Team:SDU CHINA/Design
The light-control part:
The first part is light control part. With optogenetic switches, we can directly intervene the bacteria in the system from the outside without the need of adding inducer into the system, thus avoiding the issue of the residue of the inducer.In this part, bacteria receive the external light signals and transmit the signals to downstream.
We use the blue light sensitive protein YF1/FixJ and green light sensitive protein CcaS/CcaR[1]. YF1 is a histidine kinase ,and in the absence of blue light, it can phosphorylates the regulator FixJ, which proceeds to enhance the expression of genes downstream of the FixK2 promoter. But with blue light, it switches off the expression of genes downstream. However, since blue light is not conducive to bacterial growth, we added the NOT gate PhlF to the system to turn on the optocoupler and turn it off in the dark.
Green-light-sensitive proteins:
The two-component system consists of the membrane-associated histidine kinase CcaS and its response regulator CcaR[2]. The activating information stored in light is captured by phytochromes in situ. In phytochromes, a bilin-chromophore (In this case, we call it phycocyanobilin) binds at a conserved cysteine within an N-terminal GAF (cyclic GMP phosphodiesterase, adenylyl cyclase, FhlA) domain and imparts reversible photoactivation of signaling activity with maximal responses to 535 nm (green) and 672 nm (red) light. Absorption of green light increases the rate of CcaS autophosphorylation, phosphorylates CcaR by phosphate transferring, and transcript from the promoter of the phycobilisome linker protein cpcG2, while absorption of red light reverses this process.
Initially this system faces the problem of leakage. So we improve the promotor to decrease the leakage
The quorum sensing part:
The quorum sensing system is a vital communication device among the cells and is common in nature. It is of great significance in micro-ecosystem. In the system we designed, it not only plays an important role in transferring the light signals into the correct bacteria but also acts as a buffer to make the co-culture system more stable and its establishment easier.In order to transmit the light signals to two different bacteria, we design a bidirection communication system after the light sensing system. Quenching sensing is a naturally occurring mechanism that certain strains of bacteria change their population density by regulating gene expression. These bacteria secrete signalling molecules such as N-acyl homoserine lactones, or AHLs, which bind to transcription factors to alter gene expression. We use this system to convert the light signals into a specific AHL concentration, which in turn transmits the signals to each bacteria.
We chose the Las system and the Tra system provided by Dr. Jiang Wei, which have good orthogonality. In the Las system, the LasI protein produces 3OC6HSL which can binds to the LasR protein. When LasR binds to AHL, it specifically activates Plas, which in turn enhances the expression of downstream genes. In the Tra system, the EsaI protein produces 3OC12HSL[3]. TraR protein specifically binds to this AHL, which in turn initiates Ptra and expresses downstream proteins. The component has been fully characterized in the laboratory in previous studies to confirm its orthogonality. So in our experiments, we directly combined the optical element with the quorum sensing part to characterize it to confirm the feasibility of our system.
The toxin/antitoxin system:
The third part is the toxin/antitoxin system. We use CcdA/CcdB as our toxin/antitoxin moiety, which is responsible for the regulation of bacterial amounts. They have unique advantages in regulating the number of bacteria[4,5]. With the continued activation of the second part,the two bacteria in the system receives the signals, one of which produces the CcdB protein and the other produces the CcdA protein. The CcdB protein is a toxic protein that causes genomic breaks in bacteria and ultimately kills bacteria. The CcdA protein is a small peptide that can be detoxified by binding to CcdB.
The final system:
This is the final system we build. When the blue light is illuminated, the blue light system activates the Tra part of the quorum sensing part, and the resulting 3O12HSL causes a decrease in the number of bacteria1. Similarly, when the green light is illuminated, the green light system activates the Las portion of the quorum sensing system, and then produces 3OC6HSL. This leads to the decrease of bacteria 2 and the increase of bacteria 1.
We add GFP and RFP tags to the bacterial genome for differentiation. We first measure with a microplate reader
After that, in order to compare the ratio of bacteria to bacteria in the system, we used laser confocal technology to directly observe the bacterial co-culture system. The data are as follows:
Reference
[1]Fernandez-Rodriguez J, Moser F, Song M, et al. Engineering RGB color vision into Escherichia coli[J]. Nature Chemical Biology, 2017, 13(7):706-708.
[2]Schmidl, S. R., Sheth, R. U., Wu, A., & Tabor, J. J. (2014). Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems. ACS Synthetic Biology, 3(11), 820-831.
[3]Scott, S. R., & Hasty, J. (2016). Quorum Sensing Communication Modules for Microbial Consortia. ACS Synthetic Biology, 5(9), 969-977.
[4]Balagadde, F.K., et al., A synthetic Escherichia coli predator-prey ecosystem. Molecular Systems Biology, 2008. 4.
[5]You, L.C., et al., Programmed population control by cell-cell communication and regulated killing. Nature, 2004. 428(6985): p. 868-871.