Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 1063
Promoter Engineering for
Cyanobacteria
An Essential Step
HSIN-HO HUANG
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2013
ISSN 1651-6214
ISBN 978-91-554-8724-9
urn:nbn:se:uu:diva-206188
Dissertation presented at Uppsala University to be publicly examined in Polacksbacken,
Pol_2146, Lägerhyddsvägen 1, Uppsala, Friday, September 27, 2013 at 13:15 for the degree
of Doctor of Philosophy. The examination will be conducted in English.
Abstract
Huang, H.-H. 2013. Promoter Engineering for Cyanobacteria. An Essential Step. Digital
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and
Technology 1063. 58 pp. Uppsala. ISBN 978-91-554-8724-9.
Synthetic biology views a complex biological system as an ensemble in the hierarchy of parts,
devices, systems, and networks. The practice of using engineering rules such as decoupling
and standardization to understand, predict, and re-build novel biological functions from modeldriven designed genetic circuits is emphasized. It is one of the top ten technologies that
could help solving the current and potential risks in human society. Cyanobacteria have been
considered as a promising biological system in conducting oxygenic photosynthesis to convert
solar energy into reducing power, which drives biochemical reactions to assimilate and generate
chemicals for a specific purpose such as CO2 fixation, N2 fixation, bioremediation, or fuels
production. The promoter is a key biological part to construct feedback loops in genetic circuits
for a desired biological function. In this thesis, promoters that don't work in the cyanobacterium
Synechocystis PCC 6803 in terms of promoter strength, and dynamic range of gene regulation
are identified. Biological parts, such as ribosome binding sites, and reporter genes with and
without protease tags were also characterized with the home-built broad-host-range BioBrick
shuttle vector pPMQAK1. The strong L03 promoter, which can be tightly regulated in a
wide dynamic range by the foreign Tet repressor, was created through an iterative promoter
engineering cycle. The iteration cycle of DNA breathing dynamic simulations and quantification
of a reporting signal at a single-cell level should guide through the engineering process of
making promoters with intended regulatory properties. This thesis is an essential step in creating
functional promoters and it could be applied to create more diverse promoters to realize the
emphasized practices of synthetic biology to build synthetic cyanobacteria for direct fuel
production and CO2 assimilation.
Keywords: synthetic biology, cyanobacteria, promoter, engineering, TetR, DNA breathing
dynamics, transcription, regulation
Hsin-Ho Huang, Department of Chemistry - Ångström, Box 523, Uppsala University,
SE-75120 Uppsala, Sweden.
© Hsin-Ho Huang 2013
ISSN 1651-6214
ISBN 978-91-554-8724-9
urn:nbn:se:uu:diva-206188 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-206188)
What I cannot create, I do not understand.
– Richard Feynman
List of papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Huang HH, Camsund D, Lindblad P, Heidorn T. (2010). Design and
characterization of molecular tools for a Synthetic Biology approach
towards developing cyanobacterial biotechnology. Nucleic Acids
Research, 38: 2577-2593.
II
Heidorn T, Camsund D, Huang HH, Lindberg P, Oliveira P, Stensjö K,
Lindblad P. (2011). Synthetic Biology in Cyanobacteria: Engineering
and Analyzing Novel Functions. Methods in Enzymology, 497:
539-579.
III
Huang HH, Lindblad P. (2013) Wide-dynamic range promoters
engineered for cyanobacteria. Journal of Biological Engineering, 7:10.
IV
Huang HH, Seeger C, Danielson H, Lindblad P. (2013) A point
mutation downstream of the -10 promoter element does not exhibit
long-range effect on TetR binding. Manuscript.
Reprints were made with permission from the publishers.
Contents
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Synthetic biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1 Biological parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2 Genetic circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 Biological parts - Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4 Promoter engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4.1 Transcription initiation and its regulation . . . . . . . . . . . . . . . . . . . . . .
1.5 Aims and approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
10
10
11
11
14
14
14
15
2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1 Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1 The assembly of standardized biological parts . . . . . . . . . . . . . .
2.1.2 Introducing novel biological parts into cyanobacteria . .
2.1.3 Cultivation – an in-house developed photobioreactor . . .
2.1.4 Single cell measurements – flow cytometry . . . . . . . . . . . . . . . . . .
2.1.5 Molecular interactions – surface plasmon resonance . . . .
2.2 Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1 DNA breathing dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17
17
17
19
20
20
22
23
23
3
Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1 Synthetic biology and cyanobacteria (I and II) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1 Characterization of functional parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2 Characterization of non-functional parts . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Wide-dynamic-range promoters for cyanobacteria (III and IV) .
3.2.1 A few point mutations can change promoter strength
significantly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2 The L03 promoter is regulated by TetR and
anhydrotetracycline in a wide dynamic range . . . . . . . . . . . . . . .
3.2.3 The light-sensitive property of anhydrotetracycline . . . . . .
3.2.4 A point mutation downstream of the -10 promoter
element does not exhibit a long-range effect on TetR
binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.5 The L22 promoter is a non-leaky promoter . . . . . . . . . . . . . . . . . . .
3.3 Potential applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1 Achieving indirectly a wide dynamic range of
regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26
26
26
30
31
31
32
32
35
37
37
37
3.3.2
Expanding the design space of genetic circuits for a
balanced metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enabling modularity in cyanobacteria to realize a
central concept of synthetic biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Verifying the versatile Tet repressor (TetR)-regulation
system in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
More promoters for implementing a heterogenous
dynamic sensor-regulator system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3
3.3.4
3.3.5
4
Conclusions
5
Future outlook
................................................................................................
............................................................................................
Summary in Swedish
.......................................................................................
Summary in Traditional Chinese
38
38
39
40
42
43
....................................................................
46
..........................................................................................
48
........................................................................................................
50
Acknowledgements
References
38
Abbreviations
aTc
CDS
E. coli
EPBD model
EYFP
GFPmut3B
LAHG
PB model
PBD model
PCR
RBS
revTetR
RNAP
SNP
SPR
Synechocystis
TAP
TCP
tetO
TetR
TIP
TSS
anhydrotetracycline
coding DNA sequence
Escherichia coli
extended Peyrard-Bishop-Dauxois model
Enhanced Yellow Fluorescent Protein
Green Fluorescent Protein mut3B
Light-Activated Heterotrophic Growth
Peyrard-Bishop model
Peyrard-Bishop-Dauxois model
polymerase chain reaction
ribosome binding site
reverse TetR
RNA polymerase
single-nucleotide polymorphism
surface plasmon resonance
Synechocystis PCC 6803
TetR anti-inducing peptide
TetR corepressing peptide
tet operator
Tet repressor
TetR inducing peptide
transcription start site
9
1. Introduction
1.1 Challenges
The human population on this planet increases every single day. According
to a United Nations report [1], the current world population has reached 7.2
billion and is expected to increase by 1 billion over the next 12 years and hit
9.6 billion by 2050. With the population explosion, the demand of all kinds of
resources has never been higher than today. In the Global Risks 2012 report
of the World Economic Forum [2], the six most likely risks for our future
society with high impact are water supply crises, food shortage crises, extreme
volatility in energy and agriculture prices, rising greenhouse gas emissions,
failure of climate change adaption, and antibiotic-resistant bacteria.
Among all the measures for solving these multiple crises, Synthetic biology
is considered one of the top ten technologies that can provide solutions to
the current and potential risks in human society. With the engineering tools
of synthetic biology, novel biological functions could be implemented from
well-charactized biological parts to deal with the respective crisis, such as
desalination of sea water, fast accumulation of biomass, direct fuel production
from sunlight, assimilation of excess CO2 , and novel drugs. Among these,
global warming and energy shortage problems motivate the present study to
choose cyanobacteria as the subject for introducing novel biological functions
with the engineering principles of synthetic biology.
1.2 Synthetic biology
Synthetic biology is a research field that combines different sciences to understand complex natural biological systems and uses engineering to create complex artificial biological systems. It has been reviewed intensively in many
aspects such as general introductions [3, 4], applications [5], designs [6], possible utilization from natural resources [7, 8], and genetic circuits [9]. Engineering rules such as abstraction, standardization, and decoupling are applied
in studying complex biological systems [10]. Biological systems through abstraction are viewed as hierarchy structures as parts, devices, systems, and
networks. A biological part is defined as a genetically encoded object that
exhibits a biological function via DNA or via molecules such as RNA and
protein [11]. One or more parts can physically connect as a device. The function of the device is well-defined by the functions encoded in these parts from
10
the bottom of the hierarchy structure. When advancing to the next hierarchy
level, the higher-order function should be predictable from the well-defined,
and well-characterized parts and devices. Therefore, the behaviors of complex biological systems should be predictable from biological parts. A standard, set by the BioBrick foundation (http://parts.igem.org/Main_Page), uses
defined prefix and suffix sequences, which contain specific restriction sites
on the sides of a part. These are standard interfaces used to assemble parts.
The standardization makes biological parts interchangeable and reusable and
makes the assembly process reliable. The most important part of decoupling
is that the constituent parts are independent. So, there is no functional crosstalk. Hence, novel functions of artificial biological systems may be created
and synthesized from well-characterized biological parts.
1.2.1 Biological parts
Biological parts, such as promoters, ribosome binding sites, coding DNA sequences, and terminators, compose a functional device to generate a DNAencoded product, which is either an RNA or a protein (Figure 1.1). Promoters
and ribosome binding sites are crucial parts for regulation. Large collections
of standardized parts are kept in two major databases: BioBrick (iGEM registry, MIT) and BioFab (Stanford University and UC Berkeley). However, not
all of them are well-characterized, especially parts for cyanobacteria. Without
the experimental data to describe the properties of each part individually, engineering with these assembled parts is very difficult. Therefore, one aim in
my study is to characterize the most common and useful parts directly in the
unicellular cyanobacterium Synechocystis PCC 6803 (Synechocystis).
Biological systems comparing to computers or machines are different in
their constituent parts are not independent. Cellular contexts in terms of biochemical processes and signaling pathways are different from different strains
or species. The properties of the same part might vary in different cellular
contexts [3]. Instead of using existing parts tested in Escherichia coli (E. coli),
considering the effects of cellular context on the individual parts, it is necessary to develop a set of parts specifically suitable for Synechocystis. This is
the second aim in my study.
1.2.2 Genetic circuits
The successful demonstration in E. coli of re-constructed biological functions
using synthetic genetic circuits motivate us to wonder whether similar strategies could be taken in Synechocystis to understand its natural designs of e.g.
gene regulation [6]. When understanding the design principles of a biological
system, naturally, it leads to apply the same principles but with new combi11
BBa_J23101 BBa_B0034
constitutive
promoter
RBS
BBa_C0040
BBa_B0015
L promoter
RBS*
BBa_E0030
BBa_B0015
TetR-LVA
terminator
inducible
promoter
RBS
reporter
EYFP
terminator
Figure 1.1. The device for characterizing the promoters designed in the present study.
Each geometric figure represents a biological part with its specification. A short description of the part is under the labeled figure. The part with an arrow sign attached
is a promoter. The gene product of BBa_C0040 can repress the L promoter. The
promoter strength and its regulation are reported by the expression of BBa_E0030.
nations to introduce novel and predictable behaviors in the same biological
system.
Synthetic circuits are composed of well-studied parts and hence, the gene
regulation at the transcription and translation level within the circuit could be
designed to achieve a certain behavior. The engineering principles of synthetic biology provide efficient methods to construct functional biological devices which exhibit essential behaviors, for example, switching, oscillating,
and sensing. In natural biological systems, these behaviors can be observed in
e.g. the lac operon, circadian rhythm, and transcriptional signal transduction
pathways [12]. Taking these as concrete examples, a synthetic genetic circuit
re-creates a natural behavior and with the quantitative measurement data, one
can setup a mathematical model to identify the design constrain of an observed
behavior.
Toggle Switch
The design of the genetic toggle switch [13] requires any two mutually repressing networks. A simple model can predict the necessary condition for
the dynamic property, called bistability. Bistability allows the switch from one
state to another state, even after the triggering signal disappears. This allows
engineer to design a cellular memory unit for controlling cell function.
Repressilator
The design of the repressilator [14] needs three transcriptional repressor systems. It exhibits an oscillating behavior from synthetic biological parts, of
which none is from components of natural biological clocks. The model suggests that in order to be oscillating, promoters must be strong and tightly regulated and repressors must have a high turnover rate that can be achieved using a protease tag. If one wants to implement this oscillating behavior in
Synechocystis, it means that one should have three strong, tightly regulated
promoters and a protease tag for enhanced protein degradation.
12
Cascade device
The cascade device [15] has employed three commonly used promoters that
are regulated by the TetR, LacI, and cI transcription factors. This cascade device is composed of three transcriptional cascades with one, two, and three repression stages. The number of cascade stages effectively affect the sensitivity
of this device to the stimulus. More cascade stages means a higher sensitivity
to the stimulus. This design would be very useful for sensing a certain stimulus. If one want to repeat this device with the same promoters and regulators
in Synechocystis, it is impossible because these parts are non-functional in
Synechocystis as shown in the results of the present study. Therefore, creating
functional promoters through promoter engineering in the present study is essential for enabling this cascade device in Synechocystis for sensing a specific
stimulus.
Metabolator
In addition to gene expressions [14], autonomous oscillations could also be
found in metabolic systems [16]. Natural oscillators feature with integrated
oscillations of gene expressions and metabolic pathways. To construct a synthetic oscillating device which can integrate transcriptional regulation and a
metabolic pathway, a metabolator was created [17]. The central design is
to use a flux-carrying metabolic network with two interconverting metabolite pools. Metabolites are signalling molecules to regulate gene expressions.
When the flux rate exceeds a critical value, the system start to oscillate. This
is analogous to the circadian clock. The strong and tightly regulated promoters and a protease tag for enhanced protein degradation are still required but
the design of metabolator allows to integrate transcriptional regulations with a
metabolic pathway. For future application of this device in Synechocystis, one
shall select a either natural or artificial metabolic pathway and integrate with a
synthetic transcriptoinal regulation which meets the required criteria to oscillate to control the selected metabolism. Therefore, developing more promoters
regulated by different ligands in a wide dynamic range is very important.
Insulation device
Modularity is a central concept in synthetic biology [9,18]. Modularity makes
sure that the interconnected genetic circuits keep their individual property unaffected. This is very important for predicting higher-order behaviors when
assembling more and more modules together. If one cannot predict the behavior after connecting two functional modules, these is no chance to develop even more complicated system. In order to achieve modularity, an insulation device is necessary to implement between the upstream and downstream interconnected devices. The model [18] has suggested that a design
which utilizes transcription regulation needs a strong, tightly regulated promoter and an enhanced protein degradation to become an functional insulation device. For example, if one wants to interconnect two or more genetic
13
circuits in Synechocystis, an insulation device is needed and therefore, there
is the need for more diverse promoters with a strong and tightly regulation
to realize a more complicated genetic network with insulated components in
Synechocystis.
All these successful and demonstrated cases expose the indispensable need
for promoters regulated in a wide dynamic range. This is the third aim in my
study.
1.3 Biological parts - Cyanobacteria
Several aspects should be considered when choosing the biological system for.
First, solar energy has been calculated as a major promising energy source for
human society. The surface of planet Earth receives about 445 EJ of energy
from sun in one hour and this amount of energy is about 120 % of the total
global energy consumption in a year of 2010 [19]. Therefore, the biological
systems should be able to harness solar energy. The candidates being able to
use solar energy are purple bacteria, cyanobacteria, algae, and plants, which
are common in sustaining themselves by photosynthesis. Second, water is the
ubiquitous electron and proton donor for oxygenic photosynthesis. Hence,
purple bacteria are excluded because they do not use water as the electron
donor. Third, the biological system should be simple enough and fast growing. Plants are ruled out because of its complexity and relatively slow growth.
Fourth, the biological system should be possible to genetically modify and already have at least a platform of information available for engineering. Therefore, cyanobacteria (oxygen-evolving photosynthetic prokaryotes) are among
the most suitable biological system for addressing these issues.
Cyanobacteria range from unicellular to filamentous forms with the capacity to develop several cell types. Among cyanobacteria, Synechocystis was
selected. It has been intensively studied in metabolic pathways [20], a large
scale gene expression profile [21], a large scale quantitative proteomic analysis [22], genetic engineering [23], and light responses [24, 25]. Systems biology models are also available [26, 27]. This species can be further explored
using the notion of synthetic biology to introduce novel biological functions.
1.4 Promoter engineering
1.4.1 Transcription initiation and its regulation
Transcription initiation is a key step for regulating gene expression [28]. The
essential promoter elements participate in the interactions with the RNA polymerase (RNAP). A typical promoter contains from upstream to downstream:
UP element, -35 element, extended -10, -10 element, and transcription start
site (TSS) [29]. The first three are mainly for the binding of RNAP. The -10
14
element is most important element because it has two base-specific interactions with sigma factor [30], which is one of the subunits in RNAP. The sequence in between the -10 element and TSS is important for the lifetime of the
RNAP-promoter open complex [31]. Transcriptional initiation after the recognition at the -10 element by the RNAP involves the sequential steps as follows:
RNAP binding, formation of close complex RPc , formation of the bent and
wrapped close complex I1 , formation of initial open complex I2 , and, finally
formation of a stable open complex RPo [29, 32]. Upon the formation of RPo ,
the RNAP continues the initiation steps through the DNA scrunching mechanism. An obligatory stress intermediate forms as an extra unwinding DNA
during the DNA scrunching has been proposed to provide the driving force in
promoter escape [33]. The abortive/productive ratio of transcription initiation
may be influenced by the three competitive pathways. They are abortive cycle, scrunching pathway, and promoter escape and are analyzed in the kinetic
model of transcription initiation [34]. Then, the RNAP escapes the promoter
and starts the transcription elongation and termination [35].
Transcription regulation includes activation and repression [28]. Activation
requires that a transcription factor binds to its cognate site and recruits the
RNAP to start transcription initiation. Repression also needs a transcription
factor binding to its cognate site in the vicinity of the core promoter (i.e. close
to the -35 element or to the -10 element). Wen binding, it creats a steric hindrance to prevent the RNAP’s binding and therefore, repress the transcriptional
initation.
Not only proteins have active roles in the DNA-protein interactions but also
the DNA has an active role in recruiting the protein. DNA breathing dynamaics have been proposed as an another basal transcription factor in positioning and regulating transcription initiation [36]. A strong correlation between
DNA breathing dynamics and binding affinity of a transcription factor was
also proposed and verified in the examples of eukaryotic transcription factor
YY1 [37] and Fis [38]. The DNA breathing dynamics of a promoter sequence
or a regulatory sequence can be presented as the DNA opening probability
profile and a characteristic peak corresponding to the binding site can be observed. Because a strong correlation between the DNA opening probability
and the corresponding binding affinity could be qualitatively observed, the
present study utilizes the computed DNA breathing dynamics to simulate a
promoter sequence how likely to open in a certain region and compare to the
experimental data and aim for create a promoter with a desired regulatory
property.
1.5 Aims and approaches
The aims of the present study are to characterize biological parts, to identify functional parts, and to engineer parts for desired properties in the frame15
work of synthetic biology. The present thesis focus on developing parts for
cyanobacteria with a focus on promoters and the unicellular strain Synechocystis PCC 6803. Both theoretical and experimental approaches were employed
in an iteration cycle to simulate the promoter sequence with its DNA breathing dynamics and test the model prediction experimentally. In addition, the
promoters are verified with a standardized reporter construct and examined
for performance in vivo by quantification using a flow cytometer at single cell
level. Further, kinetics and affinities of the DNA-protein interactions were determined using a Biacore biosensor. This strategy creates and characterizes
selected promoters with a desired regulatory property.
16
2. Methods
2.1 Experiments
2.1.1 The assembly of standardized biological parts
Standardization
DNA sequences are fundamental parts and their assembly are very important
in synthetic biology. The standardization of biological parts is to introduce
the BioBrick prefix and suffix sequences on the sides of the part, respectively.
One can also use other standards than BioBricks. The restriction endonuclease
sites EcoRI and XbaI in the prefix and SpeI and PstI in the suffix determine
the orientation of part to connect. Therefore, the part to be standardized must
not have these restriction sites. The convenient and easy way to standardize
is a polymerase chain reaction (PCR)-based method. The forward and reverse
primers including the prefix and suffix sequences respectively amplify the part
from a DNA template.
Standard assembly
Standard assembly (Figure 2.1) [39] utilizes the compatible overhangs of XbaI
and SpeI restrictions to join two parts and the EcoRI and PstI restrictions determine the orientation of the joint parts. This method requires the purification
of each part and the destination vector.
3A assembly
3A assembly (Figure 2.2) [40] relies on the toxicity of the ccdB gene (BBa_P10101) on the host cells. It is important to choose a non-ccdB resistant strain
for assembly and to choose a ccdB resistant strain to store a vector containing
the ccdB gene. The restrictions determine the orientation of joint two parts.
After inactivation of the restriction enzymes, no purification of each digested
DNA fragments is required. After ligation and transformation, the unwanted
assembly will be eliminated by the toxicity of the ccdB gene and the correct
assembly will be selected by the selection marker of the destination vector.
Therefore, it is crucial that the selection markers of the vectors harboring the
individual part must be different from the one of the destination vector.
Gibson assembly
Gibson assembly (Figure 2.3) [41] does not rely on the ligation of restriction
sites but on the complementary sequences of the parts to be connected. Another distinction from the previous assembly methods is that this assembly
17
Figure 2.1. The standard assembly method.
Figure 2.2. The 3A assembly method.
can simultaneously assemble more than two parts. Three enzymes enable this
isothermal assembly at 50 ◦C: T5 exonuclease, Phusion polymerase, and Tag
ligase. The selection and design of overlap region is important. My experience
is that the length of the overlap region should be decided by the melting temperature (Tm ) of the overlap region. The (Tm ) should be above the isothermal
temperature of 50 ◦C, for example, around 65 ◦C to 72 ◦C. Because the parts
to be connected are prepared with a PCR-based method, the overlap sequence
will be included in the design of primers. Therefore, avoiding secondary struc18
Overlap
3'
5'
5'
3'
50 3'
5'
5'
3'
3'
5'
Chewing-back with T5 exonuclease
Phusion polymerase
3'
Taq ligase
5'
3'
5'
50 Annealing
5'
3'
Phusion polymerase
Taq ligase
T5 exonuclease
3'
5'
5' 3'
50 5'
3'
3' 5'
5'
3'
Repairing with Phusion polymerase and Taq ligase
3'
5'
Figure 2.3. The Gibson assembly. The isothermal reaction at 50 ◦C contains T5 exonuclease, Phusion polymerase, and Taq ligase for the chewing-back, annealing, and
repairing reactions simultaneously. Adapted from [41].
tures of primers which contain the partial overlap sequence is also important.
When encountering no successful assembly, the titration of T5 exonuclease
can be optimized. It is also important to use the Phusion polymerase, but not
use its Hot Start version. The hot start requires a 95 ◦C step to activate the
enzyme and there is no such temperature during the isothermal assembly.
DNA synthesis
DNA synthesis is the ultimate method to create the engineered DNA sequences.
You order the designed sequences directly from e.g. a company. However,
still, there would be a need to assemble the synthesized DNA fragments together.
2.1.2 Introducing novel biological parts into cyanobacteria
The standardization and assembly processes were done with E. coli because
of its fast growth speeding up the assembling steps. To introduce novel biological functions into cyanobacteria, the assembled DNA needs to be transformed into Synechocystis. The three major methods, natural transformation,
conjugation, and electroporation, are reviewed in Paper II. In order to avoid
the preparation of large quantities of DNA from the low-copy-number shuttle
vector from the present work, natural transformation and electroporation were
not considered. Conjugation, in addition to the E. coli harboring the assembled device in the shuttle vector, also needs the E. coli harboring the conjugal
19
plasmid and the Synechocystis cells to be transformed. By simply incubating
three different cells (Table 2.1) together, the assembled device will transfer
into Synechocystis. A selection marker, e.g. kanamycin, is used to screen for
the correct construct. Please note that ampicillin cannot effectively select the
targeted Synechocystis cells.
Table 2.1. Cells used when introducing biological parts into the unicellular cyanobacterium Synechocystis PCC 6803 through conjugation
Strain
Plasmid
Role in conjugation
E. coli DH5α
E. coli HB101
Synechocystis
cargo plasmid (pPMQAK1)
conjugal plasmid (pRL443)
none
donor cells
conjugal cells
recipient cells
2.1.3 Cultivation – an in-house developed photobioreactor
In order to simultaneously test Synechocystis cells in different growth conditions, a photobioreactor comprised of 12 tissue culture plates (6-wells) and
12 LED light modules right above each plate was developed. The modular
design allows the photobioreactor further partitioning into three chambers.
This enables simultaneously to test three light intensities, controlled by the
LED-dimmer. The LED light modules are shown in Figure 2.4. In this thesis, two kinds of LED (red and white) were used and the respective spectrum
are shown in Figure 2.5. This photobioreactor can perform batch cultivation.
The working volume of each well is up to 6mL and each plate was covered
with a lid secured by a gas-permeable surgical tape. Simultaneously, the two
photobioreactors were stacked on the horizontally orbiting shaker in the same
temperature. The light sources could have a combination in maximum of two
colors (red and white) and each color in three different intensities. Up to 144
wells can be examined in an identical shaking condition.
2.1.4 Single cell measurements – flow cytometry
Flow cytometry is the technique employing the fluidics, optic, and electronic
systems in a flow cytometer to measure the scattered light and fluorescence
from a single cell [42]. The hydrodynamic focusing allows a single cell passing through a laser beam at a given time. The optic system can illuminate cells
and detect the signals. The electronic system converts signals to results for the
data analysis by the software. The cell-to-cell variations in a population [43]
can be statistically analyzed. In general, it provides better resolution in quantifying promoter strength reported by emission level of fluorescent proteins,
especially for weak signals. The measurement were done by using a BDT M
20
Figure 2.4. The in-house developed LED light modules. Up, back side. Middle, front
side. Down, LED light in the box.
21
absorption
white LED
red LED
Figure 2.5. The absorption spectrum (solid line) of anhydrotetracycline (aTc) in BG11
medium supplemented with kanamycin and the spectra of photons emitted from white
LED (dash line) and from red LED (dot line). Adapted from Paper III.
LSRII flow cytometer controlled by the BD FACSDivaT M software. Usually,
50,000 events are collected. Obtained data were analyzed using FlowJo 7.6.5
software. The population of singlet cells was gated and the averaged emission of Enhanced Yellow Fluorescent Protein (EYFP) expressed in a cell was
analyzed.
2.1.5 Molecular interactions – surface plasmon resonance
The kinetics and affinity of the DNA-protein interactions were investigated
by surface plasmon resonance (SPR) biosensor technology. This technology
employs the physical phenomenon of SPR to monitor the interactions between
molecules in real time [44]. In the present study, the DNA fragments were
immobilized to the surface of a sensor chip via biotin-streptavidin capture.
The transcription factors were then injected over the sensor surface. When the
transcription factors bind to the DNA fragments on the surface, a SPR response
is generated and the response is directly proportional to the bound mass. A
typical sensorgram, i.e. binding curve, is illustrated in Figure Figure 2.6. The
prepared surface sets the baseline of response in the absolute unit. When the
transcription factors in the running buffer pass over the surface and interact
with the immobilized DNA fragments, an association curve is monitored in
real time. When the running buffer without the transcription factor is injected,
a dissociation curve can be observed. Before the next cycle, a regeneration
step is needed to remove remaining ligands (transcription factors) from the
target (DNA fragments) in order for the baseline to return to the level before
22
the ligand injection. In a cycle, the recorded binding was analyzed by global
non-linear regression analysis and fitting of an appropriate interaction model
in order to determine the kinetic and affinity constants of the DNA-protein
interactions.
Resonance signal (RU)
Association
Dissociation
Regeneration
0
90
180
Time (s)
Figure 2.6. A schematic sensorgram obtained from SPR based interaction analysis.
The different phases can be divided into association, dissociation and regeneration.
During the association phase, injected ligands interact with the immobilized target
and lead to an increase of the signal as a function of time. During the dissociation
phase, the ligands dissociate from the target leading to an decrease of the signal. An
optional regeneration phase completely removes remaining ligands from their target
and the signal returns to the initial baseline level. Adapted from [45].
2.2 Simulations
2.2.1 DNA breathing dynamics
The denaturing of the DNA double helix was modeled with the Peyrard-Bishop
model (PB model) [46], and the Peyrard-Bishop-Dauxois model (PBD model)
[47], and then the extended Peyrard-Bishop-Dauxois model (EPBD model)
[48]. The first two models evolves from considering hydrogen bonding first,
and then including aromatic base stacking described by sequence-independent
force constants. The third model is the extension of the second model to use
sequence-dependent force constants. The EPBD model can describe DNA
melting in a single nucleotide resolution.
In the EPBD model, the two sides (left - vn and u right - un ) of the DNA
double strand describes the transverse opening motion of the complementary
strands. The potential surface V EPBD of the EPBD model (equation 2.1) is:
23
n
VEPBD = ∑U [un ; vn ] +W [un ; un−1 ; vn ; vn−1 ]
(2.1)
2
U [un ; vn ] = Dn e−an (un −vn ) − 1
(2.2)
i=1
Kv
Ku
W [un ; un−1 ; vn ; vn−1 ] = n;n−1 (un − un−1 )2 + n;n−1 (vn − vn−1 )2 +
2
2
2
ρ −β [(un −vn )+(un−1 −vn−1 )] u
v
e
Kn;n−1 (un − un−1 ) − Kn;n−1
(vn − vn−1 )
4
(2.3)
All N base pairs in the DNA sequence were summed up. Two independent
degree of freedom,un and vn are on each base pair. They represent the relative displacement from the equilibrium of the respective nucleotide, situated
in the right or left strand of DNA double helix. The transverse displacement,
un − vn
(Figure 2.7) representing the hydrogen bonds between the compleyn = √
2
mentary nucleotides was calculated. The first term, U[un ;vn ] (equation 2.2),
is the Morse potential for the nth base pair. U[un ;vn ] describes the combined
effects of the hydrogen bonds between the complementary bases and electrostatic repulsion of the backbone phosphates [46]. The parameters Dn and an
are sequence dependent. The second term W[un ;un−1 ;vn ;vn−1 ] (equation 2.3)
approximates to the stacking interactions between consecutive nucleotides,
which influences their transverse stretching motion. The exponential term effectively decreases the stacking interaction when one of the nucleotides is displaced away from its equilibrium position, e.g., when one of the nucleotides
is out of the DNA stack. The stacking force constants K un;n−1 (K vn;n−1 ) depend
on the nature of the base, on its closest neighbor, and on the location of the
nucleotide - the right or left DNA strand. The dinucleotide stacking force
constants were determined in [48].
Monte Carlo simulations on the EPBD model and its parameters describing
DNA breathing dynamics [38] were performed with MATLAB (MathWorks,
Natick, USA) using the same set as my previous study [49] of 2000 different
seeds from a random number generator and with parallel computing with its
distributed computing toolbox . A DNA sequence containing clamp sequences
on each end of a strand was simulated at 303 K with periodic boundary conditions to prevent the end effect. Each realization takes 2.1 × 107 steps and as the
1 × 106 steps reaches the initial equilibrium and then record every 500 steps
to have 40 000 snap shots of the displacements of a base pair. Every accepted
configuration in an advanced step is determined by the standard Metropolis
algorithm. From 40 000 recorded displacements of each base pair in the DNA
sequence, if the displacements at a base pair and its following consecutive 3
to 10 base pairs are larger than 2.8 Å, it counts one opening event at the first
24
base in the defined DNA bubble length (Figure 2.7) from 4 to 11 bp. The
opening probability of a base pair is the ratio of summed opening events to
40 000 recorded displacements. The DNA opening probability profile is averaged from 2000 realizations.
Length
y
n-2 n
n+2
Figure 2.7. Illustration of a DNA bubble with length L and the displacement y between
the nucleotides of the base pair at the position n. One opening event at the nth base pair
is defined as when the displacement y is wider than 2.8 Å in a defined DNA bubble
length L.
25
3. Results and Discussion
3.1 Synthetic biology and cyanobacteria (I and II)
Standardization and characterization of parts are the foundations for developing and applying the engineering rules of synthetic biology in cyanobacteria [11, 50]. However, until very recently there was a almost total lack of biological parts to be used in synthetic biology inspired engineering of cyanobacterial cells. Therefore, my initial focus was on developing general molecular
tools that do function in cyanobacteria. As model organism the well studied unicellular cyanobacterium Synechocystis PCC 6803 (Synechocystis) was
chosen. Biological parts such as promoters, ribosome binding site (RBS)s,
coding DNA sequence (CDS)s, and terminators were selected and assembled
in a developed shuttle vector before further used in the cyanobacterial cells and
development. Inspiration of the parts to be used came from the large collection
at the BioBrick Registry (http://parts.igem.org/Main_Page).
3.1.1 Characterization of functional parts
Shuttle vector
To characterize the standardized biological parts for cyanobacteria, the new
pPMQAK1 plasmid (Figure 3.1) for assembling and transferring parts between different bacterial strains was developed. The presence of the BioBrick
prefix and suffix sequences enable the standard assembly method to connect
parts to a device. Due to the RSF1010 origin [51], this plasmid can selfreplicate in enteric bacterium E. coli, unicellular cyanobacterium Synechocystis,
filamentous cyanobacteria Nostoc PCC 7120 and Nostoc punctiforme ATCC
29133. Potentially, many different species of cyanobacteria [52–54] or other
model organisms [55,56] can be transformed and harbor this broad-host-range
BioBrick shuttle vector. This might enable synthetic biology in these biological systems. The copy number of this plasmid in Synechocystis might range
from 10 [52] to 30 [57] per cell. Kanamycin and ampicillin are the selection
markers. Because of the inserted ccdB gene (BBa_P1010), the more efficient
3A assembly method (Figure 2.2) is enabled to connect the standardized parts.
Promoters
The native rnpB and rbcL promoters of Synechocystis were characterized and
showed promoter strengths after standardized with BioBrick prefix and suffix
and assembled into the reporter construct. The rnpB promoter can serve as
26
pPMQAK1-BBa_P1010
8372 bp
Figure 3.1. The shutte vector pPMQAK1. It features with the BioBrick interface and
the inserted ccdB gene for the standard or 3A assembly and the RSF1010 origin for
the broad-host-range. Adapted from Paper I.
a reference promoter [58] for comparing promoter strength because the rnpB
gene expression is stable when shifting culture from dark to light or treating culture with electron transport inhibitor DCMU or DBMIB under normal
growth conditions [59, 60]. The rbcL promoter region contains two putative
promoters, of which one is a type 1 promoter and one is a type 2 promoter [61].
The upstream one could be a type 2 promoter, which has a putative transcription activator NtcA binding site and a putative -10 element [62]. The downstream one could be a type 1 promoter, which has a putative -35 and a -10
element. In order to develop constitutive native promoters, the rbcL promoter
region was truncated into the six different promoter sub-regions: rbcL1A,
rbcL1B, rbcL1C, rbcL2A, rbcL2B, and rbcL2C to remove the regulatory binding site. In this study, a putative NdhR motif [63, 64] was proposed in the type
1 promoter region. Therefore, the rbcL1C promoter could be a suitable constitutive promoter because the regulatory NtcA and NdhR binding sites are
removed. Its strength is about 4-times stronger than the rnpB promoter.
The non-native LacI-regulated trc1O promoter shows very strong promoter
strength after standardization but cannot be repressed below 60 % of its strength
in Synechocystis. In order to improve the repression, an additional LacI binding site Oid [65], on top of the O1 that is already in the trc1O promoter, was
added in the distal position, creating the trc2O promoter. The repression is
27
largely improved. However, the trc2O promoter cannot be induced back to
its non-repressed activity, even after adding 10 mM inducer IPTG. The trc1O
and trc2O promoters are very strong constitutive promoters in the absence of
LacI repressor. But, in the presence of LacI repressors, the trc1O cannot be
repressed and the trc2O cannot be induced back to its high strength. Neither
of them can be a good promoter in terms of regulation range.
RBS
The ribosome binding sites selected from the BioBrick Registry, such as BBa_B0030, BBa_B0032, BBa_B0034 and the engineered RBS∗ all function in
Synechocystis. They contribute to different translation efficiency: RBS∗ >
BBa_B0030 > BBa_B0032 ∼ BBa_B0034 (Figure 3.2). The translation efficiency is cellular context [3] dependent: the ranking are different in E. coli
and in Synechocystis.
CDS/Reporter genes
Different fluorescent proteins such as cerulean, Green Fluorescent Protein
mut3B (GFPmut3B) (BBa_E0040), and EYFP (BBa_E0030) were all detectable
by subtracting high auto-fluorescence background of Synechocystis and the
respective excitation and emission spectra were obtained. For reporting dual
colors, cerulean and EYFP are suitable because of less overlap of spectra. At
optimal excitation and emission wavelengths (Figure 3.3), the emission intensity from high to low is EYFP, GFPmut3B, and cerulean.
Protease/degradation tags
Different E. coli protease tags such as ASV, AAV, and LVA for efficient degradation of a protein were tagged to EYFP, which has strongest emission level
Figure 3.2. The ribosome binding sites characterized in Synechocystis (black bar) and
in E. coli (white bar). The data presents as the emission level per averaged optical
density of a cell culture. Adapted from Paper II.
28
Figure 3.3. Excitation and emission spectra of fluorescent proteins expressed in
Synechocystis. Auto-fluorescence has been subtracted. Adapted from Paper I.
in Synechocystis. The efficiency of degradation is LVA > AAV >ASV (Figure 3.4). [66, 67]. Comparing to native protease tags IAA in Synechocystis,
the native tags provides even more efficient degradation about 1 % of untagged
EYFP [68].
Figure 3.4. The enhanced protein degradation of EYFP. The protein tagged with an
ASV, AAV, or LVA tag was constitutively expressed in Synechocystis for 48 hours and
detected. Adapted from Paper I.
Terminator
The terminator (BBa_B0015) in the Registry was used directly in all characterization constructs presuming that it is functional. At the moment, terminators
have been characterized in E. coli [69] but not in cyanobacteria.
29
3.1.2 Characterization of non-functional parts
Three important and commonly used BioBrick promoters, the LacI-regulated
lac promoter (BBa_R0010), the λ cI-regulated PR promoter (BBa_R0051), and
the TetR-regulated PL promoter (BBa_R0040) were characterized and none of
them show any detectable activity in Synechocystis.
The LacI-regulated lac promoter (BBa_R0010) contains a transcription activator Crp binding site upstream of the -35 and -10 elements. The possible reason causing this promoter non-functional in Synechocystis is discussed
with the relative position of the Crp binding site. For Crp-dependent promoters to activate transcription initiation, the helical turn spacing and helical
phasing between the Crp binding site and the -10 element are essential for
the contact of the Crp and the RNAP. The typical spacing is 5 helical turns
in E. coli [70, 71]. In Synechocystis, the transcription activator SYCRP1 and
its binding sites were identified [71–73]. For the murFP3 promoter, the helical spacing is 5.7 helical turns. The 0.7 helical turns difference not only
has different helical distance, but also has probably opposite helical phasing.
Assuming the SYCRP1 can associate with the Crp binding site, the different
required helical spacing for activating the transcription might explain why the
lac promoter (BBa_R0010) does not transcribe in Synechocystis. This explanation can be further supported by the observation that the Crp-independent
lacUV5 promoter can transcribe in the cyanobacterium Calothrix sp. strain
PCC 7601, but not the Crp-dependent lac promoter [74]. In additon to different promoter sequence arrangements, enteric and cyanobacterial RNAP have
different structures [61, 74].
The λ cI-regulated PR promoter (BBa_R0051) is from bacteriophage λ and
has the conserved -35 and -10 elements of E. coli σ 70 . There is no cI repressor in Synechocystis. The inactivity of this promoter might result from the
lack of the DksA protein in Synechocystis [75, 76]. The DksA protein directly
binds to the secondary channel of the RNAP and then stimulate the transcription initiation of the PR promoter [77, 78]. The TetR-regulated PL promoter
(BBa_R0040) is also from bacteriophage λ and has the conserved -35 and -10
element. There is no conclusive evidence to explain why this promoter does
not work in Synechocystis. Therefore, the L promoter library modified from
the PL promoter (BBa_R0040) was characterized and promoters with wide dynamic range of transcriptional regulation by the TetR binding were identified
(Paper III).
30
3.2 Wide-dynamic-range promoters for cyanobacteria
(III and IV)
Strong and tightly regulated promoters for cyanobacteria are, in general, still
missing. Therefore, a second focus in my thesis was to develop promoters
with a wide-dynamic range in regulation.
3.2.1 A few point mutations can change promoter strength
significantly
The TetR-regulated PL promoter (BBa_R0040) was selected as the template
to be modified because its sequence arrangement fits the type 1 promoter [61]
and a particular factor for transcription initiation might not be needed. First,
the -10 element was changed to the consensus sequence of Synechocystis [79].
Second, the nucleotides at 2 bp and 3 bp immediately downstream of the 10 element were systematically mutated with adenine, thymine, cytosine, and
guanine to generate 16 promoters. As the promoters were modified from the
PL promoter, this library is named as the L promoter library (Table 3.1).
Table 3.1. The TetR-regulated L promoter library developed and used for wide dynamic range of regulation. Adapted from Paper III.
The L12 promoter differs from BBa_R0040 promoter only in its consensus
-10 element. The consensus -10 element cannot increase but further decrease
the promoter strength. This reflects the fact that when the contacts between
the RNAP and the promoter are too many or too strong due to the conserved
sequence, it stalls the transcription initiation [29, 80]. The non-functionality
31
of the L12 needs a further experiment ( section 3.2.4) to be confirmed. In
addition to the L12 promoter, all other 15 L promoters show strong promoter
strength about 10 to 20-fold stronger than the reference rnpB promoter. This
indicates that a few mutations in the region between the -10 element and the
TSS change promoter strength effectively. Especially, when a guanine is located at 2 bp downstream of the -10 element, the L promoters show stronger
promoter strengths. This is consistent with the essential role of this nucleotide
at this position in causing a long-lived RNAP-promoter open complex [81,82].
The L09 promoter is unique in its high leaky gene expression in the repressed
condition. It was further examined in section 3.2.4.
3.2.2 The L03 promoter is regulated by TetR and
anhydrotetracycline in a wide dynamic range
Among 15 strong L promoters when the L12 promoter is excluded due to
its non-function confirmed in the section 3.2.4, the L03 promoter can be effectively repressed by TetR and induced by TetR’s inducer anhydrotetracycline (aTc) to show the widest dynamic range of transcriptional regulation
(Table 3.1). Further, by exploring the dose-dependence of the inducer, wide
range of promoter strengths up to about 200-fold can be fine-tuned by up
to 10 μg mL−1 aTc in 24 to 72 hours in the Light-Activated Heterotrophic
Growth (LAHG) mode.
The regulation of the L03 promoter was tested in different growth conditions including the LAHG mode in darkness, and photoautotrophic mode in
white or red light (Figure 3.5). The effect of glucose on the regulation in the
respective condition was also tested. Glucose in general can enhance eyfp gene
expression. This might result from the metabolic balance between the Calvin
cycle and the oxidative pentose phosphate pathway and glycolysis [20, 83].
The light condition affects the regulation severely because of light-sensitivity
of aTc. The best induction can be observed in darkness followed by induction
in red light and lastly in white light. This is due to the fact that aTc is most stable in darkness, less stable in red light and least stable in white light. The best
induction is 239±16 fold change in the LAHG mode in 24 hours and 290±93
fold change in photoautotrophic growth under red light in 48 hours. To the
author’s knowledge, these are the largest fold changes of gene expression that
has been observed in cyanobacteria. This promoter achieves the goal to have
a strong and tightly regulated promoter for Synechocystis.
3.2.3 The light-sensitive property of anhydrotetracycline
Since TetR is induced by a light sensitive chemical, can this chemical limit the
applications of TetR-regulated promoters in photosynthetic microorganisms
32
Figure 3.5. The transcriptional regulation of the L03 promoter under different physiological conditions. Cells were sampled on 24, 48, and 72 hours after induced with 0
(black bar), 102 (blue bar), 103 (green bar), 104 (magenta bar) ng/mL aTc and grown
under different light conditions: light-activated darkness (upper panel), red light (middle panel), and white light (lower panel). Adapted from Paper III.
33
growing under light? The answer is No because there are more options to
choose.
The transcriptional regulation systems using TetR and its mutants are welldeveloped [84] (Figure 3.6A). Not only the chemical ligand aTc, but also an
RNA aptamer [85] and the short TetR inducing peptide (TIP) [86, 87] can
induce TetR. RNA aptamers and short peptides are not sensitive to light. Besides, RNA aptamers and short peptides can be produced in a cell. A genetic
device generating these molecules can interconnect to the device regulated by
TetR.
Reverse thinking on the light-sensitive issue of aTc is that light can control
the TetR-regulated gene expression because it counteracts the induction of aTc
by degrading it. Future work on quantifying the effect of different light colors
and intensities on reducing the induction of TetR repression and the synergy
of light and aTc to fine tune a gene expression could be explored.
The reverse TetR (revTetR) in the presence of aTc can bind to the tet operator (tetO)(Figure 3.6B). The ligand aTc becomes the co-repressor of revTetR
[88].
Hence, the use of aTc provides a convenient method to develop TetR-regulated
promoters.
A
effector-free TetR
DNA-bound
effector-free TetR
anhydrotetracycline
RNA aptamer
short peptide
B
anhydrotetracycline
(as co-repressor)
effector-free revTetR
DNA-bound
revTetR
Figure 3.6. Transcriptional regulation using TetR (A) or revTetR (B) regulated promoters. (A) A TetR-regulated promoter can be induced either by (i) anhydrotetracycle,
(ii) a RNA aptamer, or (iii) the short peptide TIP. (B) A revTetR-regulated promoter
is repressed in the presence of anhydrotetracycline as the co-repressor.
34
3.2.4 A point mutation downstream of the -10 promoter element
does not exhibit a long-range effect on TetR binding
To create strong and tightly regulated promoters for Synechocystis, the L promoter library was generated by the systematic mutations in the region between
the -10 element and the TSS. The L09 promoter is unique in the unfavorable property that it is leaky under repressed conditions. Identifying why it
is leaky can help in designing strong and tightly regulated promoters. It was
compared to the L10, L11, and L12 promoters that were point-mutated at 2
bp immediately downstream of the -10 element and showed much lower promoter strengths under the repressed conditions. DNA breathing dynamics and
the SPR based interaction analysis were used to study this leakage problem
theoretically and experimentally.
Computed DNA breathing dynamics
Inspired by the long-range effect of a flanking single-nucleotide polymorphism (SNP) on altering the binding affinity of the eukaryotic YY1 transcription factor [37], whether the similar effect would be observed on the TetR
binding to the L09 promoter when comparing to the L10, L11, and L12 promoters was investigated. DNA breathing dynamics of the four promoters were
analyzed at 303 K with the EPBD model model [38] to generate the DNA
opening probability profiles for resolving the difference introduced by a single nucleotide mutation (Figure 3.7). The difference in DNA opening probability profiles between the L09 and other three L promoters on the two TetR
binding sites is trivial. Since a strong correlation has been shown between
DNA breathing dynamics and the DNA-protein interactions [38, 89], the trivial difference in DNA breathing dynamics might cause different TetR binding
affinity to the L09, L10, L11, and L12 promoters. The TetR binding kinetics
and affinity were measured by a SPR biosensor assay.
Measured kinetics and affinity of TetR binding
The DNA fragments of the L09, L10, and L11 promoters were immobilized
on the surface of a biosensor chip, respectively. The interactions between TetR
and these promoters were clearly detected. The apparent kinetic rate constants
k1 (2 × 105 s−1 M−1 ) and k−1 (1 × 10−3 s−1 ) and the equilibrium dissociation
constant KD (≈ 6 × 10−9 M) are identical for these promoters. When TetR
being in its effector-bound conformation [90,91], no interactions with the promoters were detected. The interactions between TetR and TetR L12 promoter
are qualitatively the same as the ones of the L09, L10, and L11 promoters.
This indicates that the even lower repressed strength of the L12 promoter is
not due to the stronger TetR binding but due to being a non-functional promoter.
35
10
10
9
9
bubble length (bp)
(B) 11
bubble length (bp)
(A) 11
8
7
6
5
5
4
-80 -70 -60 -50 -40 -30 -20 -10 1
(C) 11
11 21 31 41 51 61
base pair
-80 -70 -60 -50 -40 -30 -20 -10 1
(D) 11
10
10
9
9
bubble length (bp)
bubble length (bp)
7
6
4
0.0006
0.0005
0.0004
0.0003
0.0002
0.0001
0
8
8
7
8
7
6
6
5
5
4
11 21 31 41 51 61
base pair
4
-80 -70 -60 -50 -40 -30 -20 -10 1
base pair
11 21 31 41 51 61
-80 -70 -60 -50 -40 -30 -20 -10 1
11 21 31 41 51 61
base pair
Figure 3.7. The DNA opening probability profile of the L09 (A), L10 (B), L11 (C),
and L12 (D) promoters simulated by the EPBD model at 303 K. Adapted from Paper
IV.
36
Insights from simulations and experiments
The trivial difference in DNA opening probability profiles does not lead to detectable variations in DNA-protein interactions between the L09 promoter and
TetR. The leakage problem of the L09 promoter is not caused by the different
TetR binding. If TetR binding is practically the same, what other reasons can
make L09 promoter leaky? The enhanced RNAP binding might explain because transcriptional repression is due to the steric hindrance created upon the
transcription factor’s binding in the vicinity of the core promoter to prevent
RNAP binding [28]. This proposed reason was supported by the observation
of the higher DNA opening probability in the about 20 bp downstream region
of the TSS of the L09 promoter in comparison with the L10, L11, and L12
promoters (Figure 3.7). The downstream contacts between RNAP and promoter has a critical role in the formation and stability of RNAP-downstream
fork junctions complex and the formation of promoter open complex [92].
The stronger interactions in this region might enhance RNAP binding. Further
SPR measurements could confirm this proposal with SigA, which is the major
sigma factor in Synechocystis under normal growth conditions [61]. Competition experiments between SigA and TetR to the binding sites on the L09
promoter may also be performed.
3.2.5 The L22 promoter is a non-leaky promoter
Since the L12 promoter has been confirmed as a non-functional promoter, its
promoter strength value represents the detection limit. An L22 promoter has
the same strength as this value in the repressed conditions. So, this indicated
that the L22 promoter is non-leaky.
The SPR measurement confirmed that TetR in its effector-bound conformation does not bind to its cognate site. A increased L22 promoter strength was
detectable after induction. A considerable increase in strength was observed.
Hence, the L22 promoter is functional, though its dynamic range is not as
large as the L03 promoter’s. The identification of a functional and non-leaky
promoter is valuable.
3.3 Potential applications
3.3.1 Achieving indirectly a wide dynamic range of regulation
The weak L22 promoter developed (paper III) is valuable in its non-leaky
property under the repressed conditions. Therefore, it is worth to expand its
dynamic range of the regulation. When using the L22 promoter to express the
T7 RNAP [93], the T7 RNAP can bind to its cognate T7 promoters RNAP [94]
and express the target gene. This target gene may be indirectly regulated in a
wide dynamic range by this weak and non-leaky L22 promoter.
37
3.3.2 Expanding the design space of genetic circuits for a
balanced metabolism
The importance of a strong, tightly regulated promoter was also revealed by
a mathematical model exploring the design space of genetic circuit for a balanced metabolic pathway [95]. The model uses the promoter characteristic and
the RBS strengths as the parameters to explore the design space constrained by
the satisfied conditions to prevent metabolite overflow and guarantee the stability of the network. The model suggested that a promoter with wide dynamic
range and low leaky expression enlarges the design space. Another critical parameter identified in this model was the RBS ratio in an operon. The ratio is
calculated between two RBS used in the same operon. The ratio can fine tune
the circuit design. In the present study, both promoter and RBS were identified
with this described properties. Therefore, one could expect to use the developed promoter and RBS in the present study in Synechocystis for constructing
a balanced metabolic pathway. This model is using physiologically realistic
parameter values for E. coli. The potential application is to use the values for
Synechocystis and find out the design constrain for Synechocystis and choose
and create the appropriate promoters and RBS.
3.3.3 Enabling modularity in cyanobacteria to realize a central
concept of synthetic biology
As briefly mentioned in the introduction, when interconnecting the modules,
the insulation device is the key to realize the modularity in a biological system [18]. One of the designs to construct the insulation device relies on transcription regulation. According to the model of the insulation device, a strong
and non-leaky promoter and an enhanced protein degradation are required.
From the available biological parts developed in the present study, the L03
promoter and the LVA tag might be suitable parts to realize these insulation
devices in Synechocystis.
3.3.4 Verifying the versatile TetR-regulation system in
cyanobacteria
Because TetR can also be induced by RNA aptamers and short peptides (Figure 3.6), these molecules could be used as internal in vivo-produced inducers
to induce downstream devices in the interconnected modules. For the regulation by short peptides, TIP can induce TetR and TetR anti-inducing peptide (TAP) can anti-induce TetR. The anti-induction is due to the competition
of TAP to the effector binding site against the binding of aTc. In addition to
TetR, when revTetR is used, aTc and short TetR corepressing peptide (TCP)
become co-repressors. They make the application of the L03 promoter, for
38
example, more broad because revTetR recognizes the same operator as TetR
but can regulate the promoter in opposite to what TetR does. Since versatile
regulation can be achieved by TetR and revTetR regulation systems, it is worth
to verify each regulatory component of them and to obtain more possible regulations in Synechocystis.
3.3.5 More promoters for implementing a heterogenous dynamic
sensor-regulator system
The methods used in the present study could apply for developing more promoters regulated in a wide dynamic range by different transcription factors.
First, select a transcription factor together with information of its known cognate binding site. Second, the promoter sequence design can be simulated to
check its DNA opening probability profile. Third, the kinetics of DNA-protein
interactions can be probed by a SPR-based analysis. Fourth, the regulation of
the promoter can be reported by the expression of fluorescent proteins in a cell.
The promoter sequence should be changed according to the comparisons between simulations and experiments and the optimal promoter sequence which
exhibits the desired regulatory properties should be found in the iteration cycle
of simulations and experiments. The strategy of a dynamic sensor-regulator
system [96] to develop a promoter regulated by a key intermediate metabolite
for a balanced metabolic pathway could suggest the selection of transcription
factor and range of dynamic regulation.
39
4. Conclusions
Strong and tightly regulated promoters to apply the engineering rules of synthetic biology when modifying cyanobacteria are desirable but no such promoter existed until the present study.
The L03 promoter can be repressed by the foreign transcription factor TetR
and be induced by the chemical ligand aTc to transcribe strongly. Its dynamic
range in regulation is widest among the reported literature of Synechocystis.
Three protease tags such as ASV, AAV, and LVA can control different accumulated levels of the EYFP in Synechocystis. They are important to enhance
the protein degradation, which makes some synthetic devices with a certain
behavior work.
The RBSs such as BBa_B0030, BBa_B0032, BBa_B0034, RBS∗ have different strengths in Synechocystis. They are as important as promoters when
designing the genetic circuit to have a certain behavior.
The constitutive promoters can provide a constant gene expression in different levels. The levels from high to low are the trc1O, rbcL1C, BBa_J23101,
and rnpB promoters. Though trc1O has one lac operator, when there is no
LacI repressor, it will be a very strong, constitutive promoter.
In conclusion, in this thesis I have developed the first generation of biological parts to be used in the unicellular cyanobacterium Synechocystis PCC
6803. Based on my experience, I have selected the best parts for further studies
(Table 4.1). The potential applications and designs when using these biological parts are proposed in the outlook.
40
Table 4.1. Suggested biological parts for applications in synthetic biological inspired
genetic engineering in cyanobacteria.
Promoter
constitutive
trc1O
L21
BBa_J23101
rbcL1C
rnpB
RBS
a
CDS
BBa_B0030 cerulean
BBa_B0032 GFPmut3B
BBa_B0034
EYFP
RBS*
a
Protease tag Terminator
ASV
AAV
LVA
BBa_B0015
inducible
L03
non-leaky
L22
a
, All are functioning in Synechocystis though with different efficiencies.
41
5. Future outlook
Achieving the goal of having functional parts for Synechocystis is an essential
step. It is time to see what the present study can do from now on.
• A set of functional parts have been developed for Synechocystis. Therefore, the bottom-up approaches taken in synthetic biology should be enabled by these parts.
• The methodology developed in the present study could be further applied
to create more functional biological parts for Synechocystis and for other
cyanobacteria.
• The potential applications proposed in the results and discussion would
be beneficial in this research field if they were successful. Therefore, it
is worth to verify these potential applications.
42
Summary in Swedish
Kan du se en viktig tillämpning av kunskapen om hur bakterier reglerar sin
metabolism, hur cyanobakteriers cirkadiska rytm fungerar, eller hur mikrober
förflyttar sig mot födokällor? Ett svar är att kunskapen om hur dessa levande
system fungerar kan hjälpa till att rädda vår värld en dag. I rapporten ”Global
Risks 2012” från organisationen World Economic Forum identifieras sex viktiga riskfaktorer som med hög sannolikhet kan drabba vårt samhälle: brist på
dricksvatten, matförsörjningskriser, extrem volatilitet i energi- och jordbruksrelaterade priser, ökande utsläpp av växthusgaser, misslyckade anpassningar
till klimatförändringar, samt antibiotikaresistenta bakterier. Vad är den gemensamma lösningen till dessa till synes orelaterade problem? Lösningen kan vara
syntetisk biologi.
Syntetisk biologi kombinerar biologi med ingenjörsmässiga principer för att
bygga nya eller rekonstruera nya funktioner som utförs av syntetiska, levande
system med egenskaper som självassimilation, självorganisation, självreplikation samt självreparation. Att nå dessa mål är en utvecklingsprocess som
nyligen påbörjades och som går framåt med stora och snabba steg. Biologin
ger oss insikt om vad som sker i komplexa, biologiska system. Arvsmassan
DNA kodar och lagrar specifik information som sedan överförs till budbärarmolekylen RNA. Olika modifieringsprocesser kan verka på denna del av informationsflödet, vilket förändrar och berikar informationen beroende på olika
miljöfaktorer som finns vid just den tidpunkten. Denna modifierade eller ickemodifierade information i form av RNA-molekylen kan sedan översättas till
proteiner. Proteinerna kan svara på miljöfaktorerna i olika tid- eller rumskalor
genom att katalysera biokemiska reaktioner på under sekunden, genom interaktioner med DNA och RNA på minuter, eller genom att generera kemiska
gradienter i cellen. Interaktionerna mellan DNA, RNA och proteiner bestämmer de spatio-temporala koordinaterna i cellen, eller i andra ord hur cellen är
uppbyggd och förändras i tid och rum. Cellens uppbyggnad i sin tur samverkar
med DNA, RNA och proteiner till att tillsammans fungera som en levande enhet, vilket leder till liv på mikrometerskalan. Skillnaden i skala mellan dig
och en bakteriecell är lika stor som avståndet mellan en person i Uppsala och
en fågel som sitter i Kiruna, och som kanske sjunger livets musik. I naturen
existerar bakterier inte bara som ensamma celler, utan även som en population av celler. Även då dessa celler kan vara identiska genetiskt sett beter de
sig olika beroende på varje cells unika omgivning. Kommunikationen mellan
dessa celler skapar ett nätverk inom populationen, som varierar beroende på
förändringar i varje cells miljö och därför ger varje cell en chans att påverka
43
det kollektiva beteendet hos hela populationen. Hur cellernas interaktion kan
påverka hela populationens beteende kan liknas vid Eric Whitacres virtuella
körprojekt ”Fly to Paradise”, där Eric dirigerade 5905 sångare från 101 länder
över Internet. Tusentals videor från de olika sångarna sattes samman till ett
kollektivt uppträdande, vilket skapade en samverkande grupp av individer, i
detta fall i form av en kör.
Syntetiska biologer använder ingenjörsmässiga principer för att förenkla
och standardisera forskningsstrategier. Det kan hjälpa dem att förstå komplexa
biologiska system och att använda kunskapen för att konstruera nya funktioner
från systemens beståndsdelar. Genom att applicera en hierarkisk abstraktionsmodell från IT-världen, bestående av enskilda byggstenar, sammansatta funktionella moduler, system av moduler samt systemnätverk, på biologiska system kan man se en DNA-sekvens som en grundläggande byggsten. Byggstenen kan till exempel vara en promotor som driver överföringen av information från DNA till budbäraren RNA, ett ribosomalt inbindningsställe som
driver översättningen av RNA till proteiner, eller en terminator som agerar
som stoppsignal för promotorer. Då enklare biologiska byggstenar sätts ihop
till moduler eller system kan nya, sammansatta egenskaper studeras på grundval av deras enklare komponenter. Förutsägbarhet och pålitlighet är viktiga
egenskaper vid konstruktionen av nya biologiska system från grundläggande
byggstenar. Genom att dela upp naturliga och ofta ganska komplexa DNAsekvenser till enklare, funktionellt frikopplade delar kan man förenkla konstruktionen av nya system från dessa delar och förbättra systemens pålitlighet.
För att underlätta konstruktionen av nya system kan dessutom byggstenarnas
format standardiseras. Det möjliggör i sin tur en mer standardiserad metodik
för att sätta ihop dessa delar vilket påskyndar både designen och konstruktionen av nya system inom syntetisk biologi. Ytterligare en fördel med standardisering av delar och deras egenskaper är att de komplicerade detaljerna
i molekylära biologiska processer osynliggörs eller förenklas. Detta gör det
möjligt för syntetiska biologer att fokusera på den större betydelsen av varje
process och det nya systemets framträdande egenskaper. Det leder i sin tur till
en accelerering av forskningstakten, konstruktionen av nya mer komplicerade
artificiella system och bidrar dessutom till insikter om naturliga biologiska
system. Dessa system tar sig inte bara uttryck i enskilda celler, utan i hela
populationer av celler som tillsammans kan åstadkomma mer än vad som är
möjligt för en enskild cell.
De komplicerade mekanismerna som möjliggör bakteriers förmåga att förnimma näringsämnen, att uttrycka periodiskt varierande egenskaper, och att
jaga födoämnen har i princip härmats i mycket enklare artificiella biologiska
system, som kan uppvisa omställbara, oscillerande samt detekterande beteenden. Kombinationen av dessa egenskaper kan skapa nya, designade biologiska funktioner för specifika ändamål. Till exempel skulle de i rapporten
”Global Risks 2012” identifierade kriserna kunna lösas genom användningen
av syntetiska organismer som renar vatten, genererar biomassa till mat, pro44
ducerar förnyelsebar energi, binder växthusgasen koldioxid, samt dödar patogener utan behovet av antibiotika.
Denna studie siktar på att utveckla funktionella biologiska delar, speciellt
promotorer, till cyanobakterien Synechocystis PCC 6803. Cyanobakterier kan
tillgodogöra sig solenergi genom fotosyntes, vilket genererar reducerande kraft
för de biokemiska reaktioner som krävs för deras överlevnad. Denna speciella
förmåga och bindandet av koldioxid gör cyanobakterier till lovande modellorganismer som kan erbjuda en lösning till energikriser och utsläppen av växthusgaser. För syntetisk biologi är det absolut nödvändigt att ha fungerande,
standardiserade byggstenar, vilka lägger grunden för dess ingenörsmässiga
botten-upp metodik. Promotorn är den grundläggande delen som reglerar
genuttryck, i en syntetisk biologs ord är den nyckeln för att reglera uteffekten.
För att kunna göra detta på ett användbart och effektivt sätt måste promotorn
både vara hårt reglerad och kapabel till hög aktivitet.
Eftersom det saknas sådana promotorer för Synechocystis använder sig
denna studie av promotordesign för att modifiera promotorer med några få
punktförändringar i en speciell region av promotorn. Promotordesignen i
denna studie är en iterativ process som består av datorsimuleringar och laboratorieexperiment för att förstå hur DNA och proteininteraktioner påverkas då
promotorsekvensen modifieras. För detta ändamål simulerades DNA-öppningsdynamiken hos promotorns DNA-sekvens och genuttrycket som promotorn
ger upphov till kvantifierades experimentellt med hjälp av ett rapportörprotein. Genom att jämföra genuttrycket på enskild cellnivå, affiniteten och de
kinetiska konstanterna hos DNA-proteininteraktionen för en viss promotor
med simuleringsresultaten kunde värdefulla slutsatser dras om vikten av en
viss DNA-sekvens för promotorns funktion. Detta ledde till konstruktionen
av en ny promotor med en bredare regleringskapacitet än vad som tidigare har
rapporterats. Denna iterativa metod för promotordesign skulle kunna användas
för att skapa andra starka och effektivt reglerade promotorer.
De nya starka, reglerade promotorerna tillsammans med den förbättrade
proteindegraderingen och de andra verktygen som presenterats i denna avhandling kan tillämpas för att möjliggöra konstruktionen av modulära biologiska
system, vilket bidrar till att realisera ett centralt koncept inom syntetisk biologi, modularitet. Modulariteten gör det möjligt för syntetiska biologer att
använda enskilda, väl karaktäriserade byggstenar som är frikopplade från det
naturliga systemet på ett förutsägbart och pålitligt sätt för design av en given
målegenskap. Därför är promotordesignen för cyanobakterier som presenterats i denna avhandling ett viktigt steg i skapandet av en artificiell fotosyntetisk organism. En sådan organism kan bidra till utvecklingen av en effektiv
och ekonomiskt attraktiv metod för produktionen av framtidens förnyelsebara
bränslen.
45
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47
Acknowledgements
I would like to express my sincere gratitude particularly to:
Peter Lindblad, my supervisor, for accepting and welcoming me as the first
Asian PhD student into your group. Thank you for always being so scientifically open-minded, giving all the support and freedom for my scientific
explorations, and to accompany my scientific journey from the first that we
meet at Arlanda airport to the last stage of my PhD study.
Thorsten Heidorn, my co-supervisor: I very much appreciate your guidance
when I first arrived Fotomol. Thank you for all the scientific discussions we
had and your support on science and in life.
Stenbjörn Styring, for sharing your great experience and knowledge in science and support in life.
Leif Hammarström, for your kind smiles and dean’s approval to enable the
study.
Ministry of Education in Taiwan, for the support from the Studying Abroad
Scholarship.
I would also like to thank my collaborators, Christian Seeger and Helena
Danielson. Thank you for all time and effort and all the great discussions.
Daniel Camsund, for doing, discussing lots of things together in professional and daily life. Thank you for proof reading my thesis and translating
the summary into Swedish.
Paulo Oliveira, for discussions and sharing experiences in labs, in study,
and in life.
Fernando Lopes Pinto, for discussions and helping in bioinformatic analysis and your wonderful PGTX reagent.
Anja Nenninger, for being a great pleasure with you to explore the inner
universe of cells under the microscope.
48
Sven Johansson, for allowing me to use your workshop and tools to build
the LED panels and assemble the photobioreactor.
Susanne Söderberg and Åsa Furberg for the help with all the administrative
works.
All my colleagues in the department. It has been very enjoyable to work
together, especially all the members in the CyanoGroup for all the memorable
time we had in or outside the lab.
All my friends from Uppsala University Taiwanese Student Association, Vi
Taiwan for all the interesting activities and wonderful friendship. Your companion brings me so much laughter.
My cycling and hiking pals. For all the high and low; wind and rain; sweat
and weight; we have experienced together. The memory will be shining forever.
My families in Taiwan for their unconditional love support and Taiwanese
food supply.
Guei-Bau, my wife and my best friend, for your strict criticism and always
being there for me with smile and trust. Thank you. I love you.
49
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Acta Universitatis Upsaliensis
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