Application and Construction of Microbial Biosensors in Chemical Forensics Justine Couper A thesis Submitted to the Victoria University of Wellington in partial fulfillment of the requirements for the degree of Masters of Science in Molecular Microbiology School of Biological Sciences Victoria University of Wellington 2008 Abstract Forensic toxicologists are often required to rapidly determine if a suspicious substance, such as a white powder, contain toxins. Preliminary tests usually include screens for a wide range of ‘Potentially Toxic Chemicals’ (PTCs) such as cyanide, pesticides, herbicides, medicinal and illicit drugs. Subsequent analyses are generally very time-consuming and costly. Any protocol screening for a range of PTC’s, prior to more robust chemical analysis, could therefore save significant analytical time. Microbial biosensors are ideal biological tools that can be utilised for these purposes. In vivo bioassays were developed for a range of PTCs using a suite of microbial biosensors, in a variety of complex matrices including water, white powders, soils and vomit to determine the effect of matrix complexities on the biosensors, as well as the toxins. The lux biosensor , Escherichia coli HB101 pUCD607, showed an EC 50, (where EC 50 is the effective concentration of toxin causing 50% reduction in bioluminescence), of cyanide in water of 20 mg/L. This biosensor still detected cyanide, in talc and flour, at EC 50 values of 589 mg/L and 700 mg/L respectively. Vibrio harveyi showed good sensitivity to cyanide in initial water bioassays with an EC 50 of 9.66 mg/L. The V. harveyi biosensor did not detect cyanide spiked in talc or flour when tested up to a maximum concentration of 10,000 mg/L. The Mycena citricolor ATCC 34884 fungal biosensor, showed lower sensitivity levels however it detected the presence of sodium monofluoroacetate (1080) at a concentration 1000 mg/L. Preliminary investigation of a novel, faster, solid-phase sample preparation method was also undertaken and its potential proven, particularly in PTC spiked white powders. Here the biosensor showed sensitivity to arsenate, arsenite, copper, cyanide and PCP at 1000 mg/L. ii This project highlighted the inability of current biosensors to reliably detect 1080 and the difficulty in constructing a specific biosensor. The utilisation of a reliable vector and inducible promoter are pivotal in biosensor construction. iii Acknowledgements I would like to express my sincere thanks and appreciation to a fantastic and supportive team of supervisors: Dr Jacqui Horswell and Dr Ronan O’Toole. Their doors were always open for me. A special thanks to Jacqui, my day-to-day supervisor, who gave me this opportunity and didn’t laugh, sigh or cry at stupid questions. I would also like to thank the students and staff at Vic Uni Lab 817 (Shahista Nisa, Jeremy Owen, Gareth Prosser and David Ackerley) for the advice, fun and coffees. Thanks to the staff and students at the Population and Environmental Health research lab at ESR; especially my room buddies Melanie Goucher and Alice Johnstone - Alice was like the concierge in our office – she just knows everything!!!!. Professor Anne Glover & Dr Hedda Weitz deserve mention for sending me the fungi used to achieve the fungal bioassay results and answering my questions. Zac, Jake and Teagan, my three children, have sometimes felt mother-less and have only ever known their mother to be studying therefore the biggest thanks must also go to them and my husband, David, who invariably picked up the pieces and without his support I would never have tried to do this. iv Table of Contents Abstract .................................................................................................................ii Acknowledgements ..............................................................................................iv Table of Contents ................................................................................................. v List of Tables .......................................................................................................vii List of Figures.......................................................................................................ix CHAPTER ONE: INTRODUCTION ...................................................................... 1 1.2.1 Investigation of a Suite of General Biosensors.................................... 2 1.2.2 Development of a Specific Bacterial Biosensor................................... 2 1.3.1 Microbial Biosensors ........................................................................... 5 1.3.1.1 lux -Based Bacterial Biosensors (non-specific) ........................... 8 1.3.1.2 Genetically Modified Non-Specific Biosensors ......................... 11 1.3.1.3 Biosensor Case Study: Vomit Bioassay. .................................. 13 1.3.1.4 Naturally Luminescent Fungi.................................................... 14 1.3.2 The Development of a Specific Bacterial Biosensor: ‘1080-lux ’. ....... 19 CHAPTER TWO: MATERIALS AND METHODS ............................................... 28 2.1.1 Bacterial Biosensor Preparation Methods ......................................... 28 2.1.2 Fungal Biosensor Preparation Methods. ........................................... 31 2.1.3 Bioassays.......................................................................................... 35 2.1.3.1 White Powder Bioassays.......................................................... 37 2.1.3.2 Vomit Bioassays....................................................................... 39 2.2.1 Method of Solid-Phase sample Preparation...................................... 40 2.2.2 Statistical Analysis and Data Interpretation....................................... 45 2.3.1 Methods of Construction of the Specific Bacterial Biosensor. ........... 46 2.3.2 Construction of 1080-lux and JlacO-lux constructs. .......................... 51 CHAPTER THREE: RESULTS........................................................................... 60 3.1.1 Characterisation of Bacterial Biosensors........................................... 60 3.1.2 Characterisation of Fungal Biosensors.............................................. 62 3.3.1.1 PTC Methanol Extraction. ........................................................ 78 3.3.1.2 Case Study: Vomit Bioassay .................................................... 80 3.5.1 Construction of 1080-lux . .................................................................. 91 3.5.2 Screening for Positive Insert Clones. ................................................ 93 3.5.3 Construction of positive control JlacO luxCDABE constructs. ........... 98 3.5.4 Screening for JlacO promoter positive insert clones. ........................ 99 CHAPTER FOUR: DISCUSSION ..................................................................... 102 4.1.1 Bacterial Biosensors. ...................................................................... 102 4.1.1.1 Case Study: Vomit Bioassay .................................................. 106 4.1.2 Fungal Biosensors. ......................................................................... 107 4.5.1 Metabolic Biosensor Bioassays....................................................... 117 4.5.2 Solid-Phase Sample Preparation. ................................................... 118 4.5.3 Specific 1080-lux biosensor. ........................................................... 119 v REFERENCES................................................................................................. 120 vi List of Tables Table 1-1:Traditional approaches to measuring the presence of toxins. .............. 3 Table 1-2: Types and characteristics or biosensors used in environmental monitoring...................................................................................................... 4 Table 1-3: E. coli HB101 pUCD607 biosensor response to a range of toxins in comparison to concentrations found in samples from fatal poisonings........ 12 Table 1-4: Comparison of bacterial biosensors and potential of naturally luminescent fungi as biosensors. ................................................................ 17 Table 1-5: Oral Toxicity of 1080 for susceptible species. ................................... 24 Table 2-1: Basidiomycete fungi isolates chosen for investigation of bioluminescence and globular mycelial growth patterns.............................. 32 Table 2-2: PTCs used in this non-specific biosensor investigation including uses, application rates and LD 50 s where applicable. ................................... 36 Table 2-3: Optimal pH range for bioluminescence of microbial biosensors used in PTC bioassays................................................................................ 37 Table 2-4: Bacterial strains and plasmids used in the construction of specific ‘1080-lux ’ bacterial biosensor and the ‘J lac O-positive control’ bacterial biosensor..................................................................................................... 47 Table 2-5: Primers used in PCR amplification. ................................................... 52 Table 2-6: PCR protocols used for the amplification of the 1080 dehalogenase promoter and the J lac O positive control promoter fragments. ..................... 53 Table 2-7: PCR protocols used for the amplification of inserts for determination of promoter orientation in trannsformants. ............................ 58 Table 3-1: Luminescence of sourced potential fungal biosensors on three different agars