ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2013

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1063

Promoter Engineering forCyanobacteria

An Essential Step

HSIN-HO HUANG

ISSN 1651-6214ISBN 978-91-554-8724-9urn: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 degreeof Doctor of Philosophy. The examination will be conducted in English.

AbstractHuang, H.-H. 2013. Promoter Engineering for Cyanobacteria. An Essential Step. DigitalComprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 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 decouplingand standardization to understand, predict, and re-build novel biological functions from model-driven designed genetic circuits is emphasized. It is one of the top ten technologies thatcould help solving the current and potential risks in human society. Cyanobacteria have beenconsidered as a promising biological system in conducting oxygenic photosynthesis to convertsolar energy into reducing power, which drives biochemical reactions to assimilate and generatechemicals for a specific purpose such as CO2 fixation, N2 fixation, bioremediation, or fuelsproduction. The promoter is a key biological part to construct feedback loops in genetic circuitsfor a desired biological function. In this thesis, promoters that don't work in the cyanobacteriumSynechocystis PCC 6803 in terms of promoter strength, and dynamic range of gene regulationare identified. Biological parts, such as ribosome binding sites, and reporter genes with andwithout protease tags were also characterized with the home-built broad-host-range BioBrickshuttle vector pPMQAK1. The strong L03 promoter, which can be tightly regulated in awide dynamic range by the foreign Tet repressor, was created through an iterative promoterengineering cycle. The iteration cycle of DNA breathing dynamic simulations and quantificationof a reporting signal at a single-cell level should guide through the engineering process ofmaking promoters with intended regulatory properties. This thesis is an essential step in creatingfunctional promoters and it could be applied to create more diverse promoters to realize theemphasized practices of synthetic biology to build synthetic cyanobacteria for direct fuelproduction and CO2 assimilation.

Keywords: synthetic biology, cyanobacteria, promoter, engineering, TetR, DNA breathingdynamics, transcription, regulation

Hsin-Ho Huang, Department of Chemistry - Ångström, Box 523, Uppsala University,SE-75120 Uppsala, Sweden.

© Hsin-Ho Huang 2013

ISSN 1651-6214ISBN 978-91-554-8724-9urn: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 AcidsResearch, 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.1 Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.2 Synthetic biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.2.1 Biological parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.2.2 Genetic circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.3 Biological parts - Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.4 Promoter engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.4.1 Transcription initiation and its regulation . . . . . . . . . . . . . . . . . . . . . . 14

1.5 Aims and approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.1 Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.1.1 The assembly of standardized biological parts . . . . . . . . . . . . . . 17

2.1.2 Introducing novel biological parts into cyanobacteria . . 19

2.1.3 Cultivation – an in-house developed photobioreactor . . . 20

2.1.4 Single cell measurements – flow cytometry . . . . . . . . . . . . . . . . . . 20

2.1.5 Molecular interactions – surface plasmon resonance . . . . 22

2.2 Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.2.1 DNA breathing dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.1 Synthetic biology and cyanobacteria (I and II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.1.1 Characterization of functional parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.1.2 Characterization of non-functional parts . . . . . . . . . . . . . . . . . . . . . . . . 30

3.2 Wide-dynamic-range promoters for cyanobacteria (III and IV) . 31

3.2.1 A few point mutations can change promoter strength

significantly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.2.2 The L03 promoter is regulated by TetR and

anhydrotetracycline in a wide dynamic range . . . . . . . . . . . . . . . 32

3.2.3 The light-sensitive property of anhydrotetracycline . . . . . . 32

3.2.4 A point mutation downstream of the -10 promoter

element does not exhibit a long-range effect on TetR

binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.2.5 The L22 promoter is a non-leaky promoter . . . . . . . . . . . . . . . . . . . 37

3.3 Potential applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.3.1 Achieving indirectly a wide dynamic range of

regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.3.2 Expanding the design space of genetic circuits for a

balanced metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.3.3 Enabling modularity in cyanobacteria to realize a

central concept of synthetic biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.3.4 Verifying the versatile Tet repressor (TetR)-regulation

system in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.3.5 More promoters for implementing a heterogenous

dynamic sensor-regulator system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5 Future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Summary in Swedish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Summary in Traditional Chinese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Abbreviations

aTc anhydrotetracycline

CDS coding DNA sequence

E. coli Escherichia coliEPBD model extended Peyrard-Bishop-Dauxois model

EYFP Enhanced Yellow Fluorescent Protein

GFPmut3B Green Fluorescent Protein mut3B

LAHG Light-Activated Heterotrophic Growth

PB model Peyrard-Bishop model

PBD model Peyrard-Bishop-Dauxois model

PCR polymerase chain reaction

RBS ribosome binding site

revTetR reverse TetR

RNAP RNA polymerase

SNP single-nucleotide polymorphism

SPR surface plasmon resonance

Synechocystis Synechocystis PCC 6803

TAP TetR anti-inducing peptide

TCP TetR corepressing peptide

tetO tet operator

TetR Tet repressor

TIP TetR inducing peptide

TSS 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 under-

stand complex natural biological systems and uses engineering to create com-

plex artificial biological systems. It has been reviewed intensively in many

aspects such as general introductions [3, 4], applications [5], designs [6], pos-

sible utilization from natural resources [7, 8], and genetic circuits [9]. Engi-

neering rules such as abstraction, standardization, and decoupling are applied

in studying complex biological systems [10]. Biological systems through ab-

straction 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 func-

tion 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 com-

plex biological systems should be predictable from biological parts. A stan-

dard, 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 cross-

talk. 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 se-

quences, and terminators, compose a functional device to generate a DNA-

encoded 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 reg-

istry, 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, en-

gineering 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 bio-

chemical 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 neces-

sary 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 strate-

gies 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 combi-

11

BBa_C0040BBa_J23101 BBa_B0034 BBa_B0015 BBa_E0030L promoter RBS* BBa_B0015

constitutivepromoter

RBS TetR-LVA terminator induciblepromoter

RBS reporterEYFP

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 de-

scription 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 syn-

thetic biology provide efficient methods to construct functional biological de-

vices 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 SwitchThe design of the genetic toggle switch [13] requires any two mutually re-

pressing 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.

RepressilatorThe design of the repressilator [14] needs three transcriptional repressor sys-

tems. It exhibits an oscillating behavior from synthetic biological parts, of

which none is from components of natural biological clocks. The model sug-

gests that in order to be oscillating, promoters must be strong and tightly reg-

ulated and repressors must have a high turnover rate that can be achieved us-

ing 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 deviceThe cascade device [15] has employed three commonly used promoters that

are regulated by the TetR, LacI, and cI transcription factors. This cascade de-

vice is composed of three transcriptional cascades with one, two, and three re-

pression 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 stimu-

lus. 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 es-

sential for enabling this cascade device in Synechocystis for sensing a specific

stimulus.

MetabolatorIn 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 syn-

thetic 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 metabo-

lite 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 promot-

ers 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 oscil-

late to control the selected metabolism. Therefore, developing more promoters

regulated by different ligands in a wide dynamic range is very important.

Insulation deviceModularity is a central concept in synthetic biology [9,18]. Modularity makes

sure that the interconnected genetic circuits keep their individual property un-

affected. This is very important for predicting higher-order behaviors when

assembling more and more modules together. If one cannot predict the be-

havior after connecting two functional modules, these is no chance to de-

velop even more complicated system. In order to achieve modularity, an in-

sulation device is necessary to implement between the upstream and down-

stream interconnected devices. The model [18] has suggested that a design

which utilizes transcription regulation needs a strong, tightly regulated pro-

moter and an enhanced protein degradation to become an functional insula-

tion 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 - CyanobacteriaSeveral 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 grow-

ing. Plants are ruled out because of its complexity and relatively slow growth.

Fourth, the biological system should be possible to genetically modify and al-

ready have at least a platform of information available for engineering. There-

fore, 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 capac-

ity 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 analy-

sis [22], genetic engineering [23], and light responses [24, 25]. Systems biol-

ogy 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 poly-

merase (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 interac-

tions with sigma factor [30], which is one of the subunits in RNAP. The se-

quence in between the -10 element and TSS is important for the lifetime of the

RNAP-promoter open complex [31]. Transcriptional initiation after the recog-

nition 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 mech-

anism. 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 cy-

cle, 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 hin-

drance 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 dyna-

maics have been proposed as an another basal transcription factor in position-

ing 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 ob-

served. 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 iden-

tify functional parts, and to engineer parts for desired properties in the frame-

15

work of synthetic biology. The present thesis focus on developing parts for

cyanobacteria with a focus on promoters and the unicellular strain Synechocys-tis PCC 6803. Both theoretical and experimental approaches were employed

in an iteration cycle to simulate the promoter sequence with its DNA breath-

ing 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 de-

termined 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

StandardizationDNA 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 assemblyStandard assembly (Figure 2.1) [39] utilizes the compatible overhangs of XbaI

and SpeI restrictions to join two parts and the EcoRI and PstI restrictions de-

termine the orientation of the joint parts. This method requires the purification

of each part and the destination vector.

3A assembly3A 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 assemblyGibson 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. An-

other 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 Tagligase. 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 tem-

perature (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 struc-

18

3'5'

3'5'

3'5'

3'5'

3'5'

3'5'

3'5'

3'5'

3'5' 3'

5'

3'5'

3'5'

3' 5'5' 3'

Overlap

Chewing-back with T5 exonucleasePhusion polymerase

Taq ligase

Phusion polymeraseAnnealing

T5 exonuclease Taq ligase

Repairing with Phusion polymerase and Taq ligase

50

50

50

Figure 2.3. The Gibson assembly. The isothermal reaction at 50 ◦C contains T5 ex-

onuclease, 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 synthesisDNA 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 to-

gether.

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 bi-

ological functions into cyanobacteria, the assembled DNA needs to be trans-

formed 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 assem-

bled 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 cyanobac-terium Synechocystis PCC 6803 through conjugation

Strain Plasmid Role in conjugation

E. coli DH5α cargo plasmid (pPMQAK1) donor cells

E. coli HB101 conjugal plasmid (pRL443) conjugal cells

Synechocystis none recipient cells

2.1.3 Cultivation – an in-house developed photobioreactor

In order to simultaneously test Synechocystis cells in different growth condi-

tions, 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 the-

sis, 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 pass-

ing 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 quan-

tifying 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 emis-

sion 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.

Association

Dissociation

Regeneration

Re

son

an

ce

sig

na

l (R

U)

90 1800

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 VEPBD of the EPBD model (equation 2.1) is:

23

VEPBD =n

∑i=1

U [un;vn]+W [un;un−1;vn;vn−1] (2.1)

U [un;vn] = Dn

(e−an(un−vn)−1

)2(2.2)

W [un;un−1;vn;vn−1] =Ku

n;n−1

2(un −un−1)

2 +Kv

n;n−1

2(vn − vn−1)

2+

ρ4

e−β [(un−vn)+(un−1−vn−1)](√

Kun;n−1 (un −un−1)−

√Kv

n;n−1 (vn − vn−1))2

(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 rela-

tive displacement from the equilibrium of the respective nucleotide, situated

in the right or left strand of DNA double helix. The transverse displacement,

yn=un − vn√

2(Figure 2.7) representing the hydrogen bonds between the comple-

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 electro-

static repulsion of the backbone phosphates [46]. The parameters Dn and anare 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 ef-

fectively decreases the stacking interaction when one of the nucleotides is dis-

placed away from its equilibrium position, e.g., when one of the nucleotides

is out of the DNA stack. The stacking force constants Kun;n−1(Kv

n;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 condi-

tions 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 40000 snap shots of the displacements of a base pair. Every accepted

configuration in an advanced step is determined by the standard Metropolis

algorithm. From 40000 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

40000 recorded displacements. The DNA opening probability profile is aver-

aged from 2000 realizations.

y

Length

nn+2

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 devel-

oping and applying the engineering rules of synthetic biology in cyanobacte-

ria [11, 50]. However, until very recently there was a almost total lack of bio-

logical parts to be used in synthetic biology inspired engineering of cyanobac-

terial cells. Therefore, my initial focus was on developing general molecular

tools that do function in cyanobacteria. As model organism the well stud-

ied 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 vectorTo characterize the standardized biological parts for cyanobacteria, the new

pPMQAK1 plasmid (Figure 3.1) for assembling and transferring parts be-

tween 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 self-

replicate 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 biolog-

ical 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.

PromotersThe 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 rnpBgene expression is stable when shifting culture from dark to light or treat-

ing 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 transcrip-

tion activator NtcA binding site and a putative -10 element [62]. The down-

stream 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 bind-

ing 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 con-

stitutive 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 bind-

ing 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.

RBSThe 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 ef-

ficiency is cellular context [3] dependent: the ranking are different in E. coliand in Synechocystis.

CDS/Reporter genesDifferent 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 inten-

sity from high to low is EYFP, GFPmut3B, and cerulean.

Protease/degradation tagsDifferent E. coli protease tags such as ASV, AAV, and LVA for efficient degra-

dation 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 (Fig-

ure 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.

TerminatorThe terminator (BBa_B0015) in the Registry was used directly in all character-

ization 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 λcI-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 ac-

tivator Crp binding site upstream of the -35 and -10 elements. The possi-

ble reason causing this promoter non-functional in Synechocystis is discussed

with the relative position of the Crp binding site. For Crp-dependent pro-

moters 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 he-

lical 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 expla-

nation 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 differ-

ent promoter sequence arrangements, enteric and cyanobacterial RNAP have

different structures [61, 74].

The λcI-regulated PR promoter (BBa_R0051) is from bacteriophage λ and

has the conserved -35 and -10 elements of E. coli σ70. There is no cI repres-

sor 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 transcrip-

tion 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 dy-

namic 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 strengthsignificantly

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 dy-namic 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 lo-

cated 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 andanhydrotetracycline 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 ef-

fectively repressed by TetR and induced by TetR’s inducer anhydrotetracy-

cline (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−1aTc in 24 to 72 hours in the Light-Activated Heterotrophic

Growth (LAHG) mode.

The regulation of the L03 promoter was tested in different growth condi-

tions 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 sta-

ble 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 physi-

ological 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 (mid-

dle 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 well-

developed [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. Be-

sides, 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 oper-

ator (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.

anhydrotetracycline(as co-repressor)

anhydrotetracycline short peptideRNA aptamer

effector-free TetR

DNA-bound

effector-free TetR

effector-free revTetRDNA-bound

revTetR

A

B

Figure 3.6. Transcriptional regulation using TetR (A) or revTetR (B) regulated pro-

moters. (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 elementdoes not exhibit a long-range effect on TetR binding

To create strong and tightly regulated promoters for Synechocystis, the L pro-

moter library was generated by the systematic mutations in the region between

the -10 element and the TSS. The L09 promoter is unique in the unfavor-

able 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 pro-

moter 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 dynamicsInspired by the long-range effect of a flanking single-nucleotide polymor-

phism (SNP) on altering the binding affinity of the eukaryotic YY1 transcrip-

tion 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 pro-

moters 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 sin-

gle nucleotide mutation (Figure 3.7). The difference in DNA opening proba-

bility 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 triv-

ial 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 bindingThe 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 pro-

moters 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 pro-

moter.

35

base pair

base pair

bubb

le le

ngth

(bp)

bubb

le le

ngth

(bp)

0.00010.00020.00030.00040.00050.0006

0

bubb

le le

ngth

(bp)

base pair

base pair-80 -70 -60 -50 -40 -30 -20 -10 1 11 21 31 41 51 61

bubb

le le

ngth

(bp)

4

5

6

7

8

9

10

11

4

5

6

7

8

9

10

11

4

5

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9

10

11

4

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11

-80 -70 -60 -50 -40 -30 -20 -10 1 11 21 31 41 51 61 -80 -70 -60 -50 -40 -30 -20 -10 1 11 21 31 41 51 61

-80 -70 -60 -50 -40 -30 -20 -10 1 11 21 31 41 51 61

(A) (B)

(C) (D)

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 experimentsThe trivial difference in DNA opening probability profiles does not lead to de-

tectable 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 be-

cause 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 pro-

moter 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]. Com-

petition 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 conforma-

tion 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 abalanced 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 bal-

anced 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 sta-

bility of the network. The model suggested that a promoter with wide dynamic

range and low leaky expression enlarges the design space. Another critical pa-

rameter 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 devel-

oped 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 centralconcept 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 sys-

tem [18]. One of the designs to construct the insulation device relies on tran-

scription 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 incyanobacteria

Because TetR can also be induced by RNA aptamers and short peptides (Fig-

ure 3.6), these molecules could be used as internal in vivo-produced inducers

to induce downstream devices in the interconnected modules. For the reg-

ulation by short peptides, TIP can induce TetR and TetR anti-inducing pep-

tide (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 reg-

ulations in Synechocystis.

3.3.5 More promoters for implementing a heterogenous dynamicsensor-regulator system

The methods used in the present study could apply for developing more pro-

moters regulated in a wide dynamic range by different transcription factors.

First, select a transcription factor together with information of its known cog-

nate 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 be-

tween 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 syn-

thetic biology when modifying cyanobacteria are desirable but no such pro-

moter 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 accu-

mulated 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 dif-

ferent 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 differ-

ent 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 bio-

logical 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 biologi-

cal parts are proposed in the outlook.

40

Table 4.1. Suggested biological parts for applications in synthetic biological inspiredgenetic engineering in cyanobacteria.

Promoter RBS CDS Protease tag Terminator

constitutivetrc1O

L21BBa_J23101

rbcL1CrnpB

inducible

L03

non-leakyL22

BBa_B0030BBa_B0032BBa_B0034

RBS*

ceruleanGFPmut3B

EYFP

ASVAAVLVA

BBa_B0015

a, All are functioning in Synechocystis though with different efficiencies.

a a

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. There-

fore, the bottom-up approaches taken in synthetic biology should be en-

abled 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 vik-

tiga riskfaktorer som med hög sannolikhet kan drabba vårt samhälle: brist på

dricksvatten, matförsörjningskriser, extrem volatilitet i energi- och jordbruk-

srelaterade priser, ökande utsläpp av växthusgaser, misslyckade anpassningar

till klimatförändringar, samt antibiotikaresistenta bakterier. Vad är den gemen-

samma 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, självreplika-

tion 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 budbärar-

molekylen RNA. Olika modifieringsprocesser kan verka på denna del av in-

formationsflödet, vilket förändrar och berikar informationen beroende på olika

miljöfaktorer som finns vid just den tidpunkten. Denna modifierade eller icke-

modifierade 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 inter-

aktioner med DNA och RNA på minuter, eller genom att generera kemiska

gradienter i cellen. Interaktionerna mellan DNA, RNA och proteiner bestäm-

mer 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 en-

het, 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 popula-

tion 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 abstraktions-

modell från IT-världen, bestående av enskilda byggstenar, sammansatta funk-

tionella moduler, system av moduler samt systemnätverk, på biologiska sys-

tem kan man se en DNA-sekvens som en grundläggande byggsten. Bygg-

stenen kan till exempel vara en promotor som driver överföringen av infor-

mation 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 grund-

val 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 DNA-

sekvenser till enklare, funktionellt frikopplade delar kan man förenkla kon-

struktionen 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 konstruk-

tionen av nya system inom syntetisk biologi. Ytterligare en fördel med stan-

dardisering 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 förn-

imma 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 beteen-

den. Kombinationen av dessa egenskaper kan skapa nya, designade biolo-

giska 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, pro-

44

ducerar förnyelsebar energi, binder växthusgasen koldioxid, samt dödar pato-

gener 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 modellor-

ganismer som kan erbjuda en lösning till energikriser och utsläppen av väx-

thusgaser. 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 labo-

ratorieexperiment för att förstå hur DNA och proteininteraktioner påverkas då

promotorsekvensen modifieras. För detta ändamål simulerades DNA-öppnings-

dynamiken hos promotorns DNA-sekvens och genuttrycket som promotorn

ger upphov till kvantifierades experimentellt med hjälp av ett rapportörpro-

tein. 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 avhan-

dling kan tillämpas för att möjliggöra konstruktionen av modulära biologiska

system, vilket bidrar till att realisera ett centralt koncept inom syntetisk bi-

ologi, 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 presenter-

ats i denna avhandling ett viktigt steg i skapandet av en artificiell fotosyn-

tetisk organism. En sådan organism kan bidra till utvecklingen av en effektiv

och ekonomiskt attraktiv metod för produktionen av framtidens förnyelsebara

bränslen.

45

?

?

DNARNA

DNA RNADNA RNA

DNA RNA

Eric Whitacre (Virtual Choir project) 4.0

101 5905

(standardization)

(abstraction)DNADNA

46

DNA (decoupling)

( )

(switching) (oscillating) (sensing)

Synechocystis PCC 6803

DNADNA

(DNA breathing dynamics)DNA

(modularity)

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 scien-

tifically 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 sci-

ence 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 profes-

sional 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 analy-

sis 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 com-

panion 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 for-

ever.

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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