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Single cell measurements in microfluidic chip to determine the dynamics of transcription under induction

Diemer, Jascha (2019)
Single cell measurements in microfluidic chip to determine the dynamics of transcription under induction.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Single cell experiments require a system that is capable of collecting signals on the scale of a cell. Merging a microfluidic system with a microscopic setup opens up the possibility to collect data on the single cell level in a controlled manner. A microfluidic chip can both separate and trap cells with the right design. The so called “cell traps” are perfectly suited to observe the organism Saccharomyces cerevisiae over long time periods. This work contributes to the design of traps and their fabrication process. In order to reduce the complexity, two layers of the layout are combined into one. The overall orientation of the chip is reduced in size to fit multiple copies onto one device. In a first drawing, the total number of cell chambers could be increased from one to five, in a second iteration even further to twelve. Furthermore, different designs are used for different purposes. One design enables the simultaneous measurement of up to four yeast strains with the same environment and conditions. Another design allows to image again four chambers, but with different media compositions. Both designs were planned to reveal variations between the strains and conditions used. In addition, the structure of the cell trap was optimized for the fabrication devices, as those could not be generated with the same quality. A big oval shape seemed to solve the problems as it has no sharp angles and a big surface to be washed out. All other shapes of traps could only be produced with the use of a laser direct writer. Thereby, differences in the catch rate could be observed. The L-formed traps had the highest catch rate with 90%. The drawback is the limited number of traps that can be placed in close proximity. Smaller trap designs could catch higher numbers of cells as they were more densely packed. In two collaborations both chip designs could be used successfully. The characteriza- tion of a genetic logic gate based on two aptamers was performed with the same yeast strain under four different conditions: (I) positive control with inducer of gene expres- sion, (II) exposure of cells with neomycin after 2 h of induction, (III) cells were exposed to tetracycline and (IV) cells had both ligands in the media. The increase in GFP signal generated from cells with at least one repressor was stopped after 70 min. The second collaboration was about a light-sensitive potassium channel. Here, different strains with alternative DNA sequences for the GFP-tagged channel have been compared. The chip with four chambers for parallel experiment was best suited to keep the media as well as the light conditions identical. Interestingly, the non-codon optimized strain showed the best signal. The temporal development of the relative signal was in all strains the same. In the microscope images the localization of the channel in the ER membrane was visible. Transcription of mRNA is a stochastic process. So called bursts give rise to the mRNA distribution in the cell. In the case of stimulus dependent transcription, the parameters of the bursting are subject of change. This work investigates the question, which parameters change based on the intensity of activation.

The genetic background of the yeast cell line to perform measurements on the tran- scription dynamics consists of two parts: a PP7-based system to tag mRNA directly during synthesis and the GEV transcription factor to respond to β-estradiol. The coat protein of PP7 recognizes a stem loop in the mRNA and strongly binds this motif. Mul- tiple stem loops in repetition lead to an accumulation of PP7-GFP on the mRNA, which is visible as a spot in the nucleus. The duration and intensity are directly related to the transcription dynamics. The GEV transcription factor consists of a Gal4 DNA binding do- main, an estrogen receptor and a viral activation domain. GEV relocates into the nucleus during induction and activates gene expression of Gal1 and Gal10, when β-estradiol is present. As expected, the number of cells that respond to the input rose with higher concentrations of the inducer. Interestingly, the number of responders did not reach a plateau, when induced with 500 nM over 4 h, but continuously increased over time. This finding might be explained by an increased re-initiation rate of polymerases. As GEV gets accumulated in the nucleus, it is more likely to observe an actively transcribing cell. The number of mRNA synthesized is not correlated with the induction dose. Once tran- scription is started, the cell produces a number of mRNA molecules following the same distribution for any β-estradiol concentration. In addition, a GC-rich sequence should give knowledge about the influence of DNA template properties on the elongation rate. As the GC pair forms a stronger bond, it can slow down polymerases as indicated by literature. During the design of a suitable fragment, the melting temperature was chosen to be constant for a window of 14 base pairs. Although the cloning of such a construct was successful, the integration into the yeast genome was not. Furthermore, the plasmid had repeats of another coat protein, MS2, in the 3’ UTR. Such a dual tagging strategy would have simplified the determination of the elongation speed. In conclusion, the optimizations of the chip design reduced the waste and increased the number of usable chips after one fabrication run. All tested traps were able to catch and keep cells for longer time periods. Different designs had different advantages and disadvantages. The chips could be utilized in three projects. The main results were: the neomycin-tetracycline gate responds to both repressors; the light-inducible potassium channel relocates into the ER membrane and the number of responding cells increases with higher concentration of β-estradiol.

Typ des Eintrags: Dissertation
Erschienen: 2019
Autor(en): Diemer, Jascha
Art des Eintrags: Erstveröffentlichung
Titel: Single cell measurements in microfluidic chip to determine the dynamics of transcription under induction
Sprache: Englisch
Referenten: Koeppl, Professor Heinz ; Süß, Professor Beatrix
Publikationsjahr: Januar 2019
Ort: Darmstadt
Datum der mündlichen Prüfung: 20 Dezember 2018
URL / URN: https://tuprints.ulb.tu-darmstadt.de/8345
Kurzbeschreibung (Abstract):

Single cell experiments require a system that is capable of collecting signals on the scale of a cell. Merging a microfluidic system with a microscopic setup opens up the possibility to collect data on the single cell level in a controlled manner. A microfluidic chip can both separate and trap cells with the right design. The so called “cell traps” are perfectly suited to observe the organism Saccharomyces cerevisiae over long time periods. This work contributes to the design of traps and their fabrication process. In order to reduce the complexity, two layers of the layout are combined into one. The overall orientation of the chip is reduced in size to fit multiple copies onto one device. In a first drawing, the total number of cell chambers could be increased from one to five, in a second iteration even further to twelve. Furthermore, different designs are used for different purposes. One design enables the simultaneous measurement of up to four yeast strains with the same environment and conditions. Another design allows to image again four chambers, but with different media compositions. Both designs were planned to reveal variations between the strains and conditions used. In addition, the structure of the cell trap was optimized for the fabrication devices, as those could not be generated with the same quality. A big oval shape seemed to solve the problems as it has no sharp angles and a big surface to be washed out. All other shapes of traps could only be produced with the use of a laser direct writer. Thereby, differences in the catch rate could be observed. The L-formed traps had the highest catch rate with 90%. The drawback is the limited number of traps that can be placed in close proximity. Smaller trap designs could catch higher numbers of cells as they were more densely packed. In two collaborations both chip designs could be used successfully. The characteriza- tion of a genetic logic gate based on two aptamers was performed with the same yeast strain under four different conditions: (I) positive control with inducer of gene expres- sion, (II) exposure of cells with neomycin after 2 h of induction, (III) cells were exposed to tetracycline and (IV) cells had both ligands in the media. The increase in GFP signal generated from cells with at least one repressor was stopped after 70 min. The second collaboration was about a light-sensitive potassium channel. Here, different strains with alternative DNA sequences for the GFP-tagged channel have been compared. The chip with four chambers for parallel experiment was best suited to keep the media as well as the light conditions identical. Interestingly, the non-codon optimized strain showed the best signal. The temporal development of the relative signal was in all strains the same. In the microscope images the localization of the channel in the ER membrane was visible. Transcription of mRNA is a stochastic process. So called bursts give rise to the mRNA distribution in the cell. In the case of stimulus dependent transcription, the parameters of the bursting are subject of change. This work investigates the question, which parameters change based on the intensity of activation.

The genetic background of the yeast cell line to perform measurements on the tran- scription dynamics consists of two parts: a PP7-based system to tag mRNA directly during synthesis and the GEV transcription factor to respond to β-estradiol. The coat protein of PP7 recognizes a stem loop in the mRNA and strongly binds this motif. Mul- tiple stem loops in repetition lead to an accumulation of PP7-GFP on the mRNA, which is visible as a spot in the nucleus. The duration and intensity are directly related to the transcription dynamics. The GEV transcription factor consists of a Gal4 DNA binding do- main, an estrogen receptor and a viral activation domain. GEV relocates into the nucleus during induction and activates gene expression of Gal1 and Gal10, when β-estradiol is present. As expected, the number of cells that respond to the input rose with higher concentrations of the inducer. Interestingly, the number of responders did not reach a plateau, when induced with 500 nM over 4 h, but continuously increased over time. This finding might be explained by an increased re-initiation rate of polymerases. As GEV gets accumulated in the nucleus, it is more likely to observe an actively transcribing cell. The number of mRNA synthesized is not correlated with the induction dose. Once tran- scription is started, the cell produces a number of mRNA molecules following the same distribution for any β-estradiol concentration. In addition, a GC-rich sequence should give knowledge about the influence of DNA template properties on the elongation rate. As the GC pair forms a stronger bond, it can slow down polymerases as indicated by literature. During the design of a suitable fragment, the melting temperature was chosen to be constant for a window of 14 base pairs. Although the cloning of such a construct was successful, the integration into the yeast genome was not. Furthermore, the plasmid had repeats of another coat protein, MS2, in the 3’ UTR. Such a dual tagging strategy would have simplified the determination of the elongation speed. In conclusion, the optimizations of the chip design reduced the waste and increased the number of usable chips after one fabrication run. All tested traps were able to catch and keep cells for longer time periods. Different designs had different advantages and disadvantages. The chips could be utilized in three projects. The main results were: the neomycin-tetracycline gate responds to both repressors; the light-inducible potassium channel relocates into the ER membrane and the number of responding cells increases with higher concentration of β-estradiol.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Mikrofluidische Chips eignen sich hervorragend für Einzelzellmessungen. Spezielle De- signs ermöglichen das Separieren und Einfangen von einzelnen Saccharomyces cere- visiae. Das Mikroskop als leistungsstarkes Instrument zur Bestimmung von temporalen und spartialen Abläufen gewährleistet die Aufnahme von relevanten Informationen. Meine Arbeit wird durch zwei Themen motiviert. Zum einen wird ein mikrofluidischer Chip weiterentwickelt, welcher einzelne Zellen einfängt um langandauernde Experimente unter gleichbleibenden Bedingungen zu erlauben. Zum anderen wird untersucht, wie sich die Transkriptionsdynamik unter Induktion verändert. Zunächst zum Chip: Die erste Verbesserung betrifft eine wesentliche Verkleinerung des Layouts. Nun finden bis zu fünf Zellobservationskammern auf einem Chip Platz. Zudem wird die Herstellung erleichtert indem zwei von vier Masken zu einer zusammen- gelegt werden. Drei verschiedene Layouts werden entworfen, um entsprechende Experi- mente durchführen zu können. Das erste Design bietet Platz für vier unterschiedliche Hefestämme, die in einem Experiment verglichen werden sollen. Das zweite hat vier eng aneinanderliegende Zellkammern in denen unabhängige Messungen in einem Lauf aufgenommen werden können. Das letzte Design erhöht vornehmlich die Wiederverwert- barkeit eines Chips mit fünf Kammern. Die zweite Optimierung steigert nicht nur die Produktionsrate, sondern testet auch an- dere Formen der Zellfallen. Die Platznutung wird weiter verbessert und die Chips bieten nun zwischen sieben und zwöf Kammern. Da die Herstellung mit den bisherigen Fallen an ihre Grenze stößt, werden neben bereits publizierten Fallen auch ovale Formen mit erhöhter Fläche entworfen und getestet. Hierbei zeigt sich, dass nur die größten Fallen mit gleichbleibender Qualität hergestellt werden können. Erst mit der Verwendung eines Laser-Direkt-Writers kann die Fabrikation mit allen Formen vollendet werden. Die aus der Literatur bekannten L-förmigen Fallen weisen die beste Fangrate (90%) auf. Kleinere Fallen haben geringere Abstände und kommen trotz 70%iger Fangrate auf die gleiche Anzahl an aufgenommenen Zellen. Die Kooperation mit Dr. Christopher Schneider (AG Süß) kann auf den Chip mit vier unabhängigen Kammern zurückgreifen. Als genetischer Schalter wird ein Aptamer kon- struiert, welcher sowohl auf Neomycin als auch auf Tetrazyklin die Translation inhibiert (yCS). In den vier Kammern können daher zeitgleich die Positiv-Kontrolle, Neomycin, Tetrazyklin und die Kombination beider aufgenommen und ausgewertet werden. Dies gewährt eine Zeitersparnis von rund drei Tagen gegenüber der Verwendung des ur- sprünglichen Chips. Der genetische Schalter zeigt Wirkung: Neo und Tc reprimieren beide die Produktion von GFP 70 Minuten nach Zugabe. Der Chip mit vier parallelen Zellkammern kommt in der Kooperation mit Sebstian Höler (AG Thiel) zum Einsatz. Hierbei wird die Genexpression eines Licht-abhängigen Kaliumkanals untersucht (ySH). Mehrere verschiedene Sequenzoptimierungen werden vergleichend aufgenommen. Dabei stellt sich heraus, dass die einfache Sequenz das beste Signal liefert. Der Kaliumkanal lagert sich in allen Varianten im endoplasmatischen Retikulum ein. Der zweite Forschungsschwerpunkt meiner Dissertation dreht sich um die Transkrip- tionsdynamik. Auch hier werden einzelne Zellen unter dem Mikroskop mit dem mikroflu- idischen Chip gemessen. Die Visualisierung der Transkription in Echtzeit wird mittels dem Coat-Proteins des Phagen PP7 realisiert. Eine Stammschleife in der mRNA-Sequenz wird erkannt und durch Repititionen kann eine Akkumulation von PP7-GFP herbeigeführt werden, welche als Punkt gemessen wird. 14 solcher Stammschleifen vor dem Gal10-Gen werden mit dem Transkriptionsfaktor GEV unter Zugabe von β-Estradiol synthetisiert (yJD). Da Transkription als stochastischer Prozess großen Schwankungen unterworfen ist (sog. Bursts), ist es von Interesse, wie sich Burstintensität und -frequenz durch unter- schiedlich starke Stimulation beeinflussen lassen. Der erste Fund betrifft die Anzahl an S. cerevisiae Zellen, welche auf die Stimulation reagieren. Mit der höchsten verwendeten Konzentration von 500 nM β-Estradiol hat man die höchste Fraktion an Respondern mit ca. 45% gefunden. Zudem steigt die Anzahl über die Dauer des Experimentes stetig an, was mit einer Steigerung der Re-Initiationsrate erklärt werden kann. Die hohe Konzentra- tion an vorliegendem GEV im Nukleus führt zu einer höheren Wahrscheinlichkeit aktive Zellen zu beobachten. Entgegen der Erwartung und Literaturerkenntnisse ist dies die einzige Variable, welche mit der Dosis korreliert. Die Transkriptionsdynamik als solche bleibt in diesem Assay unberührt. Exprimiert eine Zelle das Konstrukt, so folgt die pro- duzierte mRNA Menge über alle Konzentrationen der gleichen Verteilung. In einer weiterführenden Studie soll der Einfluss des GC-Gehalts des DNA-Templates auf die Elongationsgeschwindigkeit ermittelt werden. Die Klonierung eines Plasmides mit 10 Stammschleifen für PP7, 1000 Basenpaaren einer 66% GC-haltigen Sequenz und anschließend 24 Stammschleifen des MS2 Coat-Proteins ermöglichen eine genauere Analyse der Elongationsrate durch die Aufnahme in zwei Farbkanälen. Dabei sollte zuerst PP7-GFP an die mRNA binden und den Begin des GC-haltigen Gen markieren, während MS2-RFP gegen Ende der Transkription sich an die mRNA lagert. Die Integration dieses Konstruktes ist nicht rechtzeitig vollendet worden und keine Aufnahme mit zwei Farben kann ausgewertet werden. Ich fasse die Kernpunkte meiner Thesis wie folgt zusammen: Die Optimierung des Chiplayouts hat den Herstellungsprozess vereinfacht und zu einer Steigerung der pro- duzierten Chips geführt. Die getesteten Fallenstukturen sind alle funktionsfähig und können Hefen über große Zeiträume hinweg kultivieren. Der genetische Schalter yCS lässt sich mit beiden Liganden reprimieren und der Licht-induzierbare Kaliumkanal ySH zeigt die gleiche Expressionsdynamik für unterschiedliche DNA-Sequenzen. Und die induzierte Transkription am Gal10 Lokus steigert die Rate mit der Zellen aktiv werden.

Deutsch
URN: urn:nbn:de:tuda-tuprints-83450
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
Hinterlegungsdatum: 03 Feb 2019 20:55
Letzte Änderung: 03 Feb 2019 20:55
PPN:
Referenten: Koeppl, Professor Heinz ; Süß, Professor Beatrix
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 20 Dezember 2018
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