國立虎尾科技大學 |

Flipping the Switch : = Engineering Inverted Function in the Lactose Repressor.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Flipping the Switch :/
Reminder of title:	Engineering Inverted Function in the Lactose Repressor.
Author:	Richards, David H.
Description:	1 online resource (114 pages)
Notes:	Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.
Contained By:	Dissertation Abstracts International78-11B(E).
Subject:	Molecular biology. -
Online resource:	click for full text (PQDT)
ISBN:	9780355027976

Flipping the Switch : = Engineering Inverted Function in the Lactose Repressor.
Richards, David H.

Flipping the Switch :Engineering Inverted Function in the Lactose Repressor. - 1 online resource (114 pages)

Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.

Thesis (Ph.D.)--Yale University, 2017.

Includes bibliographical references

An organism's ability to regulate gene expression is essential for homeostasis and viability. Likewise, our ability to manipulate gene expression in the laboratory has been a driving force for biological research. The lactose repressor (Lacl) is a genetic switch that has served as the paradigm for genetic regulation and been leveraged in a wide variety of applications. Because of its popular use, several groups have worked to expand the utility of Lacl by engineering alternate ligand specificities and functions. Most notably, two groups have been able to evolve "Anti-Lac" variants that have inverted functionality. Rather than existing in a high-repressive state that allosterically changes to a low-repressive state, the Anti-Lacs occupy a low-repressive state that converts to a high-repressive state. However, since the Anti-Lacs were generated through directed evolution, we do not yet understand how the allosteric mechanism was inverted or how to rationally design Anti-Lacs.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2018

Mode of access: World Wide Web

ISBN: 9780355027976Subjects--Topical Terms:

583443
Molecular biology.
Index Terms--Genre/Form:

554714
Electronic books.

Flipping the Switch : = Engineering Inverted Function in the Lactose Repressor.
LDR:04050ntm a2200337Ki 4500 001 918827
005 20181106104112.5
006 m o u
007 cr mn||||a|a||
008 190606s2017 xx obm 000 0 eng d
020 $a 9780355027976
035 $a (MiAaPQ)AAI10632548
035 $a AAI10632548
040 $a MiAaPQ $b eng $c MiAaPQ $d NTU
100 1 $a Richards, David H. $3 1193258
245 1 0 $a Flipping the Switch : $b Engineering Inverted Function in the Lactose Repressor.
264 0 $c 2017
300 $a 1 online resource (114 pages)
336 $a text $b txt $2 rdacontent
337 $a computer $b c $2 rdamedia
338 $a online resource $b cr $2 rdacarrier
500 $a Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.
500 $a Adviser: Corey Wilson.
502 $a Thesis (Ph.D.)--Yale University, 2017.
504 $a Includes bibliographical references
520 $a An organism's ability to regulate gene expression is essential for homeostasis and viability. Likewise, our ability to manipulate gene expression in the laboratory has been a driving force for biological research. The lactose repressor (Lacl) is a genetic switch that has served as the paradigm for genetic regulation and been leveraged in a wide variety of applications. Because of its popular use, several groups have worked to expand the utility of Lacl by engineering alternate ligand specificities and functions. Most notably, two groups have been able to evolve "Anti-Lac" variants that have inverted functionality. Rather than existing in a high-repressive state that allosterically changes to a low-repressive state, the Anti-Lacs occupy a low-repressive state that converts to a high-repressive state. However, since the Anti-Lacs were generated through directed evolution, we do not yet understand how the allosteric mechanism was inverted or how to rationally design Anti-Lacs.
520 $a Our hypothesis was that impairing the ability to allosterically change conformations via a "super-repressor" mutation is a necessary intermediate step for creating Anti-Lacs. To address this, we disrupted allostery using an assortment of different super-repressor mutations. Then using random mutagenesis and phenotypic screening, we identified a new series of Anti-Lacs that stemmed from each of the different super-repressor mutants. 3- Galactosidase screens and flow cytometry were used to characterize the gene expression profiles of the Anti-Lacs by measuring the level of reporter gene repression as a function of effector concentration. We found that the Anti-Lacs possessed idiosyncratic differences in their gene expression profiles and that these profiles can be tuned by adjusting the concentration of Anti-Lac, the time after adding effector, and the oligomeric state.
520 $a The creation of Anti-Lacs from every super-repressor mutation verified that a general impairment of allostery is a pre-requisite to inverting function in Lacl. Furthermore, we have shown that there are several distinct ways to convert the super-repressor intermediate into an Anti-Lac, which in turn can lead to different ranges of gene expression and repression. Therefore, this work served as proof of concept that it is possible to customize the expression profiles of Anti-Lacs. The Anti-Lacs produced in this work can be utilized in the creation of novel genetic circuits and expression systems. We have also laid the foundation for future research to examine the molecular mechanisms of the inverted function. First, we developed a new Lacl purification method to enable potential biophysical and structural biological experiments of Anti-Lacs. Second, we have also designed and outlined a saturation mutagenesis analysis of Anti-Lacs to identify critical residues for function.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2018
538 $a Mode of access: World Wide Web
650 4 $a Molecular biology. $3 583443
655 7 $a Electronic books. $2 local $3 554714
690 $a 0307
710 2 $a ProQuest Information and Learning Co. $3 1178819
710 2 $a Yale University. $3 1178968
773 0 $t Dissertation Abstracts International $g 78-11B(E).
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10632548 $z click for full text (PQDT)