Prior to this study it was discovered that MsrB1 from Mus musculus expressed in Escherichia Coli binds cobalt(II) (hereafter cobalt) in cobalt-supplemented growth media, and it had further been demonstrated that the His-tag was not responsible for this metal uptake. The aim of this study was to investigate the effects of cobalt on the growth of E. Coli in culture, characterize the metal-uptake and metal-binding site of cobalt-substituted MsrB1 by UV-visible spectroscopy and to gain structural information about the protein by high resolution NMR spectroscopy.The effects of cobalt on growth of E. Coli were studied by growing cultures in Lysogeny broth (LB) and minimal (M9)-media supplemented with different concentrations of cobaltdichloride (CoCl2) and monitoring culture growth by optical density (OD) measurements. Growth rates were found to decrease with increasing concentrations of CoCl2. To study the cobalt-uptake of MsrB1, the protein was recombinantly expressed in E. Coli in different cobalt-supplemented growth media, and the purified protein analyzed by UV-vis spectroscopy. Cobalt-uptake was demonstrated in all cases by characteristic absorption peaks owing to the cobalt-ligand complex, and the wavelengths of these peaks matched those of a tetrahedral four-coordinated cobalt-Cys complex. It was argued that these four Cys residues should be the same that constitute the zinc(II)-binding site of MsrB1, indicating that cobalt simply replaces zinc as a structural metal ion in cobalt-substituted MsrB1 (Co-MsrB1). It was argued that the intracellular concentration of cobalt in E. Coli should be significantly higher than zinc, and that this together with similar ionic radii for cobalt and zinc leads to the formation of Co-MsrB1. MsrB1 expressed in nickel(II)-supplemented LB did not lead to formation of Ni-MsrB1, which was argued to result from nickel not being released directly into the cytosol in E. Coli. Co-MsrB1 was produced by zinc-starvation of E. Coli followed by expressing the protein in zinc-free minimal medium supplemented with CoCl2. To investigate if pH-titratable groups could be detected, the protein was dialyzed against buffers with pH 4.9-11.5 and the molar extinction coefficient was found from UV-vis absorption spectra in the different pH. Two titration curves were observed, but assignment of the titrations to specific residues could not be made. Further, Co- and Zn-MsrB1 was dialyzed against buffers with metal chelating agents to remove the metal ions from the two proteins. Cobalt was successfully removed at pH 5.0 and 5.5, while removal of zinc from Zn-MsrB1 was not detected, demonstrating that zinc is more tightly bound to the Cys-ligands than cobalt.To study the ratio of formation of Co-MsrB1 and the native zinc-form Zn-MsrB1 in CoCl2-supplemented growth media, the molar extinction coefficient of Co-MsrB1 was determined, and the concentration of Co-MsrB1 in purified protein samples from protein expression in different growth media was determined. The Co-MsrB1:Zn-MsrB1 ratio was found to be 0.2 in M9 medium supplemented with 10 µM CoCl2, 0.1 in LB supplemented with 50 µM CoCl2 and 0.03 in LB supplemented with 10 µM CoCl2. From 2D- and 3D-NMR experiments on 13C- and 15N-enriched Co-MsrB1, a 70 % backbone assignment and 50 % side chain-assignment was accomplished using Computer Aided Resonance Assignment (CARA). The four structural Cys-residues of the native protein was not found, while the other three Cys-residues of MsrB1 were assigned, confirming that the same Cys-residues are responsible for coordination of cobalt and zinc in MsrB1. Many strongly shifted signals were observed in 1D 1H spectra of Co-MsrB1, some as far upfield as 350 ppm and downfield as -80 ppm, and it was argued that most of the unassigned residues should be found outside the spectral width of the 2D and 3D-NMR spectra.To gain further structural information about Co-MsrB1, pseudocontact shifts (PCSs) were determined for the assigned HN-atoms of Co-MsrB1 by using the published chemical shifts of for Zn-MsrB1. The PCSs were analyzed by AnisoFit using three conformers and the mean conformer of the published Zn-MsrB1 structure, and the best correlations between observed and calculated PCSs were found for conformer 3. The PCSs were plotted against their hypothetical distance to cobalt using the structure of Zn-MsrB1, and a very good PCS-distance proportionality was found, indicating that Co-MsrB1 and Zn-MsrB1 have the same overall fold and structure. The AnisoFit calculations and PCSs of the N-terminus suggested that the N-terminus spends significant time in the proximity of the metal-binding site, and it was argued that this proximity ensures high catalytic efficiency due to the short distance between the catalytic and resolving Cys-residues.

In the field of systems and synthetic biology there has been an increasing interest for the use of genetic circuits during the last decade. Several circuits have been successfully put together, many of which were based on models. During this thesis a model for a toggle switch was analysed both deterministically and stochastically.The HOM2-circuit approximation for a bistable tuneable switch from Ghim and Almaas (2009) was re-derived in order to make it asymmetrical. Deterministic analysis was conducted yielding stability diagrams, describing the phase plane showing bistability for the genetic switch. Furthermore, stochastic simulations of the approximation were conducted. This gave a somewhat narrower bistable area than the deterministic analysis, possibly due to the nature of saddle-node bifurcations. Parameter values for a switch based on experiments were estimated for the approximation, and these were used in a stochastic simulation. The result from this simulation was in correspondencewith the deterministic analysis. A stochastic simulation of the complete circuit was conducted based on parameter values found in literature. For this simulation bistability was not shown.In order to further explore the circuit, and validity of the approximation, experimental investigation is needed. This has been planned together with Rahmi Lale, PhD, and Professor Eivind Almaas at the Department of Biotechnology, NTNU.

This study has been part of the project “Development of versatile bacterial expression systems for use in recombinant protein production, metabolic engineering, and systems biology”, a collaboration between NTNU, SINTEF (Department for Biotechnology) and SU (Saarland University, Germany). The major goal of both the project and this study was to develop and apply advanced biological tools for control of gene expression. Recombinant DNA, created by artificially producing genetic sequences, can be transferred to microorganisms and change their properties. One new possible property is the production of specific recombinant proteins, which have potential for use in both industry and medicine. The most intensely studied and attractive heterologous (recombinant) protein producer is to date E. coli. Although there are several advantages using E. coli, some of its related disadvantages can cause low volumetric yield of specific proteins. When this occurs, there is a need for alternative producers and therefore a gene expression system that functions in diverse bacterial species. The Pm/XylS expression cassette, which has proven useful for industrial level production of recombinant protein, is used as a basis for this study’s expression system. Expression vectors harbouring Pm/XylS and genes for the human proteins IFN-alpha2b and GM-CSF were constructed. Protein expression from these vectors was evaluated in Pseudomonas species under different cultivation conditions. During cultivations of P. fluorescens SBW25 in shake flasks, instability of the relevant expression plasmids was detected. Evaluation of alternative Pseudomonas strains revealed that the same plasmids were stable in P. putida KT2440. Furthermore it was found that P. putida KT2440 was easy to cultivate in both rich and minimal media, and it was therefore chosen for further use in this study. Production of IFN-alpha2band GM-CSF from KT2440 was obtained under optimized shake flask experiments and fed batch fermentations, but in low quantities. To further examine KT2440s production potential, the expression plasmids was genetically engineered. This was done by incorporating a copy number mutation and codon optimizing target genes and signal sequence pelB. Exchange of trfA (the gene for replication protein TrfA) with trfAcop271C yielded approximately 3.5-fold increase of plasmid number in KT2440, the same as previously reported for E. coli. After this modification, the production of both model proteins was estimated to have increased 3.5-fold or more. Additionally, soluble IFN-alpha2b was detected, which was not reported for E. coli under the same conditions in a previous study. Codon optimization of the target genes and signal sequence did not have expected effects on protein production in KT2440 under conditions tested, when compared to wild type copy number expression plasmids. Combination of codon optimized sequences and increased copy number had negative effect under the conditions tested.Further evaluation of the genetically modified expression plasmids was performed in fed batch fermentations of KT2440. Plasmid stability was found to be high, but the protein production obtained was lower than expected from results in the previous shake flask experiment. During fed batch fermentations, an observed increase in metabolism at induction indicated that the inducer was consumed. Since induction here is performed when substrate is limited, in contrast to the conditions in shake flask experiments, it is possible that the inducer is metabolized instead of inducing protein production. This would explain the observed differences in production and should therefore be tested. P. putida KT2440 have through this study proved as a potential industrial protein producer based on its growth properties and the fact that simple genetic modifications of expression plasmids proved to positively affect the production of model proteins.

70 % of world’s oil reservoirs consist of heavy oil, and as the supply of conventional oil decreases, researchers are searching for new technologies to explore and enhance heavy oil recovery. One of the postulated technologies is microbial enhanced oil recovery (MEOR), which is predicted to be a more environmental and economical process for improving oil recovery of heavy oil. The aim of this Master’s project was to give a qualitative indication of three selected consortia’s potential to bioconvert Mariner Maureen-, Peregrino- and Bressay oil. The consortia are comprised of microorganisms isolated from oil sand, mud volcano, processing waters from water treatment plants and oil related samples from other locations. Bioconversion experiments were conducted by inoculating the three selected oils with three consortia. Two terms of different main experimental designs was used; optimal growth temperature of the consortia or the temperature at reservoir conditions, in addition to cultivation with two different growth mediums. After cultivating aerobically for seven days, the oil was separated from the water phase and analyzed by thin-layer chromatography with flame ionization detection (TLC-FID) to identify possible indication of bioconversion. The results demonstrate a reduction in either one or both saturated hydrocarbons and aromatic hydrocarbons of the oil in several of the oil samples. DNA extracted from the water phase that was analyzed with denaturing gradient gel electrophoresis (DGGE), showed several positive results with indication of high biodiversity. The overall results indicate that there has been a microbial impact on some of the heavy oil fractions in Mariner Maureen-, Peregrino- and Bressay oil. Additional experiments must be done to identify the specific changes in the oil and prove microbial impact by repeating these experiments. This could potentially lead to identification of microorganisms with the ability to bioconvert heavy oil and the development of MEOR processes.

Dårlig overlevelse av fiskelarver grunnet sykdomsutbrudd er et stort problem innen akvakultur. Dette er i stor grad forårsaket av opportunistiske bakterier som blomstrer opp når fôr tilsettes i store mengder. Et forsøk på å bekjempe sykdommene har vært bruk av antimikrobielle midler, men dette har ført til at bakterier har utviklet resistens mot slike midler. Det er derfor ønskelig å redusere bruken av disse midlene, og flere alternative metoder har blitt prøvd ut. Deriblant har bruk av både vaksiner og immunstimulerende agenter vist seg å være vellykket innen akvakultur. En annen metode, med stort potensial for å kunne kontrollere potensielle patogene stammer, er bruken av gunstige eller probiotiske bakterier. Probiotika virker ved å gi en helsefremmende effekt på vertsorganismen, og kan i tillegg utkonkurrere patogene bakterier. Tilsats av prebiotika, som er ufordøyelige komponenter, favoriserer vekst av probiotiske stammer og danner ugunstige miljø for patogene stammer.I denne oppgaven ble potensielle probiotiske bakterier for bruk til torskelarver karakterisert. Bakteriene som ble benyttet var isolert fra torskeyngel gjennom et prosjekt i SINTEF, og var karakterisert ved klassiske, mikrobiologiske metoder, i tillegg til sekvensering av deler av 16S rRNA-genet. Isolatene ble i oppgaven videre karakterisert med hensyn på probiotiske egenskaper under ulike vekstvilkår, både i renkultur, ko-kultur og blandingskultur. Denaturerende gradient gelelektroforese (DGGE), som er en metode for å analysere genetisk diversitet innen mikrobielle samfunn, ble benyttet for å karakterisere vekst i blandingskulturer. For å påvise eventuelle degraderings- og fermenteringsprodukt ble høytrykksvæskekromatografi (HPLC) benyttet. To patogene stammer ble inkludert i oppgaven; Vibrio anguillarum (V. anguillarum) HI 610 (også kalt Listonella anguillarum) og Marinomonas sp.Det ble påvist probiotiske egenskaper hos stammene som ble undersøkt i denne oppgaven, in vitro. Egenskapene inkluderte evne til vekst på prebiotiske substrat, evne til å utkonkurrere eller hemme patogene stammer, evne til adhesjon til mukus og vekst under anaerobe forhold. Ingen av stammene ble vist å være patogene for torskelarver. Kandidatene som ble vist å ha flest av de probiotiske egenskapene var av slektene Photobacterium, Pseudoalteromonas, Marinomonas, Vibrio og Shewanella. Det er ikke alltid at in vitro studier gjengis in vivo, og motsatt. Det bør derfor utføres in vivo forsøk på de beste kandidatene for å bekrefte at de virkelig er probiotiske.

The issues in new antibiotic discovery are pressing, because the frequent re-discovery of antibiotic scaffolds leads to few novel antibiotics discovered, besides, with the widespread use of antibacterial agents, multi-resistant pathogens are emerging, which poses more huge challenge in antibiotic discovery. However, next generation sequencing technology and bioinformatics have revealed that many secondary metabolite biosynthesis gene clusters possess the potential of producing new bioactive secondary metabolites (BSMs), which were ignored previously. Among the microorganisms, actinomycetes species are the best sources for those gene clusters. Synthetic biology is the enabling technology to activate those silent gene clusters, which aims to engineer organisms for expected applications with combination of various biotechnologies. The project employs the reciprocal regulation system between jadomycin (Jd) and chloramphenicol (Cm) in S. venezuelae: JadR1 activates Jd synthesis while represses Cm synthesis with ethanol shock. This system can be used to rationally engineer S. venezuelaee for heterologous production of BSMs with re-factored gene clusters containing appropriate control elements: deletion of the jadR1 gene shall lead to down-regulation of Jd production, simultaneously induce overproduction of Cm due to the relieved repression of the Cm structural genes’ promoters. Besides, the cml gene cluster should be completely deleted to avoid interfering with the introduced gene cluster. The appropriate control element is an inducible promoter screened out with GUS assay among cmlFp, cmlIp, cmlXp, jadJp. The inducible promoter would be used to construct an inducible system for industrial scale production of BSMs, because constitutive heterologous expression of BSMs is harmful for producing hosts.The jadR1- cml- mutants were successfully generated with Gibson Assembly, transconjugation, double crossover and replica plating. The gene cluster MP112-09-Lac was cloned with native promoter and ermE* respectively and transconjugated to jadR1- cml- mutant, however, cloing of MPS05-B41-Lin was hindered by wrong PCR amplification. The four promoters were tested with GUS assay, based on MYM medium and cmlF is speculated to be the most desirable inducible promoter.

Streptomyces venezuelae ATCC10712 which is an object of research investigation during this master thesis belongs to Actinomycetales which are widely recognized as a producer of antibiotics. Secondary metabolites synthesis in S. venezuelae varies depending from the conditions of incubation. If stress factors like ethanol are present in the medium, microorganism switches to production of jadomycin. In normal conditions however, nutrient depletion leads to subsequent synthesis of chloramphenicol. Regulation of aforementioned compounds is precisely, internally regulated by the set of adjacent genes. jadR1 and jadR2 were identified as genes having major influence in these pathways.The idea of this project was to re-engineer the antibiotic biosynthesis regulatory circuit of chloramphenicol and jadomycin in S. venezuelae, in order to make recombinant strain’s chloramphenicol production dependent on ethanol stress. Following steps took place to achieve this goal. Firstly, construction of recombinant strain with deletion of jadJ-E structural genes from S. venezuelae chromosome and their replacement with a reporter gene gusA. Another assembly was constructed with the aim of replacing cmlI-X promoter in deletion mutant with jadR1-jadJ promoter region in the chloramphenicol biosynthetic gene cluster. Planning was based on the fact that since those genes are under negative regulation of JadR1 regulator, exchange of the promoter will lead to its activation and subsequent chloramphenicol biosynthesis. Due to the difficulties which arose from the mutations in gusA gene, additional assembly with the deletion of of jadJ-E genes in S. venezuelae with another version of gusA region had to be constructed. Chloramphenicol production was assessed in S. venezuelae mutant carrying substituted promoter by UPLC analysis. Results revealed 5 fold increase of chloramphenicol expression under ethanol shock in recombinant strain, in comparison with wild-type S. venezuelae in the same conditions. When uninduced, mutant produced no chloramphenicol. This experiment was the main objective of this master thesis.In addition, construct for heterologous expression of chloramphenicol antibiotic gene cluster in Streptomyces albus was assembled. Strain was assessed for the production of this antibiotic by UPLC analysis.