Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

Summary

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

Abstract

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

Introduction

A biosensor is any analytical device that uses biological components (such as proteins, nucleic acids, or whole organisms) that produce a response that can be used for the detection of a chemical substance or analyte. As an example, the coal mining industry used a biosensor for much of the 20^th century to detect the presence of toxic mine gases: the canary in the coal mine¹. The biological organism's (canary's) response (death or distress) to a chemical analyte (carbon monoxide) was observed by the miners in order to protect the workers. In a more modern and sophisticated example, bacteria can be altered using synthetic biolo....

Protocol

NOTE: Synthesis of E. coli expressing RFP is presented.

1. Preparation of plasmid DNA from E. coli

1. Thaw E.coli containing a plasmid with an RFP gene and ampicillin resistance gene and grow the E.coli on Luria Broth (LB) agar plates containing 100 µg/mL ampicillin at 37 °C for 24 h. For example, use the J10060 plasmid from the registry of standard biological parts used for synthetic biology (see Table of Materials). The J10060 plasmid includes a gene for RFP (red fluorescent protein) under the control of a pBad promoter region and an ampicillin resistance gene. Altern....

Results

Fluorescence spectra for the RFP variant used in this work are shown in Figure 2. These data are from the PbRFP device as it responds to lead and the TNT-RFP device as it responds to two analytes, 2,4-DNT and 1,3-DNB. This figure shows the spectrum of a negative control (no analyte added), and the spectra at two different levels of analyte added. The maximum fluorescence signal for the RFP variant used was observed at 575 nm (excitation wavelength 500 nm). The data in

Discussion

Modifications and troubleshooting

The experiment described in Table 4 can be modified in any way appropriate to the sensors that have been designed. The most important aspect of a chemical sensor is to evaluate its sensitivity and specificity. It is beneficial to ensure that a wide range of concentrations of the analyte is analyzed to determine the useful analytical range of the sensor. It is also worth determining a maximum level of analyte for the cells. Bec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,3-dinitrobenzene, 97%	Aldrich	D194255-25G
2,4-dinitrotoluene, 97%	Aldrich	101397-5G
Agar	Fisher Scientific	BP1423-500
Ampicillin	Fisher Scientific	BP1760-5
Antimony, Reference Standard Solution (1000ppm ±1%/Certified)	Fisher Scientific	SA450-100	Standard in dilute HNO3
Cut Smart Buffer	New England BioLabs	B7204S
Duplex Buffer	Integrated DNA Technologies	11-01-03-00
EcoRI-HF Restriction Enzyme	New England BioLabs	R3101S
Ethanol, HPLC grade, denatured	Acros Organics	AC611050040	Solvents do not need to be HPLC grade, ACS or reagent grade will work.
Eurofins Genomics SimpleSeq DNA Sequencing Kits	Eurofins Genomics	SimpleSeq Kit Standard
Forward primer for colony PCR	Integrated DNA Technologies		5’- GCCGCTTGAATTCGTCATATAT-3’
Forward primer for DNA sequencing	Integrated DNA Technologies		5’- GTAAAACGACGGCCAGTG-3’
IBI Science High Speed Plasmid Mini-kit	IBI Scientific	IB47101
LB Broth, Miller	Fisher Scientific	BP1426-500
Lead, Reference Standard Solution (1000ppm ±1%/Certified)	Fisher Scientific	SL21-100	Standard in dilute HNO3
LeadOff Disposable Cleaning and Decon Wipes	Hygenall	45NRCN	Sold in canisters or individually wrapped, any alcohol based wipe will work.
Methanol, HPLC grade	Fisher Scientific	A452-4	Solvents do not need to be HPLC grade, ACS or reagent grade will work.
NEB 5-alpha Competent E. coli cells	New England BioLabs	C2987I
NheI-HF Restriction Enzyme	New England BioLabs	R3131S
Nuclease free water	New England BioLabs	B1500S
OneTaq 2X Master Mix with Standard Buffer	New England BioLabs	M0482S
Plasmids from the registry of standard biological parts used for synthetic biology	Registry of Standard Biological Parts		http://parts.igem.org/Main_Page
Promoter Sequences	Integrated DNA Technologies		Sb promoter: 5’-GCATGAATTCA GTCATATATGTTTTTGACTTATCC GCTTCGAAGAGAGAGACACTACCT GCAACAATCGCTAGCGCAT-3’ 3’-CGTACTTAAGCTCACTATA TACAAAAACTGAATAGGCGAAGC TTCTCTCTCTGTGATGGACGTTG TTAGCGATCGCGTA-5’ Pb promoter: 5’-GCATGAATTCG TCTTGACTCTATAGTAACTAAGGG TGTATAATCGGCAACGCG AGCTAGCGCAT-3’ 3’-CGTACTTAAGCAGAA CTGAGATATCATTGATCTCCCACA TCTTAGCCGTTGCGCTGCGATCGC GTA-5’ TNT promoter: 5’GCATTCTAGAT CAATTTATTTGAACAAGGCGGTCA ATTCTCTTCGATTTTATCTCTCGT AAAAAAACGTGATACTCATCACAT CGACGAAACAACGTCACTTATACA AAAATCACCTGCGAGAGATTAATT GAATTCGCAT3’ 3’CGTAAGATCTAGTTAA ATAAACTTGTTCCGCCAGTTAAGA GAAGCTAAAATAGAGAGCATTTTT TTGCACTATGAGTAGTGTAGCTGC TTTGTTGCAGTGAATATGTTTTTA GTGGACGCTCTCTAATTAACTTAA GCGTA5’
Reverse primer for colony PCR	Integrated DNA Technologies		5’- GCCGCTTGAATTCGTCTAGACT- 3’
Reverse primer for DNA sequencing	Integrated DNA Technologies		5’- GGAAACAGCTATGACCATG-3’
T4 DNA Ligase	New England BioLabs	M0202S