Team:William and Mary/Chemical
Chemically Inducible Systems
Background
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.
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_K2333428 (pTet mscarlet-I-pdt#3) and BBa_K2333434 (pLac mf-Lon) 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%. This is a negligible difference, considering how this modified protocol will allow more teams to be able to conduct FACS experiments. 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.
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 pulse, indicating IFFL behavior.
Figure 3: Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa_K2333428 (pTet mscarlet-I-pdt#3) and BBa_K2333434 (pLac mf-Lon) 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.
Developing a Method to Remove Inducer.
Since we were using chemical inducers, turning the circuit off posed a challenge. As we do not possess advanced microfluidic technology, we would need a different way to remove chemical inducers from media. In order to accomplish this, we decided to centrifuge the bacterial cultures, then wash the pellets with fresh media and resuspend. We found that a volume of 50 mL of cells worked best with a speed of 2762 RCF for 3 minutes. As can be seen in Figure 5, we were able to show 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 4: Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa_K2333428 (pTet mscarlet-I-pdt#3) and BBa_K2333434 (pLac mf-Lon) 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 of 50 mL each and and spun at 2762 RCF for 3 minutes, then washed and resuspended with prewarmed media. Half of the cells were washed with media without ATc, 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.
Demonstrating a Staircase Pulse From IFFL Activity.
With the new method to remove inducer from media, and the identification of IFFL parameters, we were able to demonstrate a stepwise pulse. As can be seen in Figure 5, we conducted an experiment to test for IFFL stepwise behavior. The result of this experiment was a characteristic staircase increase in fluorescence due to the IFFL behavior of the system. This is significant as this demonstrates the system we develop would be able to decode dynamic information via a stepwise increase in fluorescence.
Figure 5: Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa_K2333430 (pTet mscarlet-I-pdt#3b) and BBa_K2333434 (pLac mf-Lon) 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.
Limitations of the Chemically Induced System.
While we were able to obtain promising results with the staircase behavior of our IFFL system, the chemical induction system came with significant drawbacks. The primary issue is that the protocol to remove inducer from media was extremely time consuming. Having to spin and wash each bioreplicate 3 times is very time consuming. Additionally, given the nature of the dynamic system we intended our IFFL system to work in, during the time spent carrying out the protocol, the dynamics of the system could change rapidly. Due to this significant limitation, we decided that we would need a new system of gene control. Ultimately, we chose to use a heat inducible system. See our Temperature Inducible Systems page to learn about what systems we moved to.
References