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Evaluating Heterogeneity of Engineered B. subtilis Endospores for Time-Delayed Protein Production.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Evaluating Heterogeneity of Engineered B. subtilis Endospores for Time-Delayed Protein Production./
作者:	Tamiev, Denis D.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2021,
面頁冊數:	123 p.
附註:	Source: Dissertations Abstracts International, Volume: 83-08, Section: B.
Contained By:	Dissertations Abstracts International83-08B.
標題:	Biochemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28775123
ISBN:	9798780617396

Evaluating Heterogeneity of Engineered B. subtilis Endospores for Time-Delayed Protein Production.
Tamiev, Denis D.

Evaluating Heterogeneity of Engineered B. subtilis Endospores for Time-Delayed Protein Production. - Ann Arbor : ProQuest Dissertations & Theses, 2021 - 123 p.

Source: Dissertations Abstracts International, Volume: 83-08, Section: B.

Thesis (Ph.D.)--Iowa State University, 2021.

This item must not be sold to any third party vendors.

Sporulated bacterial cells have a potential to be a chassis for delayed recombinant protein expression. Spores have the ability to persist in a dormant state for prolonged periods of time, while withstanding adverse environments; they sense their surroundings for the return of conditions favorable to germination and recombinant protein expression. For these reasons, a well-studied, generally recognized as safe (GRASS) model organism of bacterial sporulation, Bacillus subtilis, has been previously engineered for various applications such as spore-display for oral vaccinations, seed coatings, and various biosensors. The work of this thesis hypothesized that the use of spores for reproducible recombinant protein expression is potentially at odds with the limitations imposed by the biochemical mechanisms of sporulation. Specifically, sporulation is initiated during the late stationary growth phase in nutrient deficient media which coincides with upregulation of low fidelity Y-family polymerases (PolY). Therefore, unfaithful replication of recombinant DNA can result in consecutive increase in the heterogeneity of recombinant proteins. Production of heterologous recombinant proteins decreases the reliability of spore-based devices.Our initial efforts were focused on developing methods that quantified heterogeneity of protein expression by subjecting B. subtilis cells to repeat cycles of sporulation and germination, and measuring the changes in the fluorescence signal produced by a recombinant DNA construct that carried a riboswitch followed by an RFP gene. In this study we also demonstrated that PolY knockouts of B. subtilis exhibit decreased heterogeneity in protein expression after sporulation or UV treatment making them more suitable as hosts for spore-based devices.This initial work clearly outlined a need for rapid, reliable and cost-effective methods of evaluating the effects of sporulation on recombinant protein expression. To address that need we developed a novel method that relied on a combination of our original protein activity-based method and Oxford Nanopore next generation sequencing (NGS) method for more direct and accurate quantification of protein heterogeneity.Another key roadblock in developing engineered biological systems, such as spore-based devices, is a slow turn-around cycle of transforming recombinant DNA into the host and conducting follow-on bioassays that characterize exogenous DNA. Cell Free Protein Synthesis (CFPS) is a rapid method for characterizing protein expression from recombinant DNA in a high-throughput screen. CFPS is an attractive alternative to the conventional transformation-bioassay cycles because it can produce assayable quantities of recombinant protein in under 2 hours without labor intensive DNA transformation and cell lysis. In contrast, transformation and screening of B. subtilis is considerably more labor intensive and can take over 2 days. This thesis presents our work on advancing the field of E. coli CFPS with anaerobically conditioned lysate that significantly outperforms conventional aerobic cell lysate. In addition, here we present our preliminary data on standardizing B. subtilis CFPS.Finally, this thesis presents our work on developing AI-enabled high-content imaging bioassays for rapid quality control and characterization of recombinant DNA transformed in B. subtilis. Our proof-of-concept system uses a deep learning network to process fluorescence microscope images of B. subtilis to classify bacterial biofilm clusters.In summary, this thesis advances the field of synthetic biology by improving our understanding of the mechanisms of bacterial sporulation and characterizing the limitations of spore-based devices. In addition, this work also advances the methods for rapid screening of recombinant DNA with Cell Free Protein Synthesis. Specifically, we developed anaerobically conditioned lysate capable of using alternative terminal electron acceptors (nitrate) that is significantly more productive than the conventional CFPS lysates when screening recombinant DNA in oxygen limited conditions such as sealed microwell plates. Finally, this thesis also presents our contributions to the field of high content imaging with the implementation of convolutional neural networks for bacterial biofilm analysis.

ISBN: 9798780617396Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Bacillus subtilis

Evaluating Heterogeneity of Engineered B. subtilis Endospores for Time-Delayed Protein Production.
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245 1 0 $a Evaluating Heterogeneity of Engineered B. subtilis Endospores for Time-Delayed Protein Production.
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300 $a 123 p.
500 $a Source: Dissertations Abstracts International, Volume: 83-08, Section: B.
500 $a Advisor: Reuel, Nigel;Honzatko, Richard.
502 $a Thesis (Ph.D.)--Iowa State University, 2021.
506 $a This item must not be sold to any third party vendors.
520 $a Sporulated bacterial cells have a potential to be a chassis for delayed recombinant protein expression. Spores have the ability to persist in a dormant state for prolonged periods of time, while withstanding adverse environments; they sense their surroundings for the return of conditions favorable to germination and recombinant protein expression. For these reasons, a well-studied, generally recognized as safe (GRASS) model organism of bacterial sporulation, Bacillus subtilis, has been previously engineered for various applications such as spore-display for oral vaccinations, seed coatings, and various biosensors. The work of this thesis hypothesized that the use of spores for reproducible recombinant protein expression is potentially at odds with the limitations imposed by the biochemical mechanisms of sporulation. Specifically, sporulation is initiated during the late stationary growth phase in nutrient deficient media which coincides with upregulation of low fidelity Y-family polymerases (PolY). Therefore, unfaithful replication of recombinant DNA can result in consecutive increase in the heterogeneity of recombinant proteins. Production of heterologous recombinant proteins decreases the reliability of spore-based devices.Our initial efforts were focused on developing methods that quantified heterogeneity of protein expression by subjecting B. subtilis cells to repeat cycles of sporulation and germination, and measuring the changes in the fluorescence signal produced by a recombinant DNA construct that carried a riboswitch followed by an RFP gene. In this study we also demonstrated that PolY knockouts of B. subtilis exhibit decreased heterogeneity in protein expression after sporulation or UV treatment making them more suitable as hosts for spore-based devices.This initial work clearly outlined a need for rapid, reliable and cost-effective methods of evaluating the effects of sporulation on recombinant protein expression. To address that need we developed a novel method that relied on a combination of our original protein activity-based method and Oxford Nanopore next generation sequencing (NGS) method for more direct and accurate quantification of protein heterogeneity.Another key roadblock in developing engineered biological systems, such as spore-based devices, is a slow turn-around cycle of transforming recombinant DNA into the host and conducting follow-on bioassays that characterize exogenous DNA. Cell Free Protein Synthesis (CFPS) is a rapid method for characterizing protein expression from recombinant DNA in a high-throughput screen. CFPS is an attractive alternative to the conventional transformation-bioassay cycles because it can produce assayable quantities of recombinant protein in under 2 hours without labor intensive DNA transformation and cell lysis. In contrast, transformation and screening of B. subtilis is considerably more labor intensive and can take over 2 days. This thesis presents our work on advancing the field of E. coli CFPS with anaerobically conditioned lysate that significantly outperforms conventional aerobic cell lysate. In addition, here we present our preliminary data on standardizing B. subtilis CFPS.Finally, this thesis presents our work on developing AI-enabled high-content imaging bioassays for rapid quality control and characterization of recombinant DNA transformed in B. subtilis. Our proof-of-concept system uses a deep learning network to process fluorescence microscope images of B. subtilis to classify bacterial biofilm clusters.In summary, this thesis advances the field of synthetic biology by improving our understanding of the mechanisms of bacterial sporulation and characterizing the limitations of spore-based devices. In addition, this work also advances the methods for rapid screening of recombinant DNA with Cell Free Protein Synthesis. Specifically, we developed anaerobically conditioned lysate capable of using alternative terminal electron acceptors (nitrate) that is significantly more productive than the conventional CFPS lysates when screening recombinant DNA in oxygen limited conditions such as sealed microwell plates. Finally, this thesis also presents our contributions to the field of high content imaging with the implementation of convolutional neural networks for bacterial biofilm analysis.
590 $a School code: 0097.
650 4 $a Biochemistry. $3 518028
650 4 $a Bioengineering. $3 657580
653 $a Bacillus subtilis
653 $a Cell free protein synthesis
653 $a Endospores
653 $a Spore-devices
690 $a 0487
690 $a 0202
710 2 $a Iowa State University. $b Biochem, Biophysics, and Molecular Biology. $3 3688747
773 0 $t Dissertations Abstracts International $g 83-08B.
790 $a 0097
791 $a Ph.D.
792 $a 2021
793 $a English
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