Restricted Evolution of Gph to Rescue ΔserB E. coli through Motility Selection 公开

Li, Aaron (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/tb09j656m?locale=zh
Published

Abstract

Under selective conditions, the promiscuous catalytic functions by enzymes can often be targets for evolution. Enzymes with beneficial mutations that affect its catalytic ability with these side-reactions can evolve to become more efficient at its new function. This phenomenon has been demonstrated in auxotrophic ΔserB Escherichia coli where the phosphoglycolate phosphatase, or Gph, protein was found to be one of three proteins that rescued the phosphoserine phosphatase ability of the E. coli. However, mutation suppression is often achieved through multiple, different mechanisms and so the evolutionary extent of any particular mechanism cannot be easily elucidated. Using plates with semisolid media and chemoattractants, the auxotrophic cells with the fittest mutations were selected for through their extent of motility. Alternating between using this motility selection to evolve the Gph protein in E.coli with mutations in serine catalysis, ΔserB, and DNA repair mechanisms, ΔmutS, and then chemically transforming its gph plasmid, we demonstrate a method to specifically evolve the gph gene without accumulating mutations in the genome, thereby restricting evolution to only Gph. A custom plasmid containing the gph gene and other inducer and antibiotic genes was first constructed and transformed into ΔserB ΔmutS E. coli before evolution on the semisolid media. We also use a similar method to show the possibility of semi-continuous evolution of Gph. Instead of selecting for and transforming the plasmids of the fittest cells into new ones, we aimed to show that conjugation can be an alternative mechanism of gene transfer to evolve the gph plasmid.

Table of Contents

Table of Contents

Introduction 1

Material and Methods 3

Figure 1 9

Figure 2 9

Figure 3 10

Figure 4 10

Figure 5 11

Results 15

Figure 6 15

Figure 7 16

Figure 8 16

Figure 9 17

Figure 10 17

Discussion 19

References 24

About this Honors Thesis

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Degree
Submission
Language
  • English
Research field
关键词
Committee Chair / Thesis Advisor
Committee Members
最新修改

Primary PDF

Supplemental Files