Surface Displayed SNAP as a New Reporter in Synthetic Biology

The field of synthetic biology has leveraged engineering tools such as molecular cloning to create new biological components, networks, and processes. While many of these components and networks have been deployed in the cytosol, there is a shortage of systems that utilize the surface of the cell. In order to address this shortcoming, we have created a synthetic, surface-displayed substrate anchor for bacteria. This approach allows us to engineer surface-based synthetic biological systems as a complement to existing intracellular approaches. We leveraged the tools of synthetic biology to display a catalytically active enzyme that covalently bonds itself to benzylguanine (BG) groups. We created a fusion protein allowing us to place human O6-alkylguanine DNA alkyltransferase (hAGT), also known as SNAP, on the surface of a bacterial cell. Initially, we used this synthetic component as a tool for spatially segregating orthogonal synthetic gene outputs by visualizing an extracellular synthetic green fluorescent reporter, SNAP-Cell® 505-Star, simultaneously with an intracellular red fluorescent protein, mCherry. Moreover, we have shown that our construct enables cells to selectively bond to BG-conjugated magnetic beads. As a result, we have demonstrated that surface displayed SNAP facilitates engineering a direct channel between intracellular gene expression and extracellular material capture. In the near future, we believe this magnetic capture can be expanded as a sortable reporter for synthetic biology as a direct extension of this work. Moreover, our work serves as an enabling technology, paving the way for extracellular synthetic biological systems that may coexist orthogonally to intracellular processes.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Biological Systems Engineering

Department dc:contributor.department

Biological Systems Engineering

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2015

Author dc:creator

• Scott, Felicia Yi Xia

Chair dc:contributor.committeechair

• Ruder, Warren Christopher

Committee members dc:contributor.committeemember

• Barone, Justin R.
• Cheng, Chao-Min

• Synthetic Biology
• Genetic Reporter
• Surface Display Proteins

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

vt_gsexam:5267

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/73915