@article{MesserschmidtHochreinDehmetal.2016, author = {Messerschmidt, Katrin and Hochrein, Lena and Dehm, Daniel and Schulz, Karina and Mueller-Roeber, Bernd}, title = {Characterizing seamless ligation cloning extract for synthetic biological applications}, series = {Analytical biochemistry : methods in the biological sciences}, volume = {509}, journal = {Analytical biochemistry : methods in the biological sciences}, publisher = {Elsevier}, address = {San Diego}, issn = {0003-2697}, doi = {10.1016/j.ab.2016.05.029}, pages = {24 -- 32}, year = {2016}, abstract = {Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular. and synthetic biology projects. (C) 2016 Elsevier Inc. All rights reserved.}, language = {en} } @article{HochreinMachensGremmelsetal.2017, author = {Hochrein, Lena and Machens, Fabian and Gremmels, Juergen and Schulz, Karina and Messerschmidt, Katrin and Mueller-Roeber, Bernd}, title = {AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies}, series = {Nucleic acids research}, volume = {45}, journal = {Nucleic acids research}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkx034}, pages = {12}, year = {2017}, abstract = {The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields.}, language = {en} } @article{HochreinMitchellSchulzetal.2018, author = {Hochrein, Lena and Mitchell, Leslie A. and Schulz, Karina and Messerschmidt, Katrin and M{\"u}ller-R{\"o}ber, Bernd}, title = {L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast}, series = {Nature Communications}, volume = {9}, journal = {Nature Communications}, publisher = {Nature Publ. Group}, address = {London}, issn = {2041-1723}, doi = {10.1038/s41467-017-02208-6}, pages = {10}, year = {2018}, abstract = {The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a lightcontrolled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome reengineering project Sc2.0 or in other recombination-based systems.}, language = {en} } @article{SedaghatmehrThirumalaikumarKamranfaretal.2021, author = {Sedaghatmehr, Mastoureh and Thirumalaikumar, Venkatesh P. and Kamranfar, Iman and Schulz, Karina and M{\"u}ller-R{\"o}ber, Bernd and Sampathkumar, Arun and Balazadeh, Salma}, title = {Autophagy complements metalloprotease FtsH6 in degrading plastid heat shock protein HSP21 during heat stress recovery}, series = {The journal of experimental botany : an official publication of the Society for Experimental Biology and of the Federation of European Societies of Plant Physiology}, volume = {72}, journal = {The journal of experimental botany : an official publication of the Society for Experimental Biology and of the Federation of European Societies of Plant Physiology}, number = {21}, publisher = {Oxford University Press}, address = {Oxford}, issn = {0022-0957}, doi = {10.1093/jxb/erab304}, pages = {7498 -- 7513}, year = {2021}, abstract = {Moderate and temporary heat stresses prime plants to tolerate, and survive, a subsequent severe heat stress. Such acquired thermotolerance can be maintained for several days under normal growth conditions, and can create a heat stress memory. We recently demonstrated that plastid-localized small heat shock protein 21 ( HSP21) is a key component of heat stress memory in Arabidopsis thaliana. A sustained high abundance of HSP21 during the heat stress recovery phase extends heat stress memory. The level of HSP21 is negatively controlled by plastid-localized metalloprotease FtsH6 during heat stress recovery. Here, we demonstrate that autophagy, a cellular recycling mechanism, exerts additional control over HSP21 degradation. Genetic and chemical disruption of both metalloprotease activity and autophagy trigger superior HSP21 accumulation, thereby improving memory. Furthermore, we provide evidence that autophagy cargo receptor ATG8-INTERACTING PROTEIN1 (ATI1) is associated with heat stress memory. ATI1 bodies co-localize with both autophagosomes and HSP21, and their abundance and transport to the vacuole increase during heat stress recovery. Together, our results provide new insights into the module for control of the regulation of heat stress memory, in which two distinct protein degradation pathways act in concert to degrade HSP21, thereby enabling cells to recover from the heat stress effect at the cost of reducing the heat stress memory.}, language = {en} } @article{ThirumalaikumarGorkaSchulzetal.2020, author = {Thirumalaikumar, Venkatesh P. and Gorka, Michal and Schulz, Karina and Masclaux-Daubresse, Celine and Sampathkumar, Arun and Skirycz, Aleksandra and Vierstra, Richard D. and Balazadeh, Salma}, title = {Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90.1 and ROF1}, series = {Autophagy}, volume = {17}, journal = {Autophagy}, number = {9}, publisher = {Taylor \& Francis}, address = {Abingdon}, issn = {1554-8635}, doi = {10.1080/15548627.2020.1820778}, pages = {2184 -- 2199}, year = {2020}, abstract = {In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS inArabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of annbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression ofHSPgenes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation ofNBR1resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.}, language = {en} }