| 1. |
- Johnsson, Ola, et al.
(författare)
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A novel feeding strategy for industrial fed-batch processes based on frequency content analysis
- 2012
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 29:Supplement, s. 11-11
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Konferensbidrag (refereegranskat)abstract
- Overflow metabolism, i.e. the production of metabolic by-products at a high glycolytic flux, is a recurring problem in fed-batch processes with many types of microorganisms. In the current study, a novel feeding strategy aimed at avoiding process failures due to overflow by-product formation was designed and implemented in a pilot-scale reactor (0.5 m3). The basic principle behind the strategy was to analyze the effects on the dissolved oxygen concentration by periodic variations in the inlet feed rate. The frequency spectrum of the dissolved oxygen signal was used to estimate the proximity of the system to the region where overflow metabolism occurs by examining the content in the relevant frequency range. A control variable based on the measured frequency content was subsequently used to control the feed rate. The only measurement required for this strategy is the dissolved oxygen level in the broth, for which robust, fast and precise probes are widely available in industrial fermentors today. The strategy was successfully implemented in pilot-scale processes for industrial enzyme production using Bacillus licheniformis. It was shown possible to run the process close to the optimal feed rate, indicated by very low amounts of acetate (the overflow metabolite) in the broth. In comparison to a reference strategy the new control strategy resulted in over 10% higher biomass yields.
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| 2. |
- Löfdahl, Per-Åke, 1959-, et al.
(författare)
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Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
- 2009
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Ingår i: New Biotechnology. - Amsterdam : Elsevier. - 1871-6784 .- 1876-4347. ; 26:5, s. 251-259
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Tidskriftsartikel (refereegranskat)abstract
- Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.
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| 3. |
- Heimersson, Sara, 1984, et al.
(författare)
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Methodological issues in life cycle assessment of mixed-culture polyhydroxyalkanoate production utilising waste as feedstock
- 2014
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 31:4, s. 383-393
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Tidskriftsartikel (refereegranskat)abstract
- Assessing the environmental performance of emerging technologies using life cycle assessment (LCA) can be challenging due to a lack of data in relation to technologies, application areas or other life cycle considerations, or a lack of LCA methodology that address the specific concerns. Nevertheless, LCA can be a valuable tool in the environmental optimisation in the technology development phase. One emerging technology is the mixed-culture production of polyhydroxyalkanoates (PHAs). PHA production by pure microbial cultures has been developed and assessed in several LCAs during the previous decade. Recent developments within mixed-culture PHA production call for environmental assessment to guide in technology development. Mixed-culture PHA production can use the organic content in wastewater as a feedstock; the production may then be integrated with wastewater treatment (WWT) processes. This means that mixed-culture PHA is produced as a by-product from services in the WWT.This article explores different methodological challenges for LCA of mixed-culture PHA production using organic material in wastewater as feedstock.LCAs of both pure- and mixed-culture PHA production were reviewed. Challenges, similarities and differences when assessing PHA production by mixed- or pure-cultures were identified and the resulting implications for methodological choices in LCA were evaluated and illustrated, using a case study with mixed- and pure-culture PHA model production systems, based on literature data.Environmental impacts of processes producing multiple products or services need to be allocated between the different products or services. Such situations occur both in feedstock production and when the studied system is providing multiple functions. The selection of allocation method is shown to determine the LCA results. The type of data used, for electricity in the energy system, is shown to be important for the results, which indicates, a strong regional dependency of results for systems with electricity use as an environmental hot spot. The importance of assessing water use, an environmental impact not assessed by any of the reviewed studies, is highlighted. © 2013 Elsevier B.V.
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| 4. |
- Gustavsson, Elin, et al.
(författare)
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Surrogate antigens as targets for proteome-wide binder selection
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 28:4, s. 302-311
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Tidskriftsartikel (refereegranskat)abstract
- In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.
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| 5. |
- McGinn, Steven, et al.
(författare)
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New Technologies for DNA analysis-A review of the READNA Project.
- 2016
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784.
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Forskningsöversikt (refereegranskat)abstract
- The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
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| 6. |
- Mezger, Anja, et al.
(författare)
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Highly specific DNA detection employing ligation on suspension bead array readout
- 2015
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 32:5, s. 504-510
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Tidskriftsartikel (refereegranskat)abstract
- We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.
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| 7. |
- Nyman, Jonas, et al.
(författare)
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Pellet formation of zygomycetes and immobilization of yeast
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:5, s. 516-522
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Tidskriftsartikel (refereegranskat)abstract
- Pelleted growth provides many advantages for filamentous fungi, including decreased broth viscosity, improved aeration, stirring, and heat transfer. Thus, the factors influencing the probability of pellet formation of Rhizopus sp. in a defined medium was investigated using a multifactorial experimental design. Temperature, agitation intensity, Ca2+-concentration, pH, and solid cellulose particles, each had a significant effect on pelletization. Tween 80, spore concentration, and liquid volume were not found to have a significant effect. All of the effects were additive; no interactions were significant. The results were used to create a simple defined medium inducing pelletization, which was used for immobilization of a flocculating strain of Saccharomyces cerevisiae in the zygomycetes pellets. A flor-forming S. cerevisiae strain was also immobilized, while a non-flocculating strain colonized the pellets but was not immobilized. No adverse effects were detected as a result of the close proximity between the filamentous fungus and the yeast, which potentially allows for co-fermentation with S. cerevisiae immobilized in pellets of zygomycetes
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| 8. |
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| 9. |
- Sjöberg, Ronald, et al.
(författare)
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Validation of affinity reagents using antigen microarrays
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 555-563
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.
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| 10. |
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