Engineering Bacterial Optogenetic Sensors
| dc.contributor.advisor | Tabor, Jeffrey J | en_US |
| dc.creator | Ong, Nicholas Ting Xun | en_US |
| dc.date.accessioned | 2019-05-16T20:52:51Z | en_US |
| dc.date.available | 2019-05-16T20:52:51Z | en_US |
| dc.date.created | 2017-12 | en_US |
| dc.date.issued | 2017-10-02 | en_US |
| dc.date.submitted | December 2017 | en_US |
| dc.date.updated | 2019-05-16T20:52:51Z | en_US |
| dc.description.abstract | Optogenetic tools use genetically encoded photoreceptors to transduce light signals and control biological processes in the cell with unprecedented spatio-temporal precision. Our lab and other groups have previously developed UV/green, blue, green/red, red/far-red and near-infrared (NIR) photosensors in E. coli to regulate gene expression. The existing NIR photosensor can be activated by shorter red wavelengths (< 700 nm), has slow response dynamics and relies on secondary messenger signaling that can affect vital cell functions. A new optogenetic tool that has a rapid photoreversible response to longer NIR wavelengths would facilitate multiplexing with existing photoreceptors to provide dynamic control of multiple genes, and enable the remote control of bacterial gene expression in the gut microbiome as NIR light has superior penetration of tissue. Here, we engineer R. palustris BphP1-PpsR2 as a photoreversible NIR/red transcriptional regulatory tool in E. coli. We also explore other photoreceptor candidates and approaches for engineering new NIR optogenetic tools, namely the R. palustris BphP4 two-component system, and swapping a NIR-absorbing cyanobacteriochrome (CBCR) minimal photosensory domain into our existing CBCR CcaS green sensor (v2.0) in E. coli. We demonstrate that BphP1-PpsR2 PBr_crtE transcriptional output can be precisely tuned by varying NIR/red light intensities. BphP1-PpsR2 has rapid photoreversible dynamics and shows changes in gene expression within minutes. BphP1-PpsR2 is the most red-shifted bacterial optogenetic tool yet reported and is strongly activated by NIR wavelengths up to 780 nm. Unlike the previously reported NIR sensor, BphP1-PpsR2 has much quicker response dynamics and does not rely on secondary messenger signaling. Additionally, based on recent literature, we apply domain truncations to our v2.0 CcaS sensor to engineer a miniaturized v3.0 CcaS sensor that retains its green/red response. We show that our miniaturized v3.0 CcaS sensor in E. coli has an enhanced dynamic range (593-fold vs. 110-fold) and lower transcriptional output when deactivated, but shares similar light sensitivity and rapid response dynamics to those of the v2.0 sensor. In sum, BphP1-PpsR2 expands the spectral boundaries of the existing bacterial optogenetic toolkit further into the NIR region, while the v3.0 CcaS sensor provides greater utility and impact with its enhanced performance characteristics. | en_US |
| dc.format.mimetype | application/pdf | en_US |
| dc.identifier.citation | Ong, Nicholas Ting Xun. "Engineering Bacterial Optogenetic Sensors." (2017) Diss., Rice University. <a href="https://hdl.handle.net/1911/105552">https://hdl.handle.net/1911/105552</a>. | en_US |
| dc.identifier.uri | https://hdl.handle.net/1911/105552 | en_US |
| dc.language.iso | eng | en_US |
| dc.rights | Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder. | en_US |
| dc.subject | synthetic biology | en_US |
| dc.subject | optogenetics | en_US |
| dc.title | Engineering Bacterial Optogenetic Sensors | en_US |
| dc.type | Thesis | en_US |
| dc.type.material | Text | en_US |
| thesis.degree.department | Bioengineering | en_US |
| thesis.degree.discipline | Engineering | en_US |
| thesis.degree.grantor | Rice University | en_US |
| thesis.degree.level | Doctoral | en_US |
| thesis.degree.name | Doctor of Philosophy | en_US |
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