Biofabrication is the assembly of biological components such as proteins, polysaccharides and nucleic acids using biological means or mimics thereof. We exploit stimuli-responsive components and deploy device born stimuli for integration with microelectronics, creating novel functional constructs. Some of the products of biofabrication include sensor “chips” for the detection of cardiac diseases, tuberculosis, drug screening in blood etc. In these chips, a scaffold interface is used to mediate the interaction between biological and electronic components and this interface is comprised of biopolymers, among them chitosan, a polysaccharide or gelatin, a protein.<br/>In this work we utilized ‘Virus like Particles’ (VLP’s) of the tobacco mosaic virus as scaffold interface to assemble a two enzyme pathway on chips. VLP’s have been well studied and are exquisitely ordered 3D structures whose composition can be tweaked through genetic engineering approaches to display desired chemical entities. We created TMV-VLP’s wherein the TMV coat protein was engineered with N terminal Cys residues enabling formation of a monolayer on gold surfaces via an Electro-Less Deposition (ELD) process. We also engineered the coat proteins to express glutamine residues on the C-termini which project outward on assembled virions. These enable the assembly of well-ordered 3D scaffold with accessible glutamines on gold surfaces. Then, to assemble biosynthetic enzymes onto VLP’s, we used a crosslinking cofactor, microbial transglutaminase that can covalently crosslink lysine and glutamine residues. Here we reengineered biosynthetic enzymes with lysine tags (7aa), enabling directed attachment to VLPs. In our test system, we assembled a two-step enzymatic synthesis pathway onto the VLP’s using this modular protein assembly approach. That is, we individually crosslinked the two enzymes onto VLP’s and then assembled the VLP-enzyme complexes onto chips via user-specified stoichiometry to create a two enzyme pathway on gold chips. VLP’s provided significant advantages over earlier techniques in that they provide spatial control over the position of enzymes by enzymatic crosslinking the rapid, facile assembly of VLPs onto surfaces enables precise control over the stoichiometries of enzymes assembled on the chips. In this study, we showed that inherent structural advantages and self-assembly properties of VLP’s can be exploited to assemble multi-component bio-devices on chips and microfluidic devices.

Biofabrication of protein networks via tobacco mosaic virus-virus like particles (TMV-VLP): 3D scaffold interfaces

251st National Meeting (2016)

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