Zinc (Zn2+) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn2+ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn2+, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn2+ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design to target the currently developed cpFP Zn2+ sensors to the lumen of the endoplasmic reticulum (ER), and expand the tool kit of cpFP Zn2+ sensors by introducing the first generation of red-shifted cpFP Zn2+ sensors. We demonstrate that not only can these Zn2+ sensors be targeted to the ER, but they can functionally be used to estimate labile ER Zn2+ concentration. We also show that red-shifted cpFP Zn2+ sensors display high sensitivity for detecting labile Zn2+, similar to the green-shifted cpFP Zn2+ sensors. These discoveries add to the current knowledge of labile Zn2+ within the lumen of the ER and introduce a new sensor that allows for the observation of labile Zn2+ in cells that was previously unavailable.
","abstract_html":"<p>Zinc (Zn<sup>2+</sup>) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn<sup>2+</sup> within the cytoplasm of cells. These sensors have proven invaluable for studying Zn<sup>2+</sup>, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn<sup>2+</sup> sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design to target the currently developed cpFP Zn<sup>2+</sup> sensors to the lumen of the endoplasmic reticulum (ER), and expand the tool kit of cpFP Zn<sup>2+</sup> sensors by introducing the first generation of red-shifted cpFP Zn<sup>2+</sup> sensors. We demonstrate that not only can these Zn<sup>2+</sup> sensors be targeted to the ER, but they can functionally be used to estimate labile ER Zn<sup>2+</sup> concentration. We also show that red-shifted cpFP Zn<sup>2+</sup> sensors display high sensitivity for detecting labile Zn<sup>2+</sup>, similar to the green-shifted cpFP Zn<sup>2+</sup> sensors. These discoveries add to the current knowledge of labile Zn<sup>2+</sup> within the lumen of the ER and introduce a new sensor that allows for the observation of labile Zn<sup>2+</sup> in cells that was previously unavailable.</p>","abstract_has_math":false,"creators":["Maslar, Drew"],"institution":null,"degree_name":"M.S.","degree_level":"Masters Thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Yan Qin","Michelle Knowles","Daniel Linseman","Dinah Loerke"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-01-01T08:00:00Z","date_published":"2021-01-01T08:00:00Z","updated_at":"2026-07-24T02:03:19Z","subjects":["Endoplasmic reticulum","Fluorescent probe","Zinc","Biochemistry, Biophysics, and Structural Biology","Biology","Cell and Developmental Biology","Cell Biology","Life Sciences","Molecular Biology"],"languages":["en"],"rights":["Copyright is held by the author. User is responsible for all copyright compliance.
"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.du.edu/etd/1959","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Yan Qin","Michelle Knowles","Daniel Linseman","Dinah Loerke"]},{"key":"dc:creator","label":"Author","values":["Maslar, Drew"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2022-02-01T08:00:00Z"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Masters Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M.S."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Endoplasmic reticulum","Fluorescent probe","Zinc","Biochemistry, Biophysics, and Structural Biology","Biology","Cell and Developmental Biology","Cell Biology","Life Sciences","Molecular Biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright is held by the author. User is responsible for all copyright compliance.
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"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Development of Endoplasmic Reticulum Targeted Probes and Red Fluorescent Probes for Detecting Zinc"]}]}],"canonical_facts":{"dc:contributor":["Yan Qin","Michelle Knowles","Daniel Linseman","Dinah Loerke"],"dc:creator":["Maslar, Drew"],"dc:date.available":["2022-02-01T08:00:00Z"],"dc:description.abstract":["Zinc (Zn2+) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn2+ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn2+, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn2+ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design to target the currently developed cpFP Zn2+ sensors to the lumen of the endoplasmic reticulum (ER), and expand the tool kit of cpFP Zn2+ sensors by introducing the first generation of red-shifted cpFP Zn2+ sensors. We demonstrate that not only can these Zn2+ sensors be targeted to the ER, but they can functionally be used to estimate labile ER Zn2+ concentration. We also show that red-shifted cpFP Zn2+ sensors display high sensitivity for detecting labile Zn2+, similar to the green-shifted cpFP Zn2+ sensors. These discoveries add to the current knowledge of labile Zn2+ within the lumen of the ER and introduce a new sensor that allows for the observation of labile Zn2+ in cells that was previously unavailable.
"],"dc:format":["application/pdf"],"dc:identifier":["https://digitalcommons.du.edu/etd/1959"],"dc:language":["en"],"dc:rights":["Copyright is held by the author. User is responsible for all copyright compliance.
"],"dc:subject":["Endoplasmic reticulum","Fluorescent probe","Zinc","Biochemistry, Biophysics, and Structural Biology","Biology","Cell and Developmental Biology","Cell Biology","Life Sciences","Molecular Biology"],"dc:title":["Development of Endoplasmic Reticulum Targeted Probes and Red Fluorescent Probes for Detecting Zinc"],"thesis:degree_level":["Masters Thesis"],"thesis:degree_name":["M.S."]},"updated_at":"2026-07-24T02:03:19Z"}