Team:Hamburg/Demonstrate
Demonstrating a working sensor
Pathogenic bacteria of the chlamydia strain have a significant negative impact on global health causing permanent disease in humans and farm animals if left untreated, with long term symptoms being infertility and irreversible loss of eyesight. The bacterial infectious disease is largely neglected and underestimated, due to a lack of symptoms in a large part of the affected populous, simplifying the spread of chlamydia over sexual transmission and smear infection – depending on the serotype.
Taz, an Tar/EnvZ fusion-protein, has been shown to detect Aspartate and subsequently activate reporter expression over the EnvZ/OmpR two-component system. Using directed mutagenesis with simulated molecular docking we created a R64K Y149S double mutant of Taz, capable of detecting meso-2,6-diaminopimelic acid (mDAP), a non-proteinogenic amino acid secreted by Chlamydia trachomatis, hereinafter called modified Taz (mTaz).
To demonstrate our receptor under working conditions, we simulated a patient sample by adding mDAP to LB broth. We incubated our diagnostic bacteria with the media for 30 minutes, followed by analysis by flow cytometry. We used Aspartate in the same experimental setup to test the receptor for specificity (Fig. 1).
Fig. 1: Flow cytometry analaysis of GFP expression induced by mDAP and aspartate. A: GFP fluorescence from diagnostic bacteria without addition of amino acid derivatives (black), addition of 10 mmol/L Aspartate (blue) and addition of 10 mmol/Lm DAP (red). B: K-S statistics indicating a significant difference between mDAP-induced expression and constitutive expression (p<0.001).
Our modified sensor worked as expected, but with a higher background activation than anticipated. It is to be noted that mDAP is still being synthesized by the detecting E. coli strain, therefore most likely also causing an interfering GFP signal.
Future Directions
We conduct that our diagnostic system is able to detect mDAP secreted by C. trachomatis but lacks sufficient specificity. This could be addressed by directed evolution of the receptor, further optimizing the planned shift of specificity.
