DOCSLIB.ORG

Redesign of Calcium-Regulated Protein Aequorin Towards the Development

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Redesign of calcium-regulated protein aequorin towards the development of a novel ion bioreporter A Thesis submitted to University College London for the degree of Doctor of Philosophy by Evlampia-Kyriaki Dimitriadou Department of Biochemical Engineering University College London UCL April 2014 I, Evlampia-Kyriaki Dimitriadou confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. - 2 - I dedicate this work to my loving parents, Sophia and Panagiotis - 3 - Abstract This thesis aimed to design novel sensor proteins that can identify and measure various metal ions in vivo and in situ . Metal ions play key role in the metabolism of the cell, and monitoring of calcium has helped interrogate cellular processes such as fertilisation, contraction and apoptosis. Real-time monitoring of more divalent metal ions like zinc and copper is required to gain much needed insight into brain function and associated disorders, such as Alzheimer’s and Parkinson’s disease. Aequorin is a calcium-regulated photoprotein originally isolated from the jellyfish Aequorea victoria . Due to its high sensitivity to calcium and its non-invasive nature, aequorin has been used as a real-time indicator of calcium ions in biological systems for more than forty years. The protein complex consists of the polypeptide chain apoaequorin and a tightly bound chromophore (coelenterazine). Trace amounts of calcium ions trigger conformational changes in the protein, which in turn facilitate the intermolecular oxidation of coelenterazine and concomitant production of CO 2 and a flash of blue light. Aequorin’s light emitting reaction can also be triggered by a range of other divalent and trivalent cations, leading however to significantly lower light yields. Based on aequorin’s promiscuity towards other ions, this project tested the hypothesis that aequorin’s preference for certain cations could be manipulated through mutations engineered in one or more of the three calcium-binding loops (EF-hands). In order to test the hypothesis, the following six stages were performed: cloning of the apoaequorin gene for expression in E. coli ; development of a high-throughput assay for expression and measurement of bioluminescent activity in microwells; design of a library containing forty eight mutant variants of aequorin; screening of the library against seven metal ions; protein purification of wild-type aequorin and one selected mutant; analysis of activity and kinetics of purified wild type and one chosen mutant against all seven ions. This work produced mutants with shifted selectivity towards new metal ions at the cost of luminescence yield. The impact of mutations is analysed and it is suggested that one of the EF-hands (EF-I) is likely to serve as a gatekeeper to aequorin’s selectivity. It was also shown that at least one mutant utilised zinc ions (that wild type failed to utilise) to achieve low levels of bioluminescent activity. - 4 - Acknowledgements I would like to thank my supervisor, Professor Paul Dalby for his valuable input throughout the duration of my project. This work would not have been possible without his continued support. I would also like to thank my advisor, Professor Gary Lye for his advice and encouragement. Special thanks go to Dr Rigini Papi, Lazaros Batsilas and Dr Jean Aucamp for providing their insights and feedback of my work. I am also grateful to Dr Martina Micheletti for training on the robotic platform I used for all the screening work described in this thesis. I would also like to thank the PhD students of Foster Court and UB18, who made the labs feel like a second home. Finally, I am most grateful to and for my parents, Sophia and Panagiotis and my dear friends Issy, Maria, Stephanos, Rigini, Lazaros, Spyros, Erato, Jean, Zehra, Rosie and Maria W. for genuine love and support. Thank you for being patient with all my absenses and for waiting on the other side. - 5 - Contents Abstract..............................................................................................................4 Acknowledgements ...........................................................................................5 Contents .............................................................................................................6 List of tables ....................................................................................................15 List of figures...................................................................................................17 Abbreviations ..................................................................................................21 1 Introduction............................................................................................24 1.1 Bioluminescence..............................................................................24 1.1.1 Definitions....................................................................................24 1.1.2 Bioluminescence in nature...........................................................26 1.1.3 Aequorea victoria , the bioluminescent jellyfish ..........................27 1.1.4 Flash- and glow-type luminescence.............................................28 1.2 Bioreporters......................................................................................29 1.2.1 Definitions....................................................................................29 1.2.2 Need for ion bioreporters .............................................................30 1.2.3 Desirable characteristics for bioreporters.....................................31 1.2.4 Signal transduction methods ........................................................32 1.2.5 Current methods for intracellular ion monitoring ........................34 1.3 Aequorin: a calcium-sensitive photoprotein ...................................36 1.3.1 Introduction..................................................................................36 1.3.2 Uses of aequorin...........................................................................40 1.3.3 Properties of aequorin ..................................................................41 1.3.4 EF-hand motif: general features..................................................44 1.3.4.1 EF-hands and cooperativity......................................................46 1.3.5 Proposed mechanism for the bioluminescence reaction ..............47 1.3.6 Regeneration of aequorin.............................................................47 1.3.7 Ion selectivity of aequorin............................................................49 1.3.8 Other photoproteins......................................................................50 1.4 Coordination of metals in proteins...................................................51 1.5 Protein engineering ..........................................................................56 1.5.1 Rational design.............................................................................56 - 6 - 1.5.2 Directed evolution........................................................................57 1.5.3 Integration of random and rational approaches............................58 1.5.4 Metalloprotein engineering..........................................................59 1.6 Overview..........................................................................................60 1.6.1 Aequorin mutants as potential bioreporters .................................60 1.6.2 Potential activator ions for aequorin mutants...............................61 1.7 Aims and organisation of thesis.......................................................62 2 Materials and Methods..........................................................................64 2.1 Preparation of buffers and media.....................................................64 2.1.1 LB medium ..................................................................................64 2.1.2 LB agar.........................................................................................64 2.1.3 YT agar.........................................................................................65 2.1.4 SOC medium................................................................................65 2.1.5 Kanamycin ...................................................................................65 2.1.6 Ampicillin ....................................................................................65 2.1.7 X-gal.............................................................................................66 2.1.8 Tris-HCl buffer.............................................................................66 2.1.9 EDTA...........................................................................................66 2.2 Cell culture.......................................................................................67 2.2.1 E. coli strains................................................................................67 2.2.1.1 TOP10 electrocompetent ..........................................................67 2.2.1.2 BL21Star™(DE3).....................................................................67 2.2.2 Streaked plates .............................................................................67 2.2.3 Overnight cultures........................................................................68 2.2.4 Shake flask cultures......................................................................68 2.2.5 Glycerol stocks.............................................................................68
Recommended publications
  • Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents

    Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents

    International Journal of Molecular Sciences Article Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents 1, 2,3, 1 2, Elena V. Eremeeva y , Tianyu Jiang y , Natalia P. Malikova , Minyong Li * and Eugene S. Vysotski 1,* 1 Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk 660036, Russia; [email protected] (E.V.E.); [email protected] (N.P.M.) 2 Key Laboratory of Chemical Biology (MOE), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; [email protected] 3 State Key Laboratory of Microbial Technology, Shandong University–Helmholtz Institute of Biotechnology, Shandong University, Qingdao 266237, China * Correspondence: [email protected] (M.L.); [email protected] (E.S.V.); Tel.: +86-531-8838-2076 (M.L.); +7-(391)-249-44-30 (E.S.V.); Fax: +86-531-8838-2076 (M.L.); +7-(391)-290-54-90 (E.S.V.) These authors contributed equally to this work. y Received: 23 June 2020; Accepted: 27 July 2020; Published: 30 July 2020 Abstract: Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial.
  • Bioluminescence Is Produced by a Firefly-Like Luciferase but an Entirely

    Bioluminescence Is Produced by a Firefly-Like Luciferase but an Entirely

    www.nature.com/scientificreports OPEN New Zealand glowworm (Arachnocampa luminosa) bioluminescence is produced by a Received: 8 November 2017 Accepted: 1 February 2018 frefy-like luciferase but an entirely Published: xx xx xxxx new luciferin Oliver C. Watkins1,2, Miriam L. Sharpe 1, Nigel B. Perry 2 & Kurt L. Krause 1 The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the frefy. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1H NMR spectroscopy. The purifed luciferase enzyme is in the same protein family as frefy luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely diferent to that of the frefy and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confrmed by chemical synthesis and bioluminescence assays. These fndings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally diferent luciferins. Glowworms are found in New Zealand and Australia, and are a major tourist attraction at sites located across both countries. In contrast to luminescent beetles such as the frefy (Coleoptera), whose bioluminescence has been well characterised (reviewed by ref.1), the molecular details of glowworm bioluminescence have remained elusive. Tese glowworms are the larvae of fungus gnats of the genus Arachnocampa, with eight species endemic to Australia and a single species found only in New Zealand2.
  • Color-Tunable Bioluminescence Imaging Portfolio for Cell

    Color-Tunable Bioluminescence Imaging Portfolio for Cell

    www.nature.com/scientificreports OPEN Color‑tunable bioluminescence imaging portfolio for cell imaging Shota Tamaki1,3, Nobuo Kitada1,3, Masahiro Kiyama1, Rika Fujii2, Takashi Hirano1, Sung Bae Kim2* & Shojiro Maki1* The present study describes a color‑tunable imaging portfolio together with twelve novel coelenterazine (CTZ) analogues. The three groups of CTZ analogues create diverse hues of bioluminescence (BL) ranging from blue to far red with marine luciferases. We found that the hue completes the whole color palette in the visible region and shows red‑shifted BL with a marine luciferase: for example, Renilla luciferase 8 (RLuc8) and Artifcial Luciferase 16 (ALuc16) show 187 nm‑ and 105 nm‑redshifted spectra, respectively, by simply replacing the substrate CTZ with 1d. The optical properties of the new CTZ analogues were investigated such as the kinetic parameters, dose dependency, and luciferase specifcity. The 2‑series CTZ analogues interestingly have specifcity to ALucs and are completely dark with RLuc derivatives, and 3d is highly specifc to only NanoLuc. We further determined the theoretical background of the red‑shifted BL maximum wavelengths (λBL) values according to the extended π conjugation of the CTZ backbone using Density Functional Theory (DFT) calculations. This color‑tunable BL imaging system provides a useful multicolor imaging portfolio that efciently images molecular events in mammalian cells. Cells provoke diverse intracellular signal transductions in response to a myriad of stimuli from the surround- ing environment1. As cellular systems are such dynamical entities, multiplex imaging is a plausible modality for spying and visualizing such molecular events in cells. To date, bioluminescence (BL) has been broadly utilized for imaging diverse molecular events in the complex context of living subjects2.
  • Construction of a Copper Bioreporter Screening, Characterization and Genetic Improvement of Copper-Sensitive Bacteria

    Construction of a Copper Bioreporter Screening, Characterization and Genetic Improvement of Copper-Sensitive Bacteria

    Construction of a Copper Bioreporter Screening, characterization and genetic improvement of copper-sensitive bacteria Puria Motamed Fath This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science With a Major in Industrial Biotechnology, 120 ECTS credits No. 9/2009 Construction of a Copper Bioreporter-Screening, characterization and genetic improvement of copper-sensitive bacteria Puria Motamed Fath, [email protected] Master thesis Subject Category: Technology University College of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640 Examiner: Dr. Elisabeth Feuk-lagerstedt Supervisor, name: Dr. Saman Hosseinkhani Supervisor, address: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University Tehran, Iran Date: 2009-12-14 Keywords: Copper Bioreporter, Luciferase assay, COP operon, pGL3, E. Coli BL-21 ii Dedicated to my parents to whom I owe and feel the deepest gratitude iii Acknowledgment I would like to express my best appreciation to my supervisor Dr. S. Hosseinkhani for technical supports, and my examiner Dr. E. Feuk-lagerstedt for her kind attention, also I want to thank Mr. A. Emamzadeh, Miss. M. Nazari, and other students of Biochemistry laboratory of Tarbiat Modares University for their kind corporations. iv Abstract In the nature, lots of organism applies different kinds of lights such as flourescence or luminescence for some purposes such as defense or hunting. Firefly luciferase and Bacterial luciferase are the most famous ones which have been used to design Biosensors or Bioreporters in recent decades. Their applications are so extensive from detecting pollutions in the environment to medical and treatment usages.
  • OREGON ESTUARINE INVERTEBRATES an Illustrated Guide to the Common and Important Invertebrate Animals

    OREGON ESTUARINE INVERTEBRATES an Illustrated Guide to the Common and Important Invertebrate Animals

    OREGON ESTUARINE INVERTEBRATES An Illustrated Guide to the Common and Important Invertebrate Animals By Paul Rudy, Jr. Lynn Hay Rudy Oregon Institute of Marine Biology University of Oregon Charleston, Oregon 97420 Contract No. 79-111 Project Officer Jay F. Watson U.S. Fish and Wildlife Service 500 N.E. Multnomah Street Portland, Oregon 97232 Performed for National Coastal Ecosystems Team Office of Biological Services Fish and Wildlife Service U.S. Department of Interior Washington, D.C. 20240 Table of Contents Introduction CNIDARIA Hydrozoa Aequorea aequorea ................................................................ 6 Obelia longissima .................................................................. 8 Polyorchis penicillatus 10 Tubularia crocea ................................................................. 12 Anthozoa Anthopleura artemisia ................................. 14 Anthopleura elegantissima .................................................. 16 Haliplanella luciae .................................................................. 18 Nematostella vectensis ......................................................... 20 Metridium senile .................................................................... 22 NEMERTEA Amphiporus imparispinosus ................................................ 24 Carinoma mutabilis ................................................................ 26 Cerebratulus californiensis .................................................. 28 Lineus ruber .........................................................................
  • New Naphthalene Whole-Cell Bioreporter for Measuring and Assessing Naphthalene in Polycyclic Aromatic Hydrocarbons Contaminated Site

    New Naphthalene Whole-Cell Bioreporter for Measuring and Assessing Naphthalene in Polycyclic Aromatic Hydrocarbons Contaminated Site

    Accepted Manuscript New naphthalene whole-cell bioreporter for measuring and assessing naphthalene in polycyclic aromatic hydrocarbons contaminated site Yujiao Sun, Xiaohui Zhao, Dayi Zhang, Aizhong Ding, Cheng Chen, Wei E. Huang, Huichun Zhang PII: S0045-6535(17)31248-1 DOI: 10.1016/j.chemosphere.2017.08.027 Reference: CHEM 19725 To appear in: ECSN Received Date: 17 April 2017 Revised Date: 22 July 2017 Accepted Date: 7 August 2017 Please cite this article as: Sun, Y., Zhao, X., Zhang, D., Ding, A., Chen, C., Huang, W.E., Zhang, H., New naphthalene whole-cell bioreporter for measuring and assessing naphthalene in polycyclic aromatic hydrocarbons contaminated site, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.08.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 New naphthalene whole-cell bioreporter for measuring and assessing 2 naphthalene in polycyclic aromatic hydrocarbons contaminated site 3 Yujiao Sun a, Xiaohui Zhao a,b* , Dayi Zhang c, Aizhong Ding a, Cheng Chen a, Wei E. 4 Huang d, Huichun Zhang a. 5 a College of Water Sciences, Beijing Normal University, Beijing, 100875, PR China 6 b Department of Water Environment, China Institute of Water Resources and 7 Hydropower Research, Beijing, 100038, China 8 c Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, UK 9 d Kroto Research Institute, University of Sheffield, Sheffield, S3 7HQ, United 10 Kingdom 11 12 Corresponding author 13 Dr Xiaohui Zhao 14 a College of Water Sciences, Beijing Normal UniversiMANUSCRIPTty, Beijing 100875, P.
  • (12) United States Patent (10) Patent No.: US 9,574.223 B2 Cali Et Al

    (12) United States Patent (10) Patent No.: US 9,574.223 B2 Cali Et Al

    USO095.74223B2 (12) United States Patent (10) Patent No.: US 9,574.223 B2 Cali et al. (45) Date of Patent: *Feb. 21, 2017 (54) LUMINESCENCE-BASED METHODS AND 4,826,989 A 5/1989 Batz et al. PROBES FOR MEASURING CYTOCHROME 4,853,371 A 8/1989 Coy et al. 4,992,531 A 2f1991 Patroni et al. P450 ACTIVITY 5,035,999 A 7/1991 Geiger et al. 5,098,828 A 3/1992 Geiger et al. (71) Applicant: PROMEGA CORPORATION, 5,114,704 A 5/1992 Spanier et al. Madison, WI (US) 5,283,179 A 2, 1994 Wood 5,283,180 A 2f1994 Zomer et al. 5,290,684 A 3/1994 Kelly (72) Inventors: James J. Cali, Verona, WI (US); Dieter 5,374,534 A 12/1994 Zomer et al. Klaubert, Arroyo Grande, CA (US); 5,498.523 A 3, 1996 Tabor et al. William Daily, Santa Maria, CA (US); 5,641,641 A 6, 1997 Wood Samuel Kin Sang Ho, New Bedford, 5,650,135 A 7/1997 Contag et al. MA (US); Susan Frackman, Madison, 5,650,289 A T/1997 Wood 5,726,041 A 3/1998 Chrespi et al. WI (US); Erika Hawkins, Madison, WI 5,744,320 A 4/1998 Sherf et al. (US); Keith V. Wood, Mt. Horeb, WI 5,756.303 A 5/1998 Sato et al. (US) 5,780.287 A 7/1998 Kraus et al. 5,814,471 A 9, 1998 Wood (73) Assignee: PROMEGA CORPORATION, 5,876,946 A 3, 1999 Burbaum et al. 5,976,825 A 11/1999 Hochman Madison, WI (US) 6,143,492 A 11/2000 Makings et al.
  • CNIDARIA Corals, Medusae, Hydroids, Myxozoans

    CNIDARIA Corals, Medusae, Hydroids, Myxozoans

    FOUR Phylum CNIDARIA corals, medusae, hydroids, myxozoans STEPHEN D. CAIRNS, LISA-ANN GERSHWIN, FRED J. BROOK, PHILIP PUGH, ELLIOT W. Dawson, OscaR OcaÑA V., WILLEM VERvooRT, GARY WILLIAMS, JEANETTE E. Watson, DENNIS M. OPREsko, PETER SCHUCHERT, P. MICHAEL HINE, DENNIS P. GORDON, HAMISH J. CAMPBELL, ANTHONY J. WRIGHT, JUAN A. SÁNCHEZ, DAPHNE G. FAUTIN his ancient phylum of mostly marine organisms is best known for its contribution to geomorphological features, forming thousands of square Tkilometres of coral reefs in warm tropical waters. Their fossil remains contribute to some limestones. Cnidarians are also significant components of the plankton, where large medusae – popularly called jellyfish – and colonial forms like Portuguese man-of-war and stringy siphonophores prey on other organisms including small fish. Some of these species are justly feared by humans for their stings, which in some cases can be fatal. Certainly, most New Zealanders will have encountered cnidarians when rambling along beaches and fossicking in rock pools where sea anemones and diminutive bushy hydroids abound. In New Zealand’s fiords and in deeper water on seamounts, black corals and branching gorgonians can form veritable trees five metres high or more. In contrast, inland inhabitants of continental landmasses who have never, or rarely, seen an ocean or visited a seashore can hardly be impressed with the Cnidaria as a phylum – freshwater cnidarians are relatively few, restricted to tiny hydras, the branching hydroid Cordylophora, and rare medusae. Worldwide, there are about 10,000 described species, with perhaps half as many again undescribed. All cnidarians have nettle cells known as nematocysts (or cnidae – from the Greek, knide, a nettle), extraordinarily complex structures that are effectively invaginated coiled tubes within a cell.
  • Molecular Mechanism of Active Photoprotein Complex Formation

    Molecular Mechanism of Active Photoprotein Complex Formation

    Molecular Mechanism of Active Photoprotein Complex Formation Elena V. Eremeeva Thesis committee Promotors Prof. dr. W.J.H. van Berkel Personal Chair at the Laboratory of Biochemistry Prof. dr. A.J.W.G. Visser Emeritus Professor of Microspectroscopy Co-promotor Dr. E.S. Vysotski Associate professor Institute of Biophysics, Russian Academy of Sciences, Krasnoyarsk Other members Prof. dr. H. van Amerongen, Wageningen University Prof. dr. S. de Vries, Delft University of Technology Dr. J.T.M. Kennis, VU University Amsterdam Dr. S. J.J. Brouns, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Molecular Mechanism of Active Photoprotein Complex Formation Elena V. Eremeeva Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 16 January 2013 at 4 p.m. in the Aula. Elena V. Eremeeva Molecular mechanism of active photoprotein complex formation 195 pages Thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in English and Dutch ISBN 978-94-6173-458-7 Table of contents Chapter 1 General Introduction 7 Chapter 2 Ligand binding and conformational states of the photoprotein 25 obelin Chapter 3 The intrinsic fluorescence of apo-obelin and apo-aequorin and 43 use of its
  • A New Jellyfish Species in the Turkish Coastal Waters - Aequorea Forskalea Péron & Lesueur, 1810 (Cnidaria: Hydrozoa)

    A New Jellyfish Species in the Turkish Coastal Waters - Aequorea Forskalea Péron & Lesueur, 1810 (Cnidaria: Hydrozoa)

    J. Black Sea/Mediterranean Environment Vol. 19, No. 3: 380˗384 (2013) SHORT COMMUNICATION A new jellyfish species in the Turkish coastal waters - Aequorea forskalea Péron & Lesueur, 1810 (Cnidaria: Hydrozoa) Mevlüt Gürlek1, Deniz Yağlıoğlu2, Deniz Ergüden1, Cemal Turan1* 1Marine Science and Technology Faculty, Mustafa Kemal University, 31220 Iskenderun, Hatay, TURKEY 2Biodiversity Implementation and Research Center (DU–BIYOM), Duzce University, Duzce, TURKEY *Corresponding author: [email protected] Abstract Aequorea forskalea Péron & Lesueur, 1810 was recorded for the first time from the Turkish coastal waters. A. forskalea was observed from January 2012 until September 2012 in Iskenderun Bay, the northeastern Mediterranean coast of Turkey. The occurrence of this species in the Turkish coastal waters may be related to its extension from the western Mediterranean waters. Keywords: Jellyfish, Aequorea forskalea, Turkish Coastal Waters. Introduction The family Aequoreidae include five genera (Aequorea, Aldersladia, Gangliostoma, Rhacostoma and Zygocanna) in the world, of which only Aequorea and Zygocanna genera are found in the Mediterranean Sea (Bouillon et al. 2004; Schuchert 2013). The genus Aequorea has 24 species in the world and 3 species in the Mediterranean Sea (Bouillon et al. 2004; WoRMS 2013). Aequorea forskalea Péron and Lesueur, 1810, common in north-western Europe, from where it extends southwards along the west coast of Africa as far as the Gulf of Guinea, on the east coast of Africa and Indian Ocean and Mediterranean. A. forskalea also occurs on the Pacific coast of North America (Kramp 1956; 1968; Navas-Pereira and Vannucci 1991; Bouillon et al. 2004; Schuchert 2013). In the Mediterranean, A. forskalea is distributed in the eastern 380 and western Mediterranean, including the Adriatic (Bouillon et al.
  • Microbial Biodegradation and Bioremediation

    Microbial Biodegradation and Bioremediation

    5 Microbial Indicators for Monitoring Pollution and Bioremediation Tingting Xua, Nicole Perryb, Archana Chuahana, Gary Saylera and Steven Rippa aThe University of Tennessee, The Center for Environmental Biotechnology, The Joint Institute for Biological Sciences, Knoxville, TN, bDepartment of Biology, Wittenberg University, Springfield, OH 5.1 Introduction Microorganisms metabolically rely upon chemicals scavenged from their environ- ment and correspondingly defend themselves against toxic chemicals to ensure their growth and survival. These essential needs can be genetically exploited to create bioreporter organisms that can rapidly and specifically indicate the presence and bio- availability of target environmental contaminants. Using the toolbox of genetic engi- neering, genes or their operon units that are activated upon exposure to a target chemical agent or chemical class can be isolated and linked to reporter genes to serve as operational switches that bioindicate chemical presence with sensitivity and reproducibility that oftentimes rivals conventional analytical methods. Reporter genes most often consist of light signaling elements such as the bioluminescent bac- terial (lux) and firefly (luc) luciferases, green fluorescent protein (gfp as well as its palette of multicolored fluorescent derivatives), and colorimetric indicators like β-galactosidase (lacZ)(Close et al., 2009). These reporter genes are widely available within numerous types of cloning vectors for straightforward manipulation and have been successfully applied toward the creation of a large number of bioreporter organisms. The challenge in bioreporter development, however, lies in discovering and isolating the appropriate promoter gene switch that activates in response to the desired chemical target or targets. Although often provided via the evolutionary adaptation of microbial populations to new chemical bioavailability, particular pro- moter elements still must be isolated and characterized, which requires significant time, effort, and cost.
  • Identification of a Fluorescent Protein from Rhacostoma Atlantica

    Identification of a Fluorescent Protein from Rhacostoma Atlantica

    Photochemistry and Photobiology, 2016, 92: 667–677 Identification of a Fluorescent Protein from Rhacostoma Atlantica† Michael R. Tota1, Jeanna M. Allen1, Jan O. Karolin2, Chris D. Geddes2 and William W. Ward*3 1Brighter Ideas, Inc., Metuchen, NJ 2Institute of Fluorescence and Department of Chemistry & Biochemistry, University of Maryland Baltimore County, Baltimore, MD 3Department of Biochemistry and Molecular Biology, Rutgers University, New Brunswick, NJ Received 21 February 2016, accepted 2 June 2016, DOI: 10.1111/php.12609 ABSTRACT been difficult to generate a sensitive far-red variant of the Aequorea victoria green FP (4,6). Improvements are also being fl fi We have cloned a novel uorescent protein from the jelly sh made to cyan FPs by altering their fluorescent lifetimes to serve fl Rhacostoma atlantica. The closest known related uorescent as better FRET donors (7,8). It would be desirable to increase fl protein is the Phialidium yellow uorescent protein, with only the repertoire of FP sequences in the hope that novel properties a 55% amino acid sequence identity. A somewhat unusual could be discovered, from either native or mutated sequences. – – alanine tyrosine glycine amino acid sequence forms the pre- Rhacostoma atlantica has been classified as a member of the sumed chromophore of the novel protein. The protein has an order Leptomedusae and family Aequoreidae (9,10). The biolu- fl absorption peak at 466 nm and a uorescence emission peak minescent jellyfish is often seen on the coasts of the northeastern fl at 498 nm. The uorescence quantum yield was measured to United States. We have observed them from the New Jersey to fi À1 À1 be 0.77 and the extinction coef cient is 58 200 M cm .