The characterization of KR showed that this protein can be used to control the number of living cells in a liquid culture using light. Indeed, we demonstrated,using (BBa_K1141001), that bacteria expressing KR can grow in the dark and are killed when illuminated. In addition, we showed that the number of viable bacteria can be stabilized at different values, using different light intensity functions predicted by our mathematical model (see here).
Our next goal was to find a way to fully automate the control of living cell density. The need to introduce IPTG into the culture was a problem that prevented our system from being fully autonomous. Automated addition of chemicals in the culture could have been a solution, but would have required using a micro pump, controlled via a computer. This approach also raised technical issues, such as the need for a reservoir containing an IPTG solution, and was consequently dropped. Using a constitutive promoter to trigger KR expression inside the cells was also initially considered. However, our KR characterization showed that KR levels had to be high enough to enable cell killing upon illumination, but had to stay below a threshold value due to its intrinsic cytotoxicity (see Determination of an optimal IPTG concentration). Thus, we decided to stick to an inducible KR expression system.
Since we were already using light for cell killing, we looked for a way to kill two birds with one stone and control KR expression with light as well. Indeed, in this approach, cell-machine communication could be mediated with light only and make our device much simpler, all the while its full utility. One important consideration was the wavelength at which the expression of the KR gene had to be induced: indeed, to produce KR without triggering its photoactivation, we had to avoid using a sensor that responded to green light. Thus we looked for a red light-inducible gene expression system. Researching the literature led to an optogenetic system that had been widely used during previous editions of the iGEM competition: the Cph8/PCB/OmpC/pompC red light-sensitive transcription system [1].
The Cph8/PCB/OmpC/pompC red-sensitive gene expression system was designed in the Voigt lab in 2010 (University of San Francisco, CA, USA). It is based on 2 switchable cyanobacterial phytochromes, named CcaS and Cph8. CcaS corresponds to a green light sensor and can be activated at 535 nm or deactivated at 672 nm. Cph8 corresponds to a red light sensor and can be activated at 705 nm or deactivated at 650 nm. These features allow control of the expression of two genes at different wavelengths. We figured that we could trigger KR expression using the red sensor and KR degradation using the green sensor, thus enabling us to fine-tune the concentration of KR in E.coli.
Voigt designed and constructed three plasmids to implement his optogenetic gene expression control system:
Figure 1.
Schematic representation of the engineered two-color light induction system.