Development and Application of Synthetic Riboswitches as Tools to Study Bacterial Pathogenesis Pubblico
Reynoso, Colleen Knight (2012)
Abstract
In the laboratory, we require tools that allow conditional
regulation of gene
expression to facilitate the study of genes with unknown or little
understood function.
The ideal tool would provide translation of external stimuli to an
internal alteration in
gene expression for any gene to be studied. Furthermore, these
tools should be
transferrable between different species of bacteria to allow
manipulation of expression in
less understood prokaryotic species. Nature has the advantage of
billions of years of
evolution to determine efficient mechanisms for gene regulation. We
have looked to
nature for inspiration and adapted natural systems to serve
alternative purposes in the
laboratory. In this thesis we describe the development of synthetic
riboswitches as
orthogonal tools to conditionally express bacterial genes of
interest. We will explore the
concept of isolating functional riboswitches from a genetically
tractable species of
bacterial and transporting these tools with limited alteration to
less genetically tractable
species. Each chapter focuses on a different bacterial species with
relevance to different
areas of microbiology and biotechnology and presents what we have
learned regarding
the portability of synthetic riboswitches.
Table of Contents
Contents
Chapter 1:
Introduction…………………………………………………………………1
1.1 Genetic Control in Response to Environmental Stimuli
………………………….......1
1.2 Sensing the Environment with RNA-Small Molecule Interactions:
Evidence of the
RNA World
……………………………………………………………………………….3
1.3 Engineered Gene Regulation
…………………………………………………………5
1.4 References
……………………………………………………………………...……14
Chapter 2: Observing Assembly of a Magnetosome protein in
Magnetospirillum
magneticum Using Synthetic
Riboswitches……………………………………………18
2.1
Introduction…...……………………………………………………………………...18
2.2 Results and
Discussion.……………………………………………………………...23
2.3
Conclusion...….……………………………………………………………………...24
2.4
Experimental.………………………………………………………………………...25
2.5
References….………………………………………………………………………...28
Chapter 3: Screening for Synthetic Riboswitches in Acinetobacter
baylyi …………32
3.1
Introduction…...……………………………………………………………………...32
3.2 Results and
Discussion……….……………………………………………………...34
3.3
Conclusion…………………………………………………………………………...44
3.4
Experimental…………………………………………………………………………45
3.5
References……………………………………………………………………………49
Chapter 4: Developing Synthetic Riboswitches for use in
Streptococcus
pyogenes…………………………………………………………………………………52
4.1
Introduction…...……………………………………………………………………...52
4.2 Results and
Discussion……….……………………………………………………...56
4.3
Conclusion…………………………………………………………………………...69
4.4
Experimental…………………………………………………………………………70
4.5
References……………………………………………………………………………74
Chapter 5: Intracellular Study of Genes Involved in Francisella
Pathogenesis……79
5.1
Introduction…...……………………………………………………………………...79
5.2 Results and
Discussion……….……………………………………………………...81
5.3
Conclusion……………………………………………………………………..….…93
5.4
Experimental…………………………………………………………………………96
5.5
References…………………………………………………………………………..103
Chapter 6: Conclusions
6.1 Summary and
Conclusions…………………………………………………………107
6.2
References…………………………………………………………………………..110
List of Figures
Figure 1.1 - Mechanisms of Natural Prokaryotic
Riboswitches………………………….4
Figure 1.2 - SELEX Aptamer Selection
Procedure………………………………………8
Figure 2.1 - Mechanism of translational theophylline-sensitive
synthetic riboswitch…..19
Figure 2.2 - Magnetosomes within M.
magneticum……………………………………..21
Figure 2.3 - Measure of reporter gene activity induced by
riboswitch F in E. coli……..22
Figure 2.4 - Riboswitch-mediated induction of
MamK-GFP…………………………...24
Figure 2.5 - Alignment of riboswitch F RBS with 16S rRNA of M.
magneticum……..25
Figure 3.1 - Diagram of the location of the N8 library in the
5'-UTR………………….34
Figure 3.2 - Effect of ACC mutation on the linker region of the
theophylline-inducible
riboswitch…………………………………………………….…………………………..35
Figure 3.3 - Schematic of the blue-white
screen………………………………………...36
Figure 3.4 - Hydrolysis of PNPG by
β-glucuronidase…………………………………..37
Figure 3.5 - β-glucuronidase activities for riboswitches
identified from the A. baylyi
library
screens……………………………………………………………………………39
Figure 3.6 - Proposed mechanism of A. baylyi riboswitch isolated
from library
screens……………………………………………………………………………………40
Figure 3.7 - Comparison of "switch pack" riboswitches in A. baylyi
and E. coli…….…41
Figure 3.8 - Comparison of 16S rRNA of A. baylyi and E. coli in
context of riboswitch
RBS
strength………………………………………………………………………….….43
Figure 4.1 - Streptococcus pyogenes
morphology………………………………………53
Figure 4.2 - Theophylline toxicity in S. pyogenes strain
JRS1278……………...………57
Figure 4.3 - β-glucuronidase activities for riboswitches with
and without the extended 5'-
UTR in E.
coli…………………………………………………………………………....59
Figure 4.4 - 3' end of S. pyogenes 16S rRNA aligned with the RBS of
riboswitches D and
E……………………………………………………………………………..…………...60
Figure 4.5 - Theophylline-dose-dependent increase of
β-glucuronidase activity……….62
Figure 4.6 - The extended 5'-UTR found downstream of the Psag
promoter allows for
more robust expression of
β-glucuronidase……………………………………………...63
Figure 4.7 - β-glucuronidase activity of cultures harvested at
mid exponential and
stationary phases of
growth……………………………………………………………...64
Figure 4.8 - The effects of cell lysate storage conditions on
β-glucuronidase activity…65
Figure 4.9 - β-glucuronidase activities for riboswitches D and
E in S. pyogenes……….66
Figure 4.10 - Theophylline-dose-dependent increase of
β-glucuronidase activity using
improved
protocol…………………………………………………………………….…67
Figure 4.11 - β-glucuronidase activities for several
riboswitches in S. pyogenes………68
Figure 4.12 - Alignment of S. pyogenes 16S rRNA to riboswitch
sequences…………..68
Figure 5.1 - Theophylline-dependent synthetic riboswitches
controlling lacZ in
F.
novicida………………………………………………………………………….……81
Figure 5.2 - Alignment of F. novicida 16S rRNA to riboswitch
sequences……….……82
Figure 5.3 - Dose-dependent riboswitch-mediated induction of
β-galactosidase……....83
Figure 5.4 - Riboswitch-mediated control of GFP in F. novicida
during macrophage
infection………………………………………………………………………………….84
Figure 5.5 - Riboswitch E controlling FTN_0818 in minimal
media…………………...86
Figure 5.6 - Riboswitch F controlling FTN_0818 in minimal
media…………………....87
Figure 5.7 - Riboswitch-mediated control of FTN_0818 facilitates
intracellular
replication………………………………………………………………………………..88
Figure 5.8 - Riboswitch E controlling β-galactosidase activity
in F. novicida and
F.
tularensis…………………………………………………………………………..…91
Figure 5.9 - Correlation of theophylline dose to theophylline
concentration in blood
serum…………………………………………………………………………………….94
List of Tables
Table 5.1 - Bacterial strains and plasmids used in Chapter
5…………………………...97
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