Optogenetic control of ER Ca2+ release and development of a mammalian cell-based library screening platform for directed evolution
Creators: Zhang, Shuce
Publisher: name: University of Alberta Library
Year: 2023

<p>Life on the earth’s surface is bathed in the white light of the Sun. Biologists have been harnessing light as a research tool to understand and manipulate many biological processes inside the cells. Optogenetics — genetically encoded proteins that enable optical visualization or manipulation of physiological states — not only provides useful tools to understand our cells and body, but also has the potential to create revolutionary therapies for nervous system disorders in humans. Calcium signalling is among the fields that have benefitted most from the development of optogenetic tools. Over the past few decades, many tools have been engineered to sense the Ca2+ concentration (genetically encoded calcium ion indicator, GECI) and manipulate the Ca2+ handling inside the cell (genetically encoded calcium ion actuator, GECA). However, tools that specifically facilitate the study of Ca2+ release from the endoplasmic reticulum (ER) have not been extensively engineered. On the other hand, although most optogenetic tools are developed for application in mammalian cells or organisms, it is cumbersome and inefficient to screen these tools in mammalian cells, and thus the prokaryotic systems are still heavily relied on for the directed evolution of optogenetic tools. A solution to achieve clonal expression in mammalian cells would be greatly helpful in achieving mammalian cell-based library screening and directed evolution.<br>
In ‎Chapter 1,  I will present a literature review of the methods for mammalian cell-based library screening. These methods are categorised as plasmid transfection, viral transduction, site-directed recombination, and in situ mutagenesis. Special attention is given to achieving variant separation in different cells when screening pooled libraries and the applications in engineering optogenetic tools, antibodies, membrane proteins, and other targets of interest for mammalian systems.<br>
In ‎Chapter 2, I will describe my original work of developing an optogenetic tool for light-induced ER Ca2+ release. This tool is a binary system consisting of an Orai channel rerouted to ER membrane by a C-terminal dilysine motif, and a STIM-based GECA that activates Orai in a light-dependent manner. We show that the ER-localised human Orai1 channels are functional and best activated by OptoCRAC (a LOV2 domain-based GECA) to mediate a mild ER Ca2+ release. This Ca2+ release can activate overexpressed RyR2 channels and elicit calcium oscillation via calcium-induced Ca2+ release (CICR). The binary system also has the potential of spatial targeting of Ca2+ release at ER-organelle and ER-plasma membrane (PM) junctions.<br>
In ‎Chapter 3, I will present my design of a mammalian cell-based library screening system for clonal expression. This system consists of an engineered HEK-293 FT landing pad stable cell line, and a pBAD-derived donor plasmid. With the help of Bxb1 recombinase, one molecule of the transfected donor plasmid in each cell is integrated into the genomic landing pad locus via specific irreversible attP × attB recombination, with the gene of interest (GOI) placed downstream of a mammalian promoter in the landing pad. This GOI thus becomes the only copy that is expressed in this cell. I report the creation and identification of a clone of stable cell harbouring a single copy of landing pad locus. I demonstrate that the variants of pooled plasmids are exclusively expressed in this stable cell line with overall expression efficiency of ~ 5 %. I will also present a design for a future generation of library screening system to facilitate screening of the linear donor DNA libraries.<br>
In ‎Chapter 4, I will describe my computational simulation on PhoCl1 dissociation pathway. Adaptive steered-molecular dynamics (ASMD) was used to sample the possible route of dissociation without presumptions of the dissociation direction. A possible route of dissociation and the dissociation-dependent pattern of conformational change of 201-207 loop were identified through the ASMD simulation. The structural and mechanistic insights facilitated the directed evolution of PhoCl2 variants with improved dissociation efficiency and kinetics.</p>