@misc{GoethelListekMesserschmidtetal.2021, author = {G{\"o}thel, Markus and Listek, Martin and Messerschmidt, Katrin and Schl{\"o}r, Anja and H{\"o}now, Anja and Hanack, Katja}, title = {A New Workflow to Generate Monoclonal Antibodies against Microorganisms}, series = {Mathematisch-Naturwissenschaftliche Reihe}, journal = {Mathematisch-Naturwissenschaftliche Reihe}, number = {20}, issn = {1866-8372}, doi = {10.25932/publishup-52334}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-523341}, pages = {17}, year = {2021}, abstract = {Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.}, language = {en} } @article{GoethelListekMesserschmidtetal.2021, author = {G{\"o}thel, Markus and Listek, Martin and Messerschmidt, Katrin and Schl{\"o}r, Anja and H{\"o}now, Anja and Hanack, Katja}, title = {A New Workflow to Generate Monoclonal Antibodies against Microorganisms}, series = {Applied Sciences}, volume = {11}, journal = {Applied Sciences}, number = {20}, publisher = {MDPI}, address = {Basel}, issn = {1454-5101}, doi = {10.3390/app11209359}, pages = {15}, year = {2021}, abstract = {Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.}, 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{RieckGeigerMunkertetal.2019, author = {Rieck, Christoph Paul Kurt and Geiger, Daniel and Munkert, Jennifer and Messerschmidt, Katrin and Petersen, Jan and Strasser, Juliane and Meitinger, Nadine and Kreis, Wolfgang}, title = {Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae}, series = {Microbiologyopen}, volume = {8}, journal = {Microbiologyopen}, number = {12}, publisher = {Wiley}, address = {Hoboken}, issn = {2045-8827}, doi = {10.1002/mbo3.925}, pages = {11}, year = {2019}, abstract = {A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5-3β-hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5-isomerase gene from Comamonas testosteronii, (c) a mutated steroid-5β-reductase gene from Arabidopsis thaliana, and (d) a steroid 21-hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed "CARD II yeast", was capable of producing 5β-pregnane-3β,21-diol-20-one, a central intermediate in 5β-cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.}, language = {en} } @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{delaCruzMachensMesserschmidtetal.2019, author = {de la Cruz, Jorge Gonzalez and Machens, Fabian and Messerschmidt, Katrin and Bar-Even, Arren}, title = {Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast}, series = {ACS synthetic biology}, volume = {8}, journal = {ACS synthetic biology}, number = {5}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.8b00464}, pages = {911 -- 917}, year = {2019}, abstract = {One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.}, language = {en} } @article{StuckasMesserschmidtPutzleretal.2009, author = {Stuckas, Heiko and Messerschmidt, Katrin and Putzler, Sascha and Baumann, Otto and Schenk, J{\"o}rg A. and Tiedemann, Ralph and Micheel, Burkhard}, title = {Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production}, issn = {1040-452X}, doi = {10.1002/Mrd.20916}, year = {2009}, abstract = {The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76\%) which explains the cross reactivity of mAb G26- AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.}, language = {en} } @article{HessBartelRothetal.2012, author = {Hess, Anne-Katrin and Bartel, Manuela and Roth, Karina and Messerschmidt, Katrin and Heilmann, Katja and Kenchington, Ellen and Micheel, Burkhard and Stuckas, Heiko}, title = {Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females}, series = {Molecular reproduction and development}, volume = {79}, journal = {Molecular reproduction and development}, number = {8}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1040-452X}, doi = {10.1002/mrd.22056}, pages = {517 -- 524}, year = {2012}, abstract = {Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females.Mol. Reprod. Dev. 79: 517-524, 2012.}, 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} } @misc{MesserschmidtMachensHochreinetal.2018, author = {Messerschmidt, Katrin and Machens, Fabian and Hochrein, Lena and Naseri, Gita}, title = {Orthogonal, light-inducible protein expression platform in yeast Sacchararomyces cerevisiae}, series = {New biotechnology}, volume = {44}, journal = {New biotechnology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1871-6784}, doi = {10.1016/j.nbt.2018.05.153}, pages = {S19 -- S19}, year = {2018}, language = {en} }