Synthetic Biology in cyanobacteria
Expression of [FeFe] hydrogenases, their maturation systems and construction of broad-host-range
vectors
Ingólfur Bragi Gunnarsson
Degree project inapplied biotechnology, Master ofScience (2years), 2011 Examensarbete itillämpad bioteknik 30 hp tillmasterexamen, 2011
Biology Education Centre and Department ofPhotochemistry and Molecular Science, Uppsala
Index
1 Introduction 1
1.1 Energy and environment 1
1.2 Current hydrogen production processes 2
1.3 Cyanobacteria 2
1.4 Hydrogenases 3
1.4.1 Cyanobacterial [NiFe] hydrogenases 4
1.4.2 Chlamydomonas reinhardtii [FeFe] hydrogenases 4
1.5 Synthetic biology 5
1.5.1 Standardized biological parts -‐ BioBricks 7
1.6 Synthetic biology in cyanobacteria 9
1.7 Project aims and goals 10
2 Results 11
2.1 Gas chromatography measurements 11
2.2 Test hydrogen electrode measurements 12
2.3 Growth characterization and hydrogen evolution measurement
in hydrogen electrode 13
2.4 Protein extraction, separation and Western Blotting 14 2.5 Discovering damage to broad-‐host-‐range vector pPMQAC1 15 2.6 Construction of new broad-‐host-‐range vectors 16
3 Discussion 20
3.1 [FeFe] hydrogenase expression in E. coli and characterization of consequent hydrogen production. 20 3.2 Conformation of [FeFe] hydrogenase expression in E. coli 20 3.3 Construction of broad-‐host-‐range vectors 21
4 Materials and methods 23
4.1 Bacterial strains, plasmids and primers 23
4.2 Growth media 24
4.3 Plasmid purification and cloning 25
4.4 Hydrogen measurements 26
4.4.1 Hydrogen electrode setup 26
4.4.2 Gas chromatography measurements 27
4.4.3 Test hydrogen electrode measurements 28
4.4.4 Growth characterization and hydrogen evolution measurement using hydrogen electrode 28
4.5 SDS-‐PAGE 29
4.6 Western blotting 30
4.7 Protein staining 30
4.8 Polymerase chain reactions (PCR) 31
4.9 Construction of new broad-‐host-‐range vectors 32
5 Acknowlegements 34
References 35
Summary
Mankind's consumption of fossil fuels is so excessive that we will most likely run out of fossil fuels this century. The depletion of fossil fuels is already causing serious con<licts and effecting the worlds economy. Global warming is already causing Earth’s climate to change fast, but the consumption of fossil fuels still increases. It is of great importance that fossil fuels are replaced by renewable energy sources so more damage to Earth’s biosphere can be prevented.
There is though one source of energy that dwarfs all other energy sources on Earth, the sun. Nature has for a long time been able to convert sunlight into energy very elegantly via photosynthesis. Mankind has not yet been able to capture the suns energy in an economical and ef<icient way. Synthetic biology de<initely has the future potential of developing photobiological systems able to produce renewable energy sources from sunlight. Photosynthetic
microorganisms e.g. cyanobacteria are able to harness sunlight and produce hydrogen in small amounts.
This project was mainly focused on two things. First, to characterize the
hydrogen production of already available [FeFe ]hydrogenase constructs (hydA2 and hydA2+fd) and their maturation systems in E. coli using gas chromatography and a Clark type electrode. Second, I was also involved in the construction of broad-‐host-‐range vectors that are able to replicate in cyanobacterial strains for the purpose of expressing productive [FeFe] hydrogenases and their maturation systems in cyanobacteria for increased hydrogen production.
1 Introduction
1.1 Energy and environment
Earth’s fossil fuel resources will run out in the not so distant future, and the release of green house gases from burning fossil fuel are causing global warming, which in turn is causing climates changes [1, 2]. The use of renewable energy sources needs to increase drastically and new ways in producing renewable energy need to be realized as soon as possible. Some renewable energy sources in use today are e.g. wind-‐, solar-‐, geothermal-‐, hydropower and biofuels such as bioethanol, biodiesel, biohydrogen. It’s important to continue ongoing research in all <ields of renewable energy because there is still no single renewable energy source that can totally replace fossil fuels, at least not when using current technologies [3]. Out of all energy sources being explored, one will eventually be able to replace fossil fuels inde<initely, this energy source is the sun. The sunlight that hits Earth’s surface contains about 103 times more energy than mankind's energy consumption [4]. Current technologies are however not able to capture this energy and transfer it into a renewable energy carriers such as electricity, hydrogen or ethanol in an economical and ef<icient way [5, 6]. Nature, on the other hand is able to do this in a very elegant way via photosynthesis. Plants, algae and some bacterial species use photosynthesis to convert sunlight into energy [7].
In the search for a way to capture sunlight and convert it into an energy carrier that can be commercially used, many possibilities are being explored. Hydrogen production using biological systems such as cyanobacteria are an interesting alternative, since they are able to naturally produce molecular hydrogen (in small amounts) from only water and sunlight via photosynthesis [8]. Molecular hydrogen contains the highest amount of energy per weight unit of all gaseous fuels and since it’s a carbon free compound, no carbon is emitted from its combustion. In fact, the combustion of hydrogen has only one combustion product, water [9].
Because of these positive attributes many believe that hydrogen will become Earth’s primary energy carrier in the future, but how that becomes reality is still unknown [5]. If biological systems like cyanobacteria are to be used for large
scale hydrogen production in the future, genetic engineering is needed to increase the production [10].
1.2 Current hydrogen production processes
Today as much as 96% of all hydrogen being produced in the world is produced directly from fossil fuels. About 4% is produced by electrolysis, where electricity generated in most cases from fossil fuels is used [10]. Steam methane reforming (SMR) is the most abundant method, as well as the cheapest way of producing hydrogen. The biggest drawback of using SMR is that the process emits large amounts of CO2 [11]. Other widely used methods for hydrogen production are e.g. coal gasi<ication, biomass pyrolysis/gasi<ication, electrolysis, photocatalytic water splitting and biological [12].
By using biological systems, hydrogen can be produced in a renewable and carbon neutral way. Biological hydrogen can be produced via photosynthesis, fermentation and microbial electrolysis cells [13]. This project was focused on hydrogen production using biological processes connected to photosynthesis.
1.3 Cyanobacteria
Cyanobacteria are photosynthetic organisms that were Earth’s <irst primary producers, and as such they play an important role in Earth’s carbon and nitrogen cycles, since many of them can <ix nitrogen from the atmosphere[14].
The mechanism of oxygenic photosynthesis is found in the thylakoid membrane.
It absorbs light with antenna complexes and Photosystem II uses the energy in the photons to split water into molecular oxygen, protons and electrons. The electrons are transferred through the electron transport chain (plastoquinone, b6f cytochrome and plastocyanin) to Photosystem I that catalyzes the membrane charge separation. This process is driving the reduction of NADP+ to NADPH (through ferrodoxin-‐NADP+ reductase) as well as providing the proton gradient necessary for producing ATP [15, 16].
The ability to split water and harvest it’s electrons via photosynthesis is a biochemical capacity that can be traced back at least 2320-‐2450 million years, when molecular oxygen was <irst found in the atmosphere. Some even believe
ago (Ma) [17, 18]. These ancient photosynthetic organisms were predecessors to currently existing cyanobacteria [19]. When oxygen became abundant in Earth’s atmosphere a new chapter in life on Earth began, aerobic organisms evolved and cellular respiration became possible [19].
Taxonomic classi<ication based on morphology and development usually divides cyanobacteria into <ive principal groups: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales. These groups are then divided into numerous sub-‐groups [8]. Cyanobacteria are found in a wide variety of habitats e.g. aquatic and terrestrial environments as well as under extreme conditions in hot springs, deserts, hydersaline alkaline lakes and polar regions [20]. Apart from the fact that cyanobacteria can grow in harsh environments, they also have some other attributes that can be bene<icial for bioindustrial processes such as simple nutrition requirements, rapid genetics and naturally produce molecular hydrogen [10].
1.4 Hydrogenases
Hydrogenases are metalloenzymes that catalyze the reversible oxidation of molecular hydrogen from protons and electrons according to this reaction:
H2⇔2H++2e−. Hydrogenases are found in many different microorganisms and are important for their energy metabolism [21]. There are three classes of hydrogenases, [NiFe] hydrogenase, [FeFe] hydrogenase and [Fe] hydrogenase.
These classes indicate what type of active site the enzyme has. Three types of Fe-‐S clusters are found in proximity to the active site, [2Fe–2S], [3Fe–4S], and [4Fe–4S]. The Fe-‐S clusters supply electrons to the active site from the enzymes redox partners (NAD, cytochrome, coenzyme F420 and ferrodoxin). In the case of uptake hydrogenase the Fe-‐S clusters guide the electrons away from the active site [22].
The enzymes however vary between species e.g. [NiFe] hydrogenases are found across a variety of organisms including cyanobacteria, while [FeFe]hydrogenases are mostly found in green algae and some anaerobic prokaryotes [23].
1.4.1 Cyanobacterial [NiFe] hydrogenases
In the hydrogen metabolism of nitrogen-‐<ixing cyanobacteria, there are three enzymes that are of high importance: 1. Nitrogenase, which produces hydrogen as a byproduct while <ixing nitrogen (will not be discussed further). 2. Uptake hydrogenase (encoded by hupSL), recycles hydrogen that is produced by the nitrogenase. 3. Bidirectional hydrogenase (encoded by hoxEFUYH), produces and consumes hydrogen [8]. Non-‐nitrogen <ixing cyanobacteria, such as Synechocystis PCC 6803 only possess the bidirectional hydrogenase [24].
In cyanobacteria all of these enzymes have [NiFe] reaction centers and are sensitive to the presence of oxygen, which will render them inactive under aerobic conditions. The inactivation of these enzymes by oxygen can however be reversed by introducing anaerobic conditions [25]. The oxygen sensitivity, and the fact that the overall productivity of the hydrogen metabolism is low means that wildtype cyanobacterial strains are not feasible for commercial hydrogen production [26].
If one would want to use cyanobacteria for industrial hydrogen production, genetic modi<ications need to be done on the hydrogen metabolism in some way e.g. by introducing more productive [FeFe] hydrogenases from green algae into cyanobacteria [27]. The cyanobacterial strain Synechocystis PCC 6803 is potentially suitable for the heterologous expression of [FeFe] hydrogenases due to its unicellular appearance, natural transformability and relatively fast growth [8]. Here is where the application of synthetic biology becomes useful.
1.4.2 Chlamydomonas reinhardtii [FeFe] hydrogenases
C. reinhardtii is a soil-‐dwelling unicellular photosynthetic green algae that possesses many different fermentation pathways [28]. Its metabolic <lexibility can be used to produce useful metabolites such as hydrogen, ethanol and organic acids. C. reinhardtii possesses very productive [FeFe] hydrogenases that could potentially be used for large scale hydrogen production, whether using
C. reinhardtii or some other host e.g. cyanobacteria.
[FeFe] hydrogenases are more productive than [NiFe] hydrogenases when it comes to hydrogen production. This makes them more desirable for hydrogen
is called the H cluster. The H cluster has a complex structure that consists of a FeFe subcluster coordinated by carbon monoxide (CO) and cyanide (CN) ligands as well as a dithiol bridge. The active site is linked to the [4Fe-‐4S] cluster by a cysteine residue [30].
[FeFe] hydrogenases are very sensitive to the presence of oxygen and very easily irreversibly inactivated [31]. Oxygen is believed to bind to the active site of the [FeFe] hydrogenases, more speci<ically at a free coordination site of the Fe atom distal to the [4Fe-‐4S] cluster (marked as Fe2 in <igure 1)[32]. In C.
reinhardtii there are two genes, hydA1 and hydA2 that encode for [FeFe]
hydrogenases, HydA1 and HydA2. The transcription of these genes is induced at anaerobic conditions. Due to the complexity of the enzyme’s active site H-‐cluster, additional maturation enzymes (HydEF and HydG) are needed for its biosynthesis and assembly [33]. These maturation enzymes are encoded by hydEF and hydG genes and when transcribed the gene products are involved in numerous reactions, such as the coupling of radical S-‐adenosyl-‐L-‐methionine (SAM) chemistry, nucleotide binding, ligand synthesis, H-‐cluster assembly as well as cluster insertion [34]. The <inal product is an active and mature [FeFe]
hydrogenase. However the details of how the [FeFe] hydrogenase maturation process works are currently unknown [30]. It is of great importance to understand the synthesis of the H-‐cluster since it could contribute signi<icant information which can help with improving genetic engineering of microorganisms used for hydrogen production as well as with the creation of hydrogen producing biomimetic catalysts [34].
1.5 Synthetic biology
With the rapidly growing knowledge of biological systems and the enormous advancements made in different <ields useful for engineering biological systems, a new multidisciplinary <ield within biology has emerged, synthetic biology.
Synthetic biology is a <ield that ties together biological science and engineering.
Different research areas within biology come together in synthetic biology: e.g.
Figure 1 -‐ Chemical structure of the [FeFe]-‐hydrogenase H-‐cluster[31].
protein engineering, systems biology, computational biology, metabolic engineering and bioinformatics. Using the knowledge from all these different
<ields, scientists are now able to design, synthesize and combine genetic material to in<luence and manipulate the cellular metabolism of unicellular and even multicellular organisms [35].
The tools and technologies that enable synthetic biology to prevail are standardized cloning, DNA synthesis and work that is being done on minimizing genomes [36].
Traditional cloning is an important tool when conducting synthetic biology, but because people use different techniques, materials and standards to conduct their cloning it is often laborious and inef<icient [37]. By using a standardized form of cloning e.g. the “BioBrick assembly standard”, where standardized cloning vectors (BioBrick vectors) and standardized genetic elements (BioBrick standard biological parts, BioBricks) are employed, the cloning process can be automated and both functionally and time optimized [37].
DNA synthesis and genome minimization both got world wide publicity at the same time in May 2010 when J. Craig Venter and his colleagues at the J. Craig Venter Institute published an article in Science [38] about the creation of a synthetic organism. Big media discussion about synthetic life and the ethical issues related to this topic followed. In the article Venter et al. announced that they were successful in designing, synthesizing and assembling a 1.08 mega-‐bp Mycoplasma mycoides genome and creating M. mycoides cells containing only the synthetic chromosome [38]. This is the <irst step in creating organisms that can be truly optimized to produce valuable compounds e.g. bioethanol, biohydrogen or pharmaceuticals. Scientists will be able to maximize the yield of their target products by creating the ideal synthetic organism for the production of that speci<ic product [39]. The synthetic genome is kept at minimum size and includes only genes that are essential for the growth of the microorganism as well as genes that are necessary for producing the target product. In this way the production of unwanted metabolites and bi-‐products can be minimized, which in turn will allow increased production of the target product [37].
1.5.1 Standardized biological parts - BioBricks
By adopting inventions and taking note of developments taking place in different
<ields of engineering, the process of engineering biology is constantly being made easier. As the goal of synthetic biology is to design and build new biological systems by assembling biological parts (or biological building blocks), challenges like the characterization and standardization of the design and assembly of these biological parts need to be overcome to make the process more ef<icient [40].
Biological parts and standard biological parts can be de<ined in the following way: “We deJine a biological part to be a natural nucleic acid
sequence that encodes a deJinable biological function, and a standard biological part to be a biological part that has been reJined in order to conform to one or more deJined technical standards” [41].
In 2003, the original BioBrick assembly standard was proposed by Thomas F.
Knight Jr. in a technical report that he wrote at MIT. In this technical report he introduces a sequence standard that requires each BioBrick component to consist of a double stranded DNA vector. The vector bears four standardized restriction sites. Two sites, EcoRI (E) and XbaI (X) are positioned upstream and the other two restriction sites, SpeI (S) and PstI (P) are positioned at the downstream end of the vector. No other copies of these restriction sites are allowed to exist on the vector. A so called pre<ix region is between the E and X restriction sites and a suf<ix region is between the S and P restriction sites. In-‐
between the restriction site pairs is the “insert” region [42].
Enzymatic digestion with X and S results in compatible sticky ends, so that they can be ligated together, the same thing applies for the ligation of the same type of sticky ends e.g. E and E sticky ends. As shown in <igure 2, BioBricks can be excised from one BioBrick vector and integrated into another BioBrick vector via ligation.
A BioBrick part (blue) is removed from its vector by cutting with E and S. In a separate
reaction a gap is induced in the vector Figure 2-‐BioBrick standard assembly example
containing the green BioBrick part, by cutting with E and X. The blue BioBrick part and the cut vector containing the green part are then puri<ied via gel electrophoresis. The two parts are then mixed together so that compatible sticky (E-‐E and S-‐X) ends can come together. When this is done the parts can be ligated together to form one vector containing a blue-‐green part. The restriction sites (S-‐
X) between two parts form a so called “scar” sequence that is not recognized by any of the four restriction enzymes, which facilitates further assembly of BioBrick parts. The resulting vector is then transformed into E. coli cells, which are then grown to produce the desired amount of the BioBrick vector [43]. After the initial proposition of the assembly standard it has been modi<ied numerous times to adapt it to new techniques.
The Registry of Biological Parts was founded in the same year as Tomas F. Knight Jr. proposed the BioBrick assembly standard. The Registry is a growing collection of standardized biological parts that can be used by scientists to design and assemble biological circuits. The standard facilitates the exchange and assembly of biological parts. The smallest unit of engineering is the part, which is represented by a DNA sequence that encodes for different functions.
These parts include several thousands of genetic elements such as: promoters, repressors, activators, terminators and ribosome binding sites (RBS). Parts can then be assembled into a “device” that performs a certain task or function with certain input and output [44].
The BioBricks Foundation is a non-‐pro<it organization that works on improving and de<ining the standards of the BioBrick assembly standard and the standardized biological parts. The foundation strives to help and support synthetic biologists by providing them with practical and theoretical knowledge through organizing workshops [45].
OpenWetWare is another example of an effort to help synthetic biologists in conducting their research by sharing information, know-‐how and wisdom to scientists to make their work easier. At their website (www.openwetware.org) one can e.g. view, download and upload protocols for all kinds of methods and experiments used in synthetic biology, as well as see the composition of numerous materials used.
1.6 Synthetic biology in cyanobacteria
Cyanobacteria have become target organisms for some scientists due to their photosynthetic abilities. To be able to apply synthetic biology in cyanobacteria, the molecular tools to do so need to be developed. It is necessary because molecular tools such as vectors that were developed for E. coli will often not work properly in cyanobacteria. This is the reason why scientists at the Department of Photochemistry and Molecular Science at Uppsala University constructed a BioBrick compatible broad-‐host-‐range shuttle vector optimized for replication in cyanobacteria [46].
T h e b r o a d -‐ h o s t -‐ r a n g e v e c t o r constructed is called pPMQAK1. The vector contains a RSF1010 replicon t h a t e n a b l e s r e p l i c a t i o n i n cyanobacteria, two antibiotic cassettes (A=Ampicillin and K=Kanamycin) and a BioBrick interface with the four standard BioBrick restriction sites (EcoRI, XbaI, SpeI and PstI). A BioBrick, BBa_P1010 was inserted into the BioBrick insertion site using the BioBrick assembly standard [46].
BBa_P1010 is the BioBrick name for
the ccdB cell death gene, which codes for the CcdB protein that kills most E. coli strains, some strains are however resistant. By having BBa_P1010 on the vector the process of inserting other BioBricks into the vector is made easier. The ccdB gene is used for positive selection of successful ligations into the BioBrick site, since all cells carrying plasmids containing BBa_P1010 will die. [43].
The RSF1010 replicon is from the broad-‐host-‐range plasmid RSF1010. It is of IncQ group and has been shown to replicate in a wide range of gram-‐negative bacteria as well as some gram-‐positive bacteria [47].
Former master students Thiyagarajan Gnanasekaran and Sean M. Gibbons at the Department of Photochemistry and Molecular Science, Uppsala University built genetic constructs e.g. hydA1 and hydA2 under PTrc1O synthetic promoter as well
Figure 3-‐Construction of pPMQAK1-‐BBa_P1010 [46]
as maturation system construct under PTrc2O synthetic promoter. They also made pPMQAC1, a modi<ied version of pPMQAK1, containing a chloramphenicol cassette instead of a kanamycin cassette. The hydrogenase constructs were inserted into pPMQAK1 and the maturation system construct into pPMQAC1 for expression in cyanobacteria.
1.7 Project aims and goals
Originally the main project goal was to <ind out why expression of already available [FeFe] hydrogenase and maturation system constructs in the cyanobacterial strain Synechocystis PCC 6803 did not result in hydrogen evolution. After that, the aim was to adjust growth conditions for Synechocystis PCC 6803 in order to optimize the hydrogen production.
The above strategy was however abandoned when discovering that one of two shuttle vectors (pPMQAC1) was not functioning properly.
A new strategy, involving the creation of new and optimized broad-‐host-‐range shuttle vectors was formed. The goal of the new strategy was to combine the RSF1010 replicon with different antibiotic cassettes and ligate it into a new and optimized BioBrick base vector obtained from The Registry of Biological Parts. If this goal was to be achieved the aim was to use the new broad-‐host-‐range shuttle vectors to express [FeFe] hydrogenases and maturation systems in Synechocystis PCC 6803.
2 Results
2.1 Gas chromatography measurements
Hydrogen measurements were tested using already available [FeFe]
hydrogenase and maturation system constructs. Hydrogen evolution from E. coli BL-‐21 (DE3) wildtype, E. coli BL-‐21 (DE3) cells carrying hydA2-‐pPMQAK1 and MatCr-‐pSB1AC3 as well as E. coli BL-‐21 (DE3) cells carrying hydA2+fd-‐pPMQAK1 and MatCr-‐pSB1AC3 was measured using gas chromatography.
No hydrogen evolution was detected from the wildtype strain. However E. coli BL-‐21 (DE3) cells carrying hydrogenase and maturation system constructs produced hydrogen. Cells carrying the hydA2 construct and maturation system produced 8.2 μmoles⋅OD-‐1 (sample 1) and 15.7 μmoles⋅OD-‐1 (sample 2), an average of 11.96 μmoles⋅OD-‐1 hydrogen was produced from cells carrying the hydA2 construct and maturation system. Cells carrying the hydA2+fd construct and maturation system produced 15.2 μmoles⋅OD-‐1 (sample 1) and 14.8 μmoles⋅OD-‐1 (sample 2), which is an average hydrogen production 15 μmoles⋅OD-‐1.
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Figure 4-‐Column chart showing the rate of hydrogen evolution (μmoles⋅ OD-‐1) when expressing HydA2 and HydA2+fd with pPMQAK1 and correct maturation system with pSB1AC3 in E. coli BL21 (DE3)cells.
2.2 Test hydrogen electrode measurements
Directly after measuring the hydrogen in the gas phase of the above cultures containing hydA2 or hydA2+fd constructs on the pPMQAK1 plasmid and maturation system construct on pSB1AC3 with the GC, the hydrogen production was measured using a hydrogen electrode.
100 μL of culture was added to 900 μL of LB medium in the electrode chamber.
The medium contained 20 mM glucose, 1 mM IPTG, 50 μg/ml kanamycin and 50 μg/ml chloramphenicol.
The hydrogen production of each culture was measured for 8 minutes in the hydrogen electrode.
The above <igures show the recorded output from the hydrogen electrode. The hydrogen production rate was calculated from the slopes (colored red) of increasing hydrogen concentration. Other artifacts in the <igures, such as decreasing hydrogen concentration, sharp peaks of signal and large deviations in signal are to be disregarded since they are the result of calibration, removal and addition of sample or signal noise.
Figure 5a shows that E. coli BL-‐21 (DE3) carrying hydA2-‐pPMQAK1 and MatCr-‐
pSB1AC3 produced 2.95 H2[nmoles]⋅min-‐1 (sample 1) and 7.13 H2[nmoles]⋅min -‐1 (sample 2), an average of 5.04 H2[nmoles]⋅min -‐1.
Figure 5a-‐Hydrogen production rate of E. coli BL-‐21 (DE3) cells carrying hydA2-‐pPMQAK1 and MatCr-‐pSB1AC3.
Figure 5b-‐Hydrogen production rate of E. coli BL-‐21 (DE3) cells carrying hydA2+fd-‐pPMQAK1 and MatCr-‐pSB1AC3.
Figure 5b shows that E. coli BL-‐21 (DE3) carrying hydA2+fd-‐pPMQAK1 and MatCr-‐pSB1AC3 produced 6.72 H2[nmoles]⋅min -‐1 (sample 1) and 4.13 H2
[nmoles]⋅min -‐1 (sample 2), an average of 5.43 H2[nmoles]⋅min -‐1.
2.3 Growth characterization and hydrogen evolution measurement using hydrogen electrode
This experiment was designed to provide more information about the hydrogen production as well as how the environmental conditions change during growth.
The pH in the medium was measured at the beginning and end of inoculation. OD and glucose concentration was measured every 30 minutes. All this information was combined with the recorded output from the hydrogen electrode and illustrated in <igure 6.
The above <igure (<igure 6) shows how the hydrogen concentration in the medium starts to increase after about 45 minutes. Hydrogen is then steadily produced and the hydrogen concentration increases as the culture grows, this phase is clearly seen in the <igure as almost a linear slope, where 2.21 H2
[μmoles]⋅OD -‐1 ⋅min -‐1 is produced. There is a steady increase in hydrogen
Figure 6-‐The <igure shows how hydrogen is produced during cell growth, as well as showing how glucose is consumed.
concentration for about 100 minutes, until the increase in concentration slows down very quickly, <lattens out and then decreases.
The concentration of glucose decrease steadily through the whole experiment, but at the end of the experiment there are still about 8-‐9 mM glucose left. During this experiment the culture grew from OD=0.1 to OD≤1.3 in 270 minutes and in that time the pH changed from 6.5 to 5.25.
2.4 Protein extraction, separation and Western Blotting
For verifying the presence of HydA2 proteins in E.coli BL-‐21 (DE3) cells carrying hydA2-‐pPMQAK1 or hydA2+fd-‐pPMQAK1 and MatCr-‐pSB1AC3 the proteins were extracted from the cells . The extracted proteins were then separated by SDS-‐
PAGE and then stained using Coomassie Blue or used for Western Blotting.
The coomassie stained SDS-‐PAGE gel seen above shows that the amount of protein is similar for all samples containing the different constructs. Two strong bands are observed between 35-‐55 kDa, one band ≈50 kDa and the other ≈40 kDa. However in the E. coli BL-‐21 (DE3) wildtype sample small bands are found at similar position in the gel as the bands observed from cells containing the constructs.
Figure 7-‐This SDS-‐PAGE gel shows the separation of proteins from E. coli BL-‐21 (DE3) cultures containing two different [FeFe] hydrogenase constructs.
The Western Blot analysis shows a ≈27 kDa band in all samples, even the wildtype sample and is therefore considered to be a result of some unspeci<ic binding of antibodies. One sample containing hydA2 and MatCr constructs gave only very faint bands at ≈50 kDa and ≈40kDa, while all other three samples gave strong bands at ≈50 kDa and ≈40kDa. Samples containing hydA2+fd construct showed faint bands at ≈60 kDa. Expected size of HydA2 is 49 kDa and the expected size of HydA2+fd is 59 kDa (49+10 kDa).
2.5 Discovering damage to broad-host-range vector pPMQAC1
For successful expression of [FeFe] hydrogenase and maturation system constructs with plasmids in cyanobacterial strains such as Synechocystis PCC 6803 broad host-‐range-‐vectors are needed. [FeFe] hydrogenase and maturation system constructs have to date, not been combined on one single vector due to the size of the resulting vector (>12-‐13 kb). Two broad-‐host-‐range vectors are therefore needed to express [FeFe] hydrogenase and maturation system constructs, pPMQAK1 and pPMQAC1. However, when starting to work with the pPMQAC1 vector, an observation was made after running an agarose gel where the digestion pattern of pPMQAC1 carrying MatCr construct was analyzed. The size of pPMQAC1 was a long way from being correct. The expected size of pPMQAC1 is ≈7,5 kb without any insert in the BioBrick site and with the maturation system construct (MatCr) in the BioBrick site (<igure 7) the total size should be ≈13 kb.
Figure 8-‐Western Blot analysis of protein extract from E. coli BL-‐21 (DE3) carrying hydA2, hydA2+f and MatCr constructs as well as wildtype.
As shown in <igure 7 the total size of pPMQAC1 carrying MatCr when linearized with EcoRI is only ≈5 kb. When the insert is cut out of the BioBrick site using XbaI and SpeI one band of
≈3,5 kb and one ≈1,75 kb are observed.
All available physical DNA constructs and glycerol stocks containing pPMQAC1 were analyzed in the same way. All pPMQAC1 vectors gave a similar digestion result as shown in <igure 7. After analyzing all sources of pPMQAC1 the use of this vector was stopped.
2.6 Construction of new broad-host-range vectors
After making sure that pPMQAC1 was not working properly, the project strategy was changed. The main project goal now became to build new and improved broad-‐host-‐range vectors. The new vectors should have only one antibiotic resistance cassette, kanamycin, chloramphenicol or ampicillin (instead of two on pPMQAK1 and pPMQAC1). The new vectors should all contain the RSF1010 replicon for replication in a wide range of bacterial hosts. The construction of the new vectors was at <irst done in BioBrick vectors (pSB1A3, pSB1K3, pSB1AC3 and pSB1AK3), and later in a BioBrick base vector (BBa_I51020).
The following broad-‐host-‐range vectors were to be constructed:
F i g u r e 9 -‐ M i d d l e : M a t C r -‐
pPMQAC1 linearized with EcoRI to see total size. Right: Insert cut out of pPMQAC1 using XbaI and SpeI.
Table 4-‐Broad-‐host-‐range BioBrick shuttle vectors to be constructed
Constructs Parts
pPMQA1 RSF1010 replicon, BBa_P1002 (Ampr), BBa_I51020 pPMQK1 RSF1010 replicon, BBa_P1003 (Kanr), BBa_I51020 pPMQC1 RSF1010 replicon, BBa_P1004 (Cmr), BBa_I51020
The RSF1010 replicon was obtained by PCR using PrimeSTAR HS DNA Polymerase with RSF1010-‐BB-‐f and RSF1010-‐BB-‐r primers containing the pre<ix and suf<ix (BioBrick restrictions sites). For the PCR pPMQAK1 was used as template, since it contains the RSF1010 replicon.
Only when using RSF1010-‐BB primers PCR product of the right size of 5,3 kb is observed. At <irst, after amplifying the replicon, PCR reactions were pooled together and digested with EcoRI and PstI (allowing ligation into BioBrick vectors later). The digestion reaction was then loaded on a 0.8% agarose gel and puri<ied with electrophoresis. The band corresponding to the RSF1010 replicon was cut out of the gel and the DNA puri<ied using a gel extraction kit. This process however always resulted in very low concentrations of DNA (<6 ng/μL) which made its application for ligation reactions dif<icult. Numerous attempts were made to ligate the RSF1010 replicon into BioBrick vectors (pSB1A3, pSB1K3, pSB1AC3 and pSB1AK3) with/without an additional antibiotic cassette ligated to the RSF1010 replicon (for better selection of positive colonies) but all trials failed, or resulted in false positives. False positives would after PCR give bands of the wrong size or faint bands, which when the restriction pattern was analyzed gave wrong fragment sizes, or even showed that restriction sites were missing.
Figure 10-‐PCR reactions containing RSF1010-‐f and RSF1010-‐r speci<ic primers do not give any product. However using RSF1010-‐BB-‐f and RSF1010-‐BB-‐r primers give the right sized product (5,3 kb).
After being unsuccessful with ligating the RSF1010 replicon into BioBrick vectors the strategy was changed. Instead of trying to ligate the replicon <irst into pSB1_3 BioBrick vectors, the RSF1010 replicon was to be ligated straight into the BioBrick base vector (BBa_I51020).
The ampicillin cassette already present on the base vector sits in between two NheI restriction sites and can therefore be replaced. The ampicillin was replaced with a chloramphenicol cassette. This step was necessary, as the RSF1010 replicon PCR products (containing pPMQAK1 as template) had to be used directly (after restriction with appropriate enzymes) for ligation reactions due to low recovery of RSF1010 replicon DNA after gel puri<ication.
When the RSF1010 replicon was ligated into the BioBrick site of the base vector it replaced the high copy number replicon and ccdb “death gene” (BBa_P1010) originally sitting together in the BioBrick site of the base vector for facilitating selection of positive clones.
Figure 11-‐Examples of false positives digested with EcoRI and PstI.
Left: One thick band (≈8,5 kb) means one restriction site is missing.
Middle: Shows the pSB1A3 backbone (2,2 kb) and chloramphenicol cassette (769 bp), RSF1010 not present. Right: Shows the pSB1AC3 backbone (3 kb) and kanamycin cassette (967 bp), RSF1010 not present.
This strategy however also only resulted in false positives, where colony PCR showed that the insert present in the BioBrick site of the base vector was not the size of the RSF1010 replicon (Figure 10). Restriction analysis also showed incorrect fragment size (Figure 11).
Figure 13-‐Restriction (cut with EcoRI and PstI) analysis of plasmids from false positives. Restriction pattern was the same for all plasmids, ≈3 kb and 850bp bands.
Figure 12-‐Colony PCR results. All
observed bands are ≈1,3kb but expected size was ≈5,3 kb.
3 Discussion
3.1 [FeFe] hydrogenase expression in E. coli and characterization of consequent hydrogen production.
Genetic constructs containing [FeFe] hydrogenases, hydA2 and hydA2+fd (linked with ferrodoxin) and their maturation system from C. reinhardtii were expressed in E. coli BL-‐21 (DE3). The broad-‐host-‐range vector pPMQAK1 was used to express hydrogenase constructs while pSB1AC3 was used to express the maturation system.
Hydrogen measurements were done using gas chromatography and Clark type hydrogen electrode. The measurements clearly showed that the [FeFe]
hydrogenase constructs were working. No hydrogen production was measured coming from E. coli BL-‐21 (DE3) wildtype, while cells containing the hydrogenase and maturation system constructs were able to produce hydrogen.
There was a slight difference in average hydrogen production between cells containing hydA2 and those containing hydA2+fd constructs, where cells containing hydA2+fd produced more hydrogen. This was the case when measured with GC as well as with hydrogen electrode. The difference is however not big enough to draw any conclusions about the hydA2+fd construct being more productive when it comes to hydrogen production. The fact that there was a large difference in hydrogen production between the cultures that contained hydA2 constructs suggests that the cultures did not grow similarly. The other cultures containing hydA2+fd construct however produced similar amounts of hydrogen. Higher number of replicates should have been done to ensure that these large deviations could have been avoided.
3.2 Conformation of [FeFe] hydrogenase expression in E. coli
When analyzing the Western Blot results, bands corresponding to the expected sizes were observed. From samples containing hydA2 constructs a band was observed at ≈50 kDa which corresponds well to the expected size of HydA2, 49 kDa. This band was however also observed in samples containing hydA2+fd constructs which was unexpected since the expected size of hydA2+fd is 59 kDa.
Faint bands corresponding to the right size of 59 kDa were though also observed
sizes, the result is far from being optimal. The low strength of bands in one of the samples containing hydA2 construct suggests that the amount of [FeFe]
hydrogenase enzyme is less than in the other samples. The lesser amount of enzyme would then explain the lower production of hydrogen that was observed from this culture when measured with GC and hydrogen electrode.
A strong signal is seen at ≈27 kDa in all samples, including the wildtype sample not containing any hydrogenase constructs. This strong signal in wildtype is most likely due to unspeci<ic binding of the antibody to some other protein that is found in the E. coli BL-‐21 (DE3) wildtype stain.
3.3 Construction of broad-host-range vectors
Probably the most important and at the same time inconvenient observation that was made during this project was <inding out that one of the broad-‐host-‐range vectors was “damaged”. This observation could possibly explain why the expression of [FeFe] hydrogenase constructs (on pPMQAK1) and maturation systems (on pPMQAC1) in cyanobacteria e.g. Synechocystis PCC 6803 did not resulted in hydrogen production. The “damaged” pPMQAC1 vector might not be able to express the maturation system as it should, and therefore the [FeFe]
hydrogenase not be matured, and as a result of that no hydrogen is produced.
Why pPMQAC1 was “damaged” is unknown, it possible that a recombination event (due to homologous promotor sequences) happened somewhere in the construction process of the vector, and as a result a part of the vector was lost.
The construction of new broad-‐host-‐range vectors was initiated so that hydrogenase constructs and maturation systems constructs could be expressed on two different vectors. The goal was to construct three broad-‐host-‐range vectors under the working names: pPMQA1, pPMQK1 and pPMQC1. The new vectors should consist of the RSF1010 replicon (ampli<ied from pPMQAK1), one of three antibiotic cassettes available (ampicillin, kanamycin or chloramphenicol) and a BioBrick base vector (BBa_I51020).
Since the RSF1010 replicon is quite large (5,3 kb) and has high GC-‐content, it took some time before good PCR products were obtained. The RSF1010-‐f and -‐r speci<ic primers designed for amplifying the RSF1010 replicon did not work. The reason why the primers did not work is unknown, but it could be that the wrong
