NANOFLEX: Standardized, Flexible and Accessible Cellular Biosensor

Best New Application Project of iGEM 2020

NANOFLEX is a cellular biosensor adaptable to detect your analyte of choice and generate a colorimetric output, visible by the naked eye. The First Generation of NANOFLEX achieves this through using modified DNA binding domains (DBDs) fused to nanobodies that bind specifically to an analyte of choice, binding a specific promoter, pCadBA, which induces a reporter gene. The proof of concept of the First Generation of NANOFLEX uses anti-caffeine nanobody and mRFP1 as the reporter gene, showing the system's sensitivity at low concentrations of analyte in both analytically clean and crude samples. This was also reassured by a stochastic model predicting the same pattern for the signal in different caffeine concentrations.

The Next Generation of NANOFLEX focuses on improving the signal by amplifying it and removing the background noise. Signal amplification and noise reduction modules employ Qβ viral replicase and T7 RNA polymerase systems respectively. A switch towards an enzymatic reporter, β-galactosidase, is explored for faster and stronger signals as well.

Further focus lays on enabling detection of larger analytes that cannot cross the membrane of our biosensor cell Escherichia coli (E. coli), for example proteins. Firstly, the avenue of outer cell wall deficient E. coli was explored. Secondly, modified NarX-NarL, two-component system from E. coli, fused to nanobodies was explored as the system is rewired for a gram positive host, Bacillus subtilis. This will allow detection in the extracellular media, as this organism only has a single membrane.

Protein detection was explored in several ways. A guideline on creating new nanobodies was compiled and a Molecular Dynamics (MD) pipeline was created in order to study nanobody affinity in silico. We explored detection of HSP16.3 protein, a biomarker for tuberculosis, as a potential application of NANOFLEX. An anti-HSP16.3 nanobody was run through the MD pipeline and practical implementation information was gathered via interviews with experts in the field.

NANOFLEX's potential lays within an easy-to-use format of a cellular biosensor. In order to envision potential distribution, a prototype of a distribution kit was developed and biosensing was tested after lyophilization. Read more here.