The overall goal of the following experiment is to rapidly detect the presence of specific bacteria such as e coli using novel flu genic, DNA, Zyme probes First, complete genic DNA. Zyme probes are generated by ligation. Bacteria are then cultured to enrich the number of target molecules and accrued extracellular mixture is prepared from the supernatant.
The crude extracellular mixture is then combined with the DNA zyme probe interaction between the DNA Zyme probe and target molecules results in cleavage of the probe, which then fluoresces is using. A fluorimeter interaction between the probe and target is seen as an increase in relative fluorescence. The results are then verified using denaturing poly acrylamide gel electrophoresis analysis.
The main advantage of this technique over PCR or antibody based methods is that the procedure is simpler and easier to perform. Though this method has been developed with the model bacterium e coli, it can be applied to the detection of any other foodborne or nosocomial Bacterial pathogens. Generally, individuals new to this method will struggle because they may not be accustomed to the handling of synthetic DNA probes and bacteria Demonstrating the procedure is Sergio Aguire, a research assistant from my laboratory and Monso Ali, a postdoctoral fellow For this protocol.
R-F-D-E-C one is the featured DNA Zyme. It consists of the catalytic sequence EC one underlined in black and the substrate sequence FS one underlined in green contained within the substrate is an RNA linkage indicated by the blue r flanked by a Fluor labeled DT indicated by the F and a QUENCHER labeled DT indicated by the queue RF SS one is a scrambled version of RFD EEC one where the catalytic sequence EEC one is partially shuffled into SS one, but the FS one portion remains unchanged. RF DEC one and RF SS one are made by template mediated enzymatic ligation of the oligonucleotide FS one with oligonucleotide EC one or SS one in the presence of LT one as the ligation template to prepare crude extracellular mixtures or cems that will be used as a target for R-F-D-E-C one begin by dispensing two milliliters of LB into sterile 14 milliliter culture tubes using a pipette gun.
Next, using a sterile pipette tip, pick a single colony of e coli from an auger plate and drop it into a culture tube containing lb. Place the tubes in an incubator set at 37 degrees Celsius and shake at 250 RPM for 14 hours after the incubation. To generate a 1%re inoculation culture, dispense two milliliters of fresh LB into 14 milliliter culture tubes and add 20 microliters of the bacterial culture from the previous step.
Incubate the tubes at 37 degrees Celsius with shaking at 250 RPM until each bacterial solution reaches an OD 600 of approximately one transfer. One milliliter of each culture to a new 1.5 milliliter micro centrifuge tube and pellet the cells by centrifugation at 11, 000 G for five minutes. After the spin transfer the clear supernatant to a fresh 1.5 milliliter micro centrifuge tube.
Store the supernatant at minus 20 degrees Celsius. If it will not be immediately used to determine whether fluorescent signal is generated with interaction of the DNA enzymes with the crude cell extract, the samples are assayed by spectra Photometry. Turn on fluorescent spectrophotometer and set up data acquisition parameters with excitation at 488 nanometers and emission at 520 nanometers.
Also set up the instrument to take readings every minute for one hour. Wash three quartz crystal cuvettes first with distilled deionized water and then with 100%ethanol. Dry the cuvettes by flashing nitrogen gas.
Label the cuvettes as C one for control one C two for control two and T for test next transfer 24 microliters of distilled deionized water to C one and 24 microliters of the prepared e coli crude extracellular mixture to C two and T.Add 25 microliters of two X RB to each Q vet and place them in the fluorescent spectrophotometer. Start collecting fluorescence data after five minutes, add one microliter of five micromolar RFS S one to C two and one microliter of five micromolar, RFD eec one to T and C one ensuring that the fluorescence readings are not interrupted. Mix each solution by pipetting, then allow the reaction to continue for the remainder of the acquisition period.
Save the data in Excel file format. Then transfer the data to a personal computer and use Excel to create a graphical image. The same reaction mixtures that were used for analysis by Spectra.
Photometry can be used for analysis by gel electrophoresis. After one hour of acquisition. Remove the cuvettes from the spectrophotometer and transfer the solutions to new micro centrifuge tubes.
Quench the reactions by adding five microliters of three molar sodium acetate and 125 microliters of 100%ethanol. Mix each solution by vortexing and place the tubes in minus 20 degrees Celsius freezer for one hour following the incubation centrifuge. The reaction mixtures at 11, 000 G for 20 minutes at four degrees Celsius and carefully remove the supernatant by pipetting.
Dry the pellets using A DNA concentrator for 10 minutes. Add 20 microliters of gel loading buffer and briefly vortex. Spin down briefly with a benchtop centrifuge.
Next load 10 microliters of the reaction samples per well of 10%Dage gel after the run. Remove the glass plates and wash thoroughly with tap water. To remove any gel pieces, wipe the plates with a Kim wipe.
Scan the gel plate for fluorescence using a typhoon scanner. Analyze the resulting images using image quant software to assess the sensitivity of the system. Serially diluted cultures are grown for increasing time periods.
To determine the minimum incubation period required to detect a single bacterium, prepare 10 culture tubes containing two milliliters of lb inoculate each culture with 100 microliters of two CFU per milliliter of a glycerol stock of e coli and incubate at 37 degrees Celsius with shaking at 250 RPM at 4 8, 12 16, and 24 hours. Harvest 300 microliters from each of the inoculated culture tubes since there may be no detectable bacteria for samples harvested at time. Points 4 8 12.
Allow the remaining culture to grow for 24 hours in order to identify cultures containing bacteria. Measure the OD 600 and precipitate the cells by centrifugation at 11, 000 G for five minutes. Transfer the supernatant which contains the CEMS to fresh 1.5 milliliter micro centrifuge tubes, and store at minus 20 degrees Celsius until use the next day.
Inspect the cultures for growth. Only one or two of the 10 cultures may contain e coli after inoculation and the remaining tubes will not contain any cells. Use the CEMS recovered from positive cultures at the designated time points To prepare cleavage reactions with RFD EEC one, analyze the reaction mixtures using DAGE gel electrophoresis as described earlier, both RFD EEC one and RF SS one probes were prepared by enzymatic ligation of the DNA zyme portions to the substrate.
FS one interaction of CEM EEC and RFD EEC one or RF SS one was then assessed by spectrometry. As can be seen here. R-F-D-E-C one produced a high level of fluorescent signal upon the addition of CEM ec.
In stark contrast, R FS S one did not produce a strong fluorescent signal. Thus the fluorescence producing function of RFD EEC one. Upon contacting CEM EC is sequenced.
Specific to verify that observed fluorescence increases were due to the cleavage of the RNA linkage. The reaction mixtures were analyzed by dage cleavage of RFD eec, one by 0.25 molar sodium hydroxide is expected to generate 2D NA fragments, a five prime fragment retaining the Fluor and a three-prime fragment retaining the quencher. As can be seen here.
The RFD EEC CEM EEC reaction mixture indeed produced the expected cleavage product. Moreover, only UNC cleaved R-F-D-E-C one and the five-prime fragment could be detected by fluorescence imaging. To examine the specificity of RFD EEC one fluorescence assays were performed using CEMS collected from several other gram-negative and gram-positive bacteria only.
The sample containing CEM EC produced an increase in fluorescence. The lack of cross reactivity with CEMS from the other bacteria indicates that R-F-D-E-C one is highly selective for e coli to determine the minimum culture time required to detect a single e coli cell e coli diluted to one CFU per milliliter in 100 microliters cultured with growth medium for 4 8, 12 16, and 24 hours. The resulting CEMS mixed with R-F-D-E-C one and cleavage was assessed by dage as shown here.
An image of the Dage assay indicates R-F-D-E-C one did not produce the expected cleavage product. When reacted with CEM. ECS collected at four and eight hours of growth indicating that culturing time of 12 hours is needed Once mastered.
The key component of this technique that DA enzyme based assay can be done in 60 minutes if it is performed properly After this development. The procedure paved the way for the researchers in the field of bio analysis to explore allogenic DNA enzymes for the detection of a wide range of bacterial pathogens.
