This protocol describes the preparation and analysis of a unique biosensor that has been designed to detect the chemical components of gunshot residue. The use of biosensors for forensic applications is unique. It represents a low-cost, simple to use alternative to the highly specialized instrumentation typically used in forensic laboratories.
The synthetic biology methods described can be used for any system that uses standard synthetic biology parts. The chemical analysis described is applicable to any biosensor that expresses red fluorescent protein. Demonstrating the procedure will be Andrea Soles and Elle Richardson, undergraduate students at Longwood University.
To begin this procedure, add ten microliters of the previously isolated J10060 plasmid DNA to a microcentrifuge tube. Add eight microliters of water, and one microliter of each of the EcoRI and NHEL enzymes pre-mixed with one microliter of buffer. For the promoter DNA, add ten microliters of annealed promoter DNA sequences to a fresh microcentrifuge tube.
Add eight microliters of water, and one microliter of each of the EcoRI and NHEL enzymes pre-mixed with one microliter of buffer. Using a pipette set to ten microliters, gently pipette the samples up and down to mix. Incubate the samples at 37 degrees Celsius for 30 minutes.
Then, heat and active the enzymes at 80 degrees Celsius for five minutes. Store the digested DNA in a freezer until ready to proceed. First, use the plasmid and promoter DNA that were previously digested to set up the reaction in a microcetrifuge tube on ice, as outlined in table one of the text protocol.
Making sure to add the T4 DNA Ligase last. Pipette up and down gently to mix the reaction, and centrifuge briefly. Incubate at room temperature for ten minutes.
Then, heat and activate at 65 degrees Celsius for ten minutes. Perform the transformation as outlined in the text protocol. First, set up the reaction mixtures for PCR as outlined in table two of the text protocol.
Gently pipette up and down to mix the reactions. Using a yellow pipette tip, scrape a colony of the transformed E.coli. Transfer a swipe of this E.coli onto a new LB-ampicillin agar plate that has been sectioned off, and then insert the pipette tip into the PCR tube.
Shake the pipette tip to mix the E.Coli with the PCR mix. Repeat this process three more times for additional colonies. Then, load the PCR tubes into a PCR machine, and begin thermocycling as outlined in table three of the text protocol.
To begin label the appropriate number of sterile culture tubes as outlined in table four of the text protocol. Add two milliliters of the prepared culture broth to each tube. Add the anilite stock solution to the tubes as outlined in table four.
Then, place the snap caps on the culture tubes so that they are loose to allow air flow into the tube. Vortex each culture tube. After this, place the tubes into a shaking incubator at 37 degrees Celsius and at 220 RPM for at least 24 hours.
First, use an ethanol based wipe designed for removing lead to wipe all surfaces of the hands, including between the fingers. Store the wipes in an appropriately labeled sealable baggie until analysis. Use an alcohol based wipe to wipe down any large surfaces to be tested.
Use a cotton swap moistened with ethanol to wipe down any small surfaces to be tested. Wearing clean gloves and using scissors that have been cleaned with alcohol, cut a section that is approximately one square centimeter out of the center of the wipe. Then, place the cut piece of wipe, or cotton swab, directly into a culture tube containing two milliliters of the sensor bacteria, and ensure that it is completely submerged in the broth.
Prepare the spectrometer to collect fluorescent submission at the appropriate wavelength for the RFP variant, with an excitation wavelength of 500 nanometers. Then, use a vortex mixer to shake the tubes. Carefully transfer each supernatant to a low volume cuvette, and collect the emission intensity.
Using a vortex mixer, shake the tubes. Transfer 200 microliters of the broth to a well in the well plate. Record which samples went into each well of this plate.
Set up the flourometer to collect the emission intensity at the appropriate wavelength for the RFP variant. The fluorescence spectra for a representative RFP variant shows both the negative control and the spectra at two different levels of anilite added. The maximum fluorescent signal for the RFP variant used in this work is observed at 575 nanometers.
Representative data is collected for the same set of solutions, from both the portable spectrometer and the fluorometer. There is a general trend of the fluorescence increasing as the concentration of anilite increases. It is worth noting that at high concentrations, greater than about 800 parts per billion for the lead sensor, the response drops off due to the toxicity of lead at such a high concentration.
Analysis of ethanol swab samples from the inside of a spent 40 caliber cartridge casing provides proof of principle results. The positive results from each of the three sensor bacteria, indicates a positive test for gunshot residue. The biosensors prepared in this protocol can also be used individually to detect contamination in food, water, or environmental samples.
This technique paves the way for researchers to develop biosensors using standard synthetic biology techniques for a variety of different chemical anilites. Personal protective equipment should be worn, and analysts should wash hands and disinfect surfaces after handling E.coli. Wastes should be autoclaved, and any wastes containing heavy metal should be treated accordingly.
