Pathogen Sensing – TUDA iGEM 2021

Pathogen Sensing

A key aspect and major part of our project is a detection system for pathogens that initiates a response to kill them. This system is part of a sensing circuit in Bacillus subtilis. The detection should lead to the production of bacteriophages against the pathogen. Hence, the expression is linked to the presence of signaling molecules specific to the pathogens when they invade the biofilm, otherwise the B. subtilis cells remain silent in the biofilm. Therefore, they are called sleeper cells. As a first step and proof of concept we determined Pseudomonas aeruginosa as our main target pathogen.

Our sensing circuit is activated by a quorum sensing molecule, which is characteristic for P. aeruginosa. Therefore, an allosteric transcription factor originating from P. aeruginosa is implemented in our B. subtilis and used for our detection system.¹ The quorum sensing molecule binds to this transcription factor. Afterwards, the resulting molecule complex binds to the respective promoter and thus the production of a target protein and finally of the bacteriophages is initiated.^2–4

Overall, the detection of P. aeruginosa activates our sensing circuit, which leads to the production of bacteriophages against P. aeruginosa (Figure 1).

Figure 1. Schematic overview of our designed genetic circuit in our B. subtilis sleeper cells. The gene for the transcription factor (purple) is constitutively expressed. Signaling molecules (pink) enter a B. subtilis cell. The transcription factor binds the signaling molecule leading to the ability to bind the inducible promoter.³ This triggers the target protein production, wich then induces the phage to change from lysogenic into lytic cycle.

Background

Background of quorum sensing and acyl homoserine lactones

Circuit

Explanation of the sensing circuit in E. coli and in B. subtilis

Laboratory

Collected results and discussion of our laboratory work

1. Lequette Y, Lee J-H, Ledgham F, Lazdunski A, Greenberg EP. A Distinct QscR Regulon in the Pseudomonas aeruginosa Quorum-Sensing Circuit. Journal of Bacteriology. 2006 May:3365–3370. http://dx.doi.org/10.1128/JB.188.9.3365-3370.2006. doi:10.1128/jb.188.9.3365-3370.2006
2. Wu Y, Wang C-W, Wang D, Wei N. A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens. ACS Synthetic Biology. 2021 Jan 26:333–344. http://dx.doi.org/10.1021/acssynbio.0c00491. doi:10.1021/acssynbio.0c00491
3. Chapter One: The master elements of control. In: A Genetic Switch: Phage Lambda Revisited. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2004.
4. Vlašić I, Šimatović A, Brčić-Kostić K. Genetic Requirements for High Constitutive SOS Expression in recA730 Mutants of Escherichia coli. Journal of Bacteriology. 2011:4643–4651. http://dx.doi.org/10.1128/jb.00368-11. doi:10.1128/jb.00368-11

Team:TU Darmstadt/sensing