(1), 75-80. doi:10.1038/nchembio.2233
[3] D Ewen Cameron and James J Collins. Tunable protein degradation in bacteria. Nature biotechnology, 32(12):1276–1281, 20
[4] One-Step Cloning and Chromosomal Integration of DNA
François St-Pierre, Lun Cui, David G. Priest, Drew Endy, Ian B. Dodd, and Keith E. Shearwin
ACS Synthetic Biology 2013 2 (9), 537-541
DOI: 10.1021/sb400021j
