Morten H. H. Nørholm

Bio: Morten H. H. Nørholm is an academic researcher from Technical University of Denmark. The author has contributed to research in topics: Synthetic biology & Gene. The author has an hindex of 18, co-authored 62 publications receiving 1777 citations. Previous affiliations of Morten H. H. Nørholm include University of Copenhagen & Stockholm University.

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Abstract: The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

Generation of mutation hotspots in ageing bacterial colonies

Abstract: How do ageing bacterial colonies generate adaptive mutants? Over a period of two months, we isolated on ageing colonies outgrowing mutants able to use a new carbon source, and sequenced their genomes. This allowed us to uncover exquisite details on the molecular mechanism behind their adaptation: most mutations were located in just a few hotspots in the genome, and over time, mutations increasingly were consistent with the involvement of 8-oxo-guanosine, formed exclusively on the transcribed strand. This work provides strong support for retromutagenesis as a general process creating adaptive mutations during ageing.

A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.

Abstract: Background: The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR. Results: Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertioncompatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering. Conclusions: The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.

Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning

Abstract: Simple and reliable DNA editing by uracil excision (aka USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive Here, we use two model constructs based on expression of gfp and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.

Abstract: In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

Gene silencing in plants using artificial microRNAs and other small RNAs

Abstract: Comprehensive analysis of gene function requires the detailed examination of mutant alleles In Arabidopsis thaliana, large collections of sequence-indexed insertion and chemical mutants provide potential loss-of-function alleles for most annotated genes However, limitations for phenotypic analysis include gametophytic or early sporophytic lethality, and the ability to recombine mutant alleles in closely linked genes, especially those present as tandem duplications Transgene-mediated gene silencing can overcome some of these shortcomings through tissue-specific, inducible and partial gene inactivation, or simultaneous targeting of several, sequence-related genes In addition, gene silencing is a convenient approach in species or varieties for which exhaustive mutant collections are not yet available Typically, gene function is reduced post-transcriptionally, effected by small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules A recently introduced approach is the use of artificial microRNAs (amiRNAs) Here, we review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes

Abstract: The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

A P450-centric view of plant evolution.

Abstract: Being by far the largest family of enzymes to support plant metabolism, the cytochrome P450s (CYPs) constitute an excellent reporter of metabolism architecture and evolution. The huge superfamily of CYPs found in angiosperms is built on the successful evolution of 11 ancestral genes, with very different fates and progenies. Essential functions in the production of structural components (membrane sterols), light harvesting (carotenoids) or hormone biosynthesis kept some of them under purifying selection, limiting duplication and sub/neofunctionalization. One group (the CYP71 clan) after an early trigger to diversification, has kept growing, producing bursts of gene duplications at an accelerated rate. The CYP71 clan now represents more than half of all CYPs in higher plants. Such bursts of gene duplication are likely to contribute to adaptation to specific niches and to speciation. They also occur, although with lower frequency, in gene families under purifying selection. The CYP complement (CYPomes) of rice and the model grass weed Brachypodium distachyon have been compared to view evolution in a narrower time window. The results show that evolution of new functions in plant metabolism is a very long-term process. Comparative analysis of the plant CYPomes provides information on the successive steps required for the evolution of land plants, and points to several cases of convergent evolution in plant metabolism. It constitutes a very useful tool for spotting essential functions in plant metabolism and to guide investigations on gene function.

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities

Abstract: In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca(2+)-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca(2+)-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca(2+)-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.