Attributions and thanks

All experiments conducted in this project has been designed, executed and analysed by members of the iGEM Group Stockholm. We, as iGEM Team Stockholm, are confidently standing behind the presented data. We have put a special effort not to modify the actual picture that we got by conducting our experiments in any way.

Our project has been developed independently from other projects. We are not really building up on a single project. We discovered that the iGEM Team from Dundee 2013 has been working with a chimeric version of the EnvZ as well. Therefore, we have analysed their project and adjusted our experiments accordingly. After request, we were also very happy to receive the EnvZ deficient E.Coli strain BW25113 from Dundee, in person of Mr. John Allan.

We have been also inspired by the work that has been already conducted with the OmpR signaling cascade. Notably, the former iGEM Teams from Technion Israel.

Supervisors

• Johan Rockberg, KTH Royal Institute of Technology. He heads a research group at the department of Proteomics and nanotechnology at the KTH School of Biotechnology. His projects are centered on the studying and engineer- ing of binding surfaces of proteins for therapy and development of cell factories for sustainable production of pharmaceuticals in mammalian and microbial hosts.

• Ute Römling, Karolinska Institutet. She is Professor of Medical Microbial Physiology at the Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet. The major research topic of her group is regulation of microbial biofilm formation by cyclic di-GMP second messenger signaling and other factors. In addition, interactions of biofilm forming cells with the host organism and persistence mechanisms are investigated.

• Håkan Jönsson, KTH Royal Institute of Technology. He is an Assistant Professor at KTH Biotechnology heading the Biomicrofluidics research group at Science for Life Laboratory in Solna. His research centers on microfluidic high throughput single cell analysis and screening in biomedical and biosustainability settings.

• Teresa Frisan, Karolinska Institutet. She leads a group at the Department of Cell and Molecular Biology at Karolinska Institutet which does research on the carcinogenic properties of chronic infection with CDT-producing bacteria.

At this point, we would like to thank our supervisors who have been accompanying us throughout the entire project. They provided us with invaluable feedback at the weekly meetings, gave us new ideas and promoted our research. We are glad for their engagement for this student-driven research project and that they have been helping us not only scientifically but also administratively to get insurance and a laboratory facility in which we could perform our research in.

Advisor

• Roger Draheim, University of Portsmouth, is our primary advisor. He has been formerly working at the Stockholm University and is an expert in the field of Osmoregulation via EnvZ-OmpR pathways. He has been independently conducting similar experiments as we have and we could thus compare our data to his results. Among these experiments, you can find the prediction of EnvZ (extracellular domain) and the characterization of the EnvZ gene in his expression vector from which we derived our EnvZ biobrick (BBa_1766008). In both given examples, we got to similar results and can therefore give a confident conclusion to these results presented. He has provided us with the MDG147 and EPB30 strains which have a genetically integrated YFP/CFP reporter system depending on the phosphorylation status of OmpR. We would like to thank Roger for his advice, feedback and constructive suggestions throughout the entire project.

Others

• Gunnar von Heijne, Stockholm University, has provided us with many good contacts (among which we find Roger Draheim) and has helped us to get the answers we needed from experts in the field. His kind and immediate reactions to questions related to our project has been very much acknowledged.

• Martin Högbom, Stockholm University, is a structural biologist and has been supporting our project as we were creating our own chimeric constructs. He gave us an insight in the possibilities of protein folding and the strengths and weaknesses for structure predictions.

• Jan Willem De Gier, Stockholm University, supported our project by giving us strong advice in questions related to surface protein expression. We furthermore could discuss with him the possibility of using spheroplasts for testing gene expression after our system. He kindly provided us with a protocol for spheroplasting E.Coli strains.

• Tae Hyun Kang, The University of Texas Austin, is a PhD who has been supporting our project by sending us a protocol for the production of spheroplasts from E.Coli. We are very glad about our correspondance with him and for all the answers to our several questions.

• Francis Jingxin Hu, KTH Royal Institute of Technology, is a PhD student from the laboratory of Johan Rockberg and he has been showing us different microbiological techniques to start our project with.

• Niklas Thalén, KTH Royal Institute of Technology, working mainly with fluorescent agents. He is another PhD student of the laboratory of Johan Rockberg and has been introducing us to the plate reader we could use and has been furthermore providing us with more information related to fluorescent measurements.

• Ken Andersson, KTH Royal Institute of Technology, has provided us with advice and suggestions in the laboratory. He helped us producing big amounts of competent cells and has been, besides his work as PhD student, always willing to help us in our project.

• Matheus Dyczynski, Karolinska Institutet, is a Post-Doc who has been working with different cloning and transformation methods. We would like to thank Matheus for giving us a seminar about cloning and transformation methods and to be a contact for cloning related questions.

• Jan-Olof Höög, Karolinska Institutet, has supported this group from the very beginning before the teams was yet formed or registered. He helped to pass administrational barriers and supported the team with the right contacts and short cuts to make it to this years competition.

• Lars-Arne Haldosen, Karolinska Institutet, represents first supporters of this year’s iGEM team. He helped to make the participation in this competition popular and supported our project throughout the entire process of registration, insurance and laboratory search.

• Anders Andersson, KTH Royal Institute of Technology, is the most influential “close-to-be” supervisor. He has supported the project with new ideas and advice in its founding phase and provided us with new insights. Unfortunately, he had to drop out of the project, however we thank you for your kind discussions.

• Hans-Georg Koch, University Freiburg, has been providing us with a protocol for subcellular fragmentation, which we haven’t been able to use during the short time of our project, but we would like to acknowledge his spirit and kindness to provide the protocol.

• Agneta Richter-Dahlfors, Karolinska Institutet, has allowed us to use a plate reader capable of measuring OD and fluorescence at the same time in order to analyze the strains sent by our collaborator from ETH Zurich.

• Annika Cimdins, Karolinska Institutet, has been supervising our experiments in the very last weeks of the project in which we were working in the laboratory of our supervisor Ute Römling.

• Anna-Luisa Volk, KTH Royal Institute of Technology, has supported the team with Western blot and ELISA materials.

• Michel Sadelain, Memorial Sloan Kettering Cancer Center, has provided us a detailled insight in the design and conception of chimeric antigen receptors (CARs). We would like to thank Prof. Sadelain the valuable discussions about creating a Bacterial Antigen Receptor in a simular ways as CARs.

• Gunaratna Kuttuva Rajarao, KTH Royal Institute of Technology, has given us a proper introduction into the fully equiped student's lab that we could use during the summer to perform our experiments. Furthermore, she provided us with all information regarding good laboratory practice and biosafety.

Mitochondria class sponsor

Connecting solutions to drive healthcare performance.

IMS Health is a leading global information and technology services company providing clients in the healthcare industry with comprehensive solutions to measure and improve their performance.

End-to-end proprietary applications and configurable solutions connect 10+ petabytes of complex healthcare data through theIMS One™ cloud-based master data management platform, providing comprehensive insights into diseases, treatments, costs and outcomes.

The company’s 15,000 employees blend global consistency and local market knowledge across 100 countries to help clients run their operations more efficiently. Customers include pharmaceutical, consumer health and medical device manufacturers and distributors, providers, payers, government agencies, policymakers, researchers and the financial community.

Ribosome class sponsor

Based in Stockholm Sweden, Atlas Antibodies provide advanced research reagents targeting all human proteins to researcher world-wide. Triple A Polyclonals are advanced antibodies developed and validated in the prestigious Human Protein Atlas (HPA) project, presented with an unmatched amount of characterization data. Our in-house developed PrecisA Monoclonals are precise, accurate and targeted. We also provide QPrEST, a family of isotope labeled multipeptide Mass Spectrometry standards for absolute quantification of proteins, developed by the HPA project.

Vesicle class sponsors

HealthCap is one of the largest venture capital firms worldwide with an exclusive focus on life science and innovative medical therapies. Since 1996, we have created a strong track record through more than 100 investments, thanks to an experienced and dedicated team, bringing over 40 disruptive medical products to the global market for the benefit of patients, payers and investors.

Sobi’s mission is to develop and deliver innovative therapies and services to help improve the lives of people living with rare diseases. We are committed to co-creating with the rare disease community with the simple goal of providing people with rare diseases choices that help them live the lives they wish for.

Material sponsors

QIAGEN is the world’s leading provider of innovative Sample & Assay Technologies, products used worldwide by customers to isolate genetic material from biological samples and to reveal the information concealed within.

Eppendorf is a leading life science company that develops and sells instruments, consumables, and services for liquid handling, sample handling, and cell handling in laboratories worldwide.

Bio Basic is a life science research‐support company which is dedicated to providing the highest quality research products and services at the most affordable rates in the industry.

Since 1990, GATC Biotech has dedicated all resources to advancing DNA and RNA sequencing projects reaching from basic research to personalised medicine. Leading technologies, innovative products and expert analysis define our brand.

Sigma-Aldrich, a leading Life Science and Technology company focused on enhancing human health and safety, manufactures and distributes 230,000 chemicals, biochemicals and other essential products and 40,000 equipment products to more than 1.4 million customers globally - Sigma-Aldrich is committed to enabling science to improve the quality of life.

Designed by scientists, SnapGene is the fastest, easiest way to plan, visualize and simulate your everyday cloning experiments.

Bio-Techne the home of leading bioscience brands: R&D Systems – Renowned high-quality immunoassay, protein and antibody provider. Novus Biologicals – >200,000 antibodies and related products. Tocris – leading supplier of biologically active molecules.