The Idea:

We are aiming to use modural LucCage – LucKey system designed by Baker Lab. This new class protein biosensors use de novo design principles. These biosensors function as modular molecular devices that switch from a non-luminescent state to a luminescent state upon the binding of a target molecule. The design is simplified by requiring only one target binding domain, which allows for direct readouts in solution. The sensors are highly sensitive and can detect various clinically relevant proteins and antibodies. We decided to bring the power of this biosensor to help humanity.

Our Binders:

Bacteriocins are strong, naturally-occuring bacterial toxins. Their N-terminal domain functions as a binder, while the C-terminal domain is highly toxic. Bacteriocins are species specific – we decided to use that property to selectively detect bacteria. Two bacteriocins were picked for testing based on their shape, binding properties and the bacterial species they target (see human practices tab)

Targets:

1. Escherichia coli is a highly adaptable bacteria, E. coli strains can cause a multitude of diseases including urinary tract infections, septicemia, and neonatal meningitis. Colicin E3 - the targets would be BtuB in E. coli, outer membrane cobalamin transporter  Affinity:  Kd = 0.9 ± 0.2 nM (ka = 4.5 ± 0.1 × 105 s−1 M−1; kd = 4.1 ± 0.5 × 10−4 s−1) at pH 8, 0.1 M ionic strength. This corresponds to a free energy of binding of R135 of approximately −12 kcal mol−1 

Important notes:   Because of the predominantly β-strand structure of BtuB, it was possible using far-UV circular dichroism (CD) to measure the changes in helical content of R135, caused by binding to BtuB. Upon complex formation, the helical content of R135 decreased by 12 ± 5%. 

2. Pseudomonas aeruginosa This bacterium primarily causes respiratory and urinary tract infections, particularly in immunocompromised individuals. It is also a common cause of sepsis and burn wound infections. Key Paper:   Kurisu, Genji, et al. “The Structure of BtuB with Bound Colicin E3 R-Domain Implies a Translocon.” Nature Structural & Molecular Biology, vol. 10, no. 11, Oct. 2003, pp. 948–54, https://doi.org/10.1038/nsb997.  Pyocin S2 binding the iron transporter FpvAI. Affinity: Kd = 240 pM  Deletion of the first 45 residues, including the PRR (Δ1–45 pyoS2NTD), decreased pyoS2NTD binding ∼1,000-fold.  .

Important notes:   There is some non-specific binding to removing the ß-hairpin. Direct binding of pyoS2NTD to TonB1 was shown by ITC (Kd ∼ 1 μM), as well as by cross-linking, and was abolished when the β-hairpin was deleted (Δ1–30 pyoS2NTD)  FpvAI was expressed in E. coli TNE012 cells (ompA−, ompB−, and tsx−) transformed with pNGH183 carrying the fpvAI gene from P. aeruginosa PAO1 with an E. coli ompF signal sequence. After isolation of the OM fraction, FpvAI was purified by anion exchange chromatography and SEC.  Direct binding of pyoS2NTD to TonB1 was shown by ITC (Kd ∼ 1 μM), as well as by cross-linking, and was abolished when the β-hairpin was deleted (Δ1–29 pyoS2NTD) 

To truly help where the help is needed, our biosensor must meet some key conditions:

Specificity: The biosensor should accurately identify the specific bacterial strain it is designed to detect without cross-reactivity with other bacterial species or host biomolecules. This may require the careful selection and design of proteins that undergo unique conformational changes in response to target bacterial proteins or other biomarkers.   

Sensitivity: The biosensor should be able to detect bacterial presence even at low concentrations to diagnose infections in their early stages.     

Stability: Given the harsh conditions and lack of refrigeration in low-resource settings, the biosensor should be stable at a wide range of temperatures and humidity levels. It should also be resistant to degradation from microbial contamination or other environmental factors.     

Ease of Use: The biosensor should be designed to be user-friendly, requiring minimal training for operation. Consider simple color change signals or other clear indicators of bacterial presence that can be easily interpreted.     

Affordability: To be accessible in low-resource settings, the biosensor should be designed with low-cost materials and fabrication processes. (needs concrete value, e.g. < 1 USD / test)     

Scalability: The design should be scalable for mass production and distribution without a significant increase in unit cost.     

Rapid Response: The time from sample introduction to result should be minimized to facilitate fast diagnosis and treatment.     

Portability: The biosensor should be compact and lightweight for easy transportation and use in field settings.     

Safety: The biosensor should be safe to use and dispose of, with minimal risk of biohazard or chemical hazard.     

Robustness: The biosensor should provide reliable results across a wide range of operating conditions and should not be overly sensitive to slight variations in handling or environmental conditions.