Introduction

Foodborne illnesses are prevalent worldwide and are mainly caused by pathogenic bacterial strains that are unintentionally inoculated on food and subsequently ingested ₁. In many cases, these illnesses go unreported, are unrecognized, or are asymptomatic, and can lead to many chronic conditions including kidney and liver failure, as well as cancer₂. The problem is further exacerbated in developing nations where sanitary conditions are scarce and climates are far more conducive for bacterial growth.

Thus, it has become necessary to create a cheap, effective, and mobile screening platform for simple detection of foodborne pathogens. Escherichia coli represents a significant proportion of foodborne infections, and is frequently associated with multi-drug resistance₂. Thus the strain is a prime substrate for the development of a pre-emptive detection platform.

Current detection methods for bacterial pathogens involve techniques that are financially costly and low-throughput, or otherwise require equipment that is not simple to carry around, sell at a department store, or acquire in the household ₃. We propose a new cost-effective solution involving functional nucleic acids with the ability to cleave RNA in the presence of specific bacteria with the result of a detectable change in fluorescent signal. There is much work describing the efficacy of the RNA-cleaving fluorogenic DNAzyme (RFD) in the detection of small molecules, peptides, and unique cells ₄. RFDs have been developed and previously demonstrated to produce fluorescence specifically in the presence of small numbers of E. coli₅. The E.coli-sensitive RFD (RFD-EC1) is a long-term cost-effective product and therefore an ideal candidate for a common household bacterial screening platform.

The following experiments thus focus on optimizing the application of RFD-EC1 in an agar plate-based assay for quick, user-friendly and sensitive detection of E. coli bacteria.

Timeline of Experiments

Fluorophore

May 23, 2017

This experiment was one of the first to be completed by our team in the Li lab. That being said, the purpose of this experiment was to visualize the relative intensity of DNAzyme fluorescence without exposing it to E. coli. We tested a wide range of DNAzyme concentrations between 100-5000 uM, in a total volume of 10uL. After visualization under UV in Eppendorf tubes, we plated the DNAzyme so that we could see how it would affect our experiments. The DNAzyme exhibits minimal self-cleavage in small amounts, however we needed larger amounts to detect plate fluorescence. This can become an issue if the fluorescence of the self-cleaved DNAzyme masks the fluorescence of our actual results. Our findings in this experiment led us to restrict DNAzyme usage to under 10uL of 1000uM DNAzyme.

Medium Optimization: M9 vs LB (Luria Bertani)

June 12-13, 2017

Throughout the initial experiments, we found that it was difficult to visualize the fluorescence of the DNAzyme responding to the E. coli due to the autofluorescence caused by the LB agar. This background fluorescence affected results both under UV light as well as Typhoon Imager images. In light of this, we decided to test M9 minimal media to reduce this background fluorescence issue. To compare the two media types, we created 60mm plates with both LB or M9 with differing volumes of media. The UV results were very astonishing in the magnitude of contrast between the fluorescence given off by these two media types. For all future experiments plates containing 2.5mL of M9 media were used. More experiments and optimization were then completed to ensure growth of multiple bacterial species upon the M9 media.

Testing DNA Specificity

June 22-25, 217

The DNAzyme itself cleaves specifically in the presence of E. coli K12 RNAse I protein, some of which can be found in the extracellular matrix. The purpose of conducting this experiment was to observe the contrast between the fluorescence of the DNAzyme when dropped on E. coli K12 colonies vs E. coli K12 delta RNAse I colonies, and to test both the specificity and the sensitivity of the DNAzyme. The bacteria was incubated overnight for 16 hours before 10pmol of DNAzyme was pipetted onto the colonies. After Typhoon imaging, we observed a substantial difference in fluorescence between E. coli K12 colonies and E. coli K12 delta RNAse colonies.

Fluorescence Detecting Wavelength

July 25th, 2017

We wanted to ensure that the wavelength of our measurements using the Typhoon imager were the best possible settings that would produce the most accurate results. Our DNAzyme utilizes fluorescein which has a peak excitation at 488nm and a peak emission at 526nm, but it was in our best interest to make sure that our settings while imaging were appropriate. A variety of wavelengths were tested (see image for details) and it was found that nm was the best option.

Bacterial Autofluorescence

August 20, 2017

Bacterial autofluorescence also posed an issue as E. coli K12 itself fluorescences at a similar wavelength as the fluorophore, fluorescein. To address this problem we observed the autofluorescence produced by increasing amounts of bacteria. We plated 10^7, 10^5, 10^4, and 10^3 bacteria and incubated for 16 hours before imaging with UV light and the Typhoon imager. The results displayed helped in the next experiment which involved optimizing the bacterial cell number necessary to elicit a DNAzyme response but low enough to avoid major bacterial autofluorescence.

DNAzyme Autofluorescence

August 21, 2017

As it is difficult to view contrasting fluorescence, we worked to quantify and minimize the amount of autofluorescence produced from this DNAzyme. 4 eppendorf tubes were holding 5uL of DNAzyme diluted to different amounts (40pmol/5uL,30pmol/5uL, 20pmol/5uL, 10pmol/5uL). All tubes were then imaged to view the impact the amount of DNAzyme has on fluorescence. Results show that difference in fluorescence is significant in tubes, but show differently when dropped onto plates as it spreads.

Optimal Response

August 21, 2017

Considering DNAzyme autofluorescence, we aimed to generate the optimal response for the DNAzyme to detect E. coli K12 while minimizing all autofluorescence. 5uL of 10-40pmol of DNAzyme was dropped onto quadrants on a plate with E. coli grown in increasing amount (10^3 to 10^7 cells/quandrant). Based on results, 10^4 cells with 40pmol of DNAzyme had the optimal response while accounting for background fluorescence. It was also noticed after August 21 that reaction buffer (2X SB) was not included for this experiment. As a result, experiments after August 21 do not include reaction buffer to obtain results.

DNAzyme Specificity - 2X Selection Buffer Added

August 21, 2017

After establishing what we thought to be the optimal amount of DNAzyme and bacterial cells, we wanted to verify the specificity of the DNAzyme solely for E. coli K12, and show the inability to cleave against other bacterial species. 10^4 cells of 6 different bacterial species were plated in separate quadrants and grown overnight. 40pmol of DNAzyme was then dropped on top along with reaction buffer. Typhoon scans show DNAzyme fluoresces slightly stronger in the presence of E. coli compared to other species, but this difference is not significant enough. We hypothesized that the addition of selection buffer may cause premature cleavage.

Tracking Fluorescence Over Time

August 21, 2017

To quantify DNAzyme fluorescence over time and determine whether the addition of selection buffer impacts fluorescence results, 10^4 cells were spread and incubated for 16 hours and 40pmol of DNAzyme was dropped afterwards. Typhoon scans show that DNAzyme fluorescence fades after 2 hours and that selection buffer does not seem to make much difference in fluorescence when interacting with E. coli.

Specificity Test - Without Selection Buffer

September 28, 2017

After deciding to leave out selection buffer from plate experiments involving DNAzyme, we wanted to once again test the specificity of the DNAzyme and supply appropriate measurements for data analysis. 10^4 cells of 6 bacterial types were dropped into separate quadrants then grown for 16 hours. 40pmol of DNAzyme was dropped afterwards onto the quadrants and examined. Under UV light, DNAzyme glows bright when dropped onto E. coli and appears to show no visible fluorescence with other species.

Protocols

Testing Fluorescence of Bacterial Plates

Date: May 18
Reagents:

3x DNAzyme stock: 13.35 uL (200nM final concentration)
2x Selection Buffer (150uL)
dH2O: 136.65uL

Protocol:

The DNAzyme stock solution above was assembled in an Eppendorf tube.
100 uL of DNAzyme stock solution was pipetted into Plates 1,2, and 3. A spreading stick was used to spread the solution over the surface.
Plates 1-3 were left for 5 minutes to dry (with plate lids on).
A disposable inoculation loop was used to streak nothing onto Plates 1 and 5.
Disposable inoculation loops were used to streak E. coli on Plates 2 and 4 and B. subtilis on Plate 3.
Plates were incubated upside down at 37℃.

Plates:

Only DNAzyme
E. coli with DNAzyme
B. subtilis with DNAzyme
E. coli only
Nothing

Fluorophore Concentrations

Date: May 23

Reagents:

FDNA
dH₂O
Spreading stick
LB agar plates
UV light box

Protocol:

Seven different concentrations of fluorescent DNA with the fluorophore were assembled. These concentrations were 0, 100, 250, 500, 1000, 2500, and 5000 nM.
10 uL from each of the concentrations were dropped into the middle of a quadrant on a plate.
A spreading stick was used on each quadrant to spread the fluorescent DNA.

*Did not spread because the dot of solution had set into the agar and therefore did not spread with the spreading stick. This time we dotted the plates and immediately used the spreading stick after putting each dot onto each quadrant.

Optimizing Concentrations & Cleavage

Date of Experiment: May 26

Materials/Reagents:

3 agar plates
100uL of:

2.5uM FDNA, 5.0uM FDNA
2.5uM FQ substrate (RS28), 5.0uM FQ substrate (RS28)
Glycerol stock of E.coli

Protocol:

3 plates were split into quadrants with 25uL FDNA and FQ substrate stock on each quadrant:

Plate 1 (2.5uM): FDNA, Negative, E. coli, FDNA + E. coli
Plate 2 (2.5uM): FQ substrate, negative, FQ substrate + 0.5uM 0.2uM NaOh, FDNA
Plate 3 (5.0uM): FQ substrate, negative, FQ substrate with 0.5uM 0.2M NaOH, FDNA

E. coli was streaked onto its designated quadrants on plate 1
Plate 2 and 3 were checked under blue light for fluorescence after NaOH applied for 15 minutes
All plates were placed in 37C incubator overnight
Plates were visualized using the Typhoon. The following scans were performed using the blue light setting (488nm):

526 SP Fluorescein, Cy2, AlexaFluor488 Emission Filter
520 BP CY2, ECL+, Blue FAM Emission Filter
Emission Filter

M9 vs LB Autofluorescence via Agar Thickness

Date of Experiment: June 12-13
Goal: Testing different thicknesses of M9 minimal media and LB (Luria Bertani) plates to find the minimal autofluorescence. Determine the optimal medium (M9 vs LB) and the optimal volume/thickness
Different thicknesses by volume were spread on a 5 mL plate: 5 mL, 3.5 mL, 2.5 mL, 1 mL
Materials:

LB (12 mL) & M9 (12 mL)
Small plates (typically 5 mL agar capacity)
UV light & Typhoon Imaging
FDNA

Protocols:

Make 200 mL stock of M9 (http://www.thelabrat.com/protocols/m9minimal.shtml) (http://www.protocolsonline.com/recipes/media/m9-medium-5x/)
1. Make M9 salts:
Note: M9 media ratios are 16:4:0.4 respectively for MgSO₄ solution, M9 salts and glucose. MgSO₄+water+agar solution expires 2 weeks after first use.
Melt LB (12 mL) with microwave
Pour M9 and LB into the 60 mm diameter plates each with the following different volumes:

5 mL
3.5 mL
2.5 mL
1.5 mL

Drop 20 uL of 2.5 uM FDNA in one corner of each plate
Negative control:

blank plate, no M9 or LB
Leaving 2 plates without FDNA but with 5 mL of each medium

Observe under UV light & Typhoon Imaging

Cell Detection Using DNAzyme in M9

Date of Experiment: June 14-15, 2017
Purpose:To observe the effectiveness of DNAzyme in M9 with the pH of 8 to detect E. coli colonies. This is within the optimal range mentioned in “A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection (Aguirre et al.)

Materials & Reagents:

UV box & Typhoon imager
Incubator
60 mm diameter agar plates
Disposable wire loops
E. Coli cells from glycerol stock (K12 3)
DNAzyme (RFD-EC1) 2.245 uM
Selection buffer
FQ (2.5 uM)
NaOH (0.2 M)
HCl (0.2 M)
M9 solution

Protocol:

Prepare two M9 plates at pH 8
Streak E. coli cells onto the plate using a wire loop into 3 quadrants

The streaking pattern should look like this.

Prepare DNAzyme SB (5 uL of stock + 5 uL of 2x SB)
Place in incubator (37 degrees celsius) overnight
Drop DNAzyme and FQ into the different quadrants as shown:

Cell + DNAzyme (4.454 uL)
Cell + FQ (4 uL diluted)
Cell only
FQ (4 uL) + NaOH (enough to cover FQ ~2 uL)

Afterwards, put in box (don’t expose to light)
For quadrant 4, let FQ be cleaved over time and add HCl (2 uL) to neutralize right before screening.
After ~5 hours, observe plate under UV light and record observations
Analyze fluorescence using the typhoon imager to view any other sources of fluorescence

Differing [NaOH] for Positive Control

Date of Experiment: June 19-20, 2017
Purpose:To ensure that NaOH can cleave the FQ substrate so that we have a reliable positive control in future experiments.

Reagents:

NaOH (varying concentrations)
FQ substrate (2.5 uM)
Eppendorf tubes
UV light
Typhoon imager

Protocol:

Add 6uL of FQ substrate to 5 separate Eppendorf tubes.
Dilute the NaOH stock to the appropriate concentrations for use.
Add 4uL of NaOH to each tube with concentrations as described in the table below:

Tube #	1	2	3	4	5
[NaOH]	0.2	0.4	0.6	0.8	1.0

Let tubes sit for 30 minutes, then observe fluorescence using the UV box in the cold room.
Transfer 10 uL of NaOH+FQ sub as well as 10uL FDNA control onto labeled quadrants of M9 media plates as drawn below:

Testing DNAzyme Sensitivity

Date of Experiment: June 22-25 Purpose: Proof of concept that RFD-EC1 probe is specific only to E. coli that express the RNAse I enzyme

Setup:

E. coli K12 + DNAzyme (4.45 uL of 2.245 uM)
E. coli K12 (delta RNAseI) + DNAzyme (4.45 uL of 2.245 uM)
FDNA (4 uL of 2.5 uM)
Nothing

Bacteria was streaked on each plate in respective quadrants and grown overnight. next morning: DNAzyme was dropped on cells and FDNA was dropped on 3rd quadrant. The M9 media is pH 8, 2.5 mL.

DNAzyme Specificity

Date of Experiment:July 5

Reagents:

DNAzyme (50 pmol)
E. coli K12 glycerol stock
B. subtilis glycerol stock
Brevundimonas diminuta glycerol stock
Hafnia alvei glycerol stock
Achromobacter xylosoxidans glycerol stock
Serratia fonticola glycerol stock
Leuconostoc mesenteroides glycerol stock
Another bacteria glycerol stock
UV lightbox & Typhoon Imager
M9 Media plates 4cm diameter

Protocol:

Label 4 M9 pH 7.0-7.2, 2.5mL plates as depicted.
Streak bacteria in pattern shown with a 10uL pipette tip (exception: for plate 1 use a 2uL pipette tip) @ 4:30pm
Leave plates upside down in a 37 degree Celsius incubator overnight (until 9:30am next morning)
Drop 7.24 uL of 6.9 uM DNAzyme (effective concentration: 50pmol). To ensure less spreading drop 3.62 uL (2x).

Bacterial Mixture

Date of Experiment:July 7
Purpose:To determine whether the RFD-EC1 DNAzyme can detect E. coli while in a sample of multiple different species of bacteria.

Materials/Reagents:

E. coli (K12) cells
B. subtilis cells
Serratia Fonticola cells
Brevundimonas diminuta cells
New M9 media solution of pH 7.0-7.2
Plates (4cm diameter)
DNAzyme probe (50 picomol per quadrant)

Protocol:

Plate 2 plates (one with DNAzyme spread before cell growth, another with DNAzyme dropped after cell growth) with M9 solution and separate into 3 quadrants
- Negative control of nothing
- Positive control of E. coli + DNAzyme
- 4 distinct streaks of E. coli, Serratia Fonticola, B subtilis, and Brevundimonas diminuta
Grow the cells overnight
UV image next morning to ensure cells have grown
For plate with label “DNAzyme drop after”, drop and spread 50 picomoles of DNAzyme probe on the positive control and experiment quadrants

Plate: “DNAzyme to be dropped after"
For that, we'll need to drop a larger volume of DNAzyme so we'll use DNAzyme Eppendorf labelled 2.245 uM that's a larger volume and lighter in color (that's the one we diluted with water)
To get 50 picomoles, dilute 20.46 uL of DNAzyme with 20.46 uL of 2x buffer and pour the total volume of 40.9198 uL over the 2 non-negative control quadrants (3/4 of the plate).
(volume may be a lot but the thing is, I kind of need to flood the surface area of the quadrants since I can't spread with hockey sticks - will shift cells - and I need to cover the entire area since I drew cells in thin lines over a large surface area)
If volume is too large, we can drop half of the mixed solution (20.46 uL) first, let set, and drop the other half again as a second layer

Let sit for 30 minutes to 1 hour for DNAzyme to set
UV and Typhoon image to see which bacteria species cleaved DNAzyme probe

See if presence of other bacteria interferes with the fluorescence compared to the positive control
If brighter bc other bacterial autofluorescence or dimmer bc of lower concentration of target E. coli bacteria strain

Fluorescence detecting wavelength

Date of experiment: July 25, 2017
Purpose: To find out which wavelength best detects fluorescence from the cleavage of DNAzyme in the presence of E. coli K12 bacteria

Reagents/Materials:

13.8 uM DNAzyme
2x selection buffer
E. coli K12 and delta RNAse
100uM FDNA
2 M9 plates
5 Eppendorf tubes
UV box
Typhoon imager

Protocol:

Grow K12 and delta RNAse in liquid culture until both reach a count of 10^8 cells/mL
Label 2 plates with quadrants: K12, delta RNAse, negative, FDNA
In one eppendorf tube, insert 11.45 uL 2xSB, 10uL K12 cells, 1.45uL DNAzyme
Repeat step 3 in a separate eppendorf tube but instead inserting 10uL delta RNAse *** make sure to add DNAzyme last (SB may cause unintentional cleavage)
Place both eppendorf tubes in an incubator for at least 30 minutes
Remove tubes from incubator and drop 10uL from both tubes onto its labeled quadrant on the plate letting it slightly spread
Perform a 1:10 dilution of FDNA in an eppendorf tube using sterile water and drop 2uL onto its quadrant
Image under UV and typhoon to observe fluorescence

Optimal DNAzyme Concentration

Date of experiment: July 25, 2017
Experiment done by: Audrey
Purpose: After discovering the optimal number of bacterial cells needed for visible DNAzyme cleavage, this experiment will find the minimal number/concentration of DNAzyme to show distinguishable fluorescence

Reagents/materials:

13.8 uM DNAzyme
2x selection buffer
E. coli K12
100uM FDNA
2 M9 plates
5 Eppendorf tubes
UV box
Typhoon imager

Protocol:

Grow K12 in liquid culture until it reaches a cell count of 10^8 cells/mL
Label eppendorf tubes: 10pmol DNAzyme, 20pmol DNAzyme, 30pmol DNAzyme, 40pmol DNAzyme, FDNA In 4 separate eppendorf tubes, drop 10uL of cells and the following amount of reagent:

10pmol DNAzyme: 10.73uL SB + 0.75uL DNAzyme
20pmol DNAzyme: 11.45uL SB + 1.45uL DNAzyme
30pmol DNAzyme: 12.17uL SB + 2.174uL DNAzyme
40pmol DNAzyme: 12.90uL SB + 2.90uL DNAzyme

*** make sure to add DNAzyme last (SB may cause unintentional cleavage)

Place eppendorf tubes in an incubator for at least 30 minutes
Label 2 plates with quadrants:

10pmol, 20pmol, DNAzyme, FDNA
30pmol, 40pmol, DNAzyme, FDNA

Remove tubes from incubator and drop 10uL from tubes onto its labeled quadrant on the plate letting it slightly spread
Drop 5uL DNAzyme separately onto its quadrant (to check for autofluorescence)
Perform a 1:10 dilution of FDNA in an eppendorf tube using sterile water and drop 2uL from the tube onto the plate with the lower cell count. Drop 4uL onto plate with the higher cell count.
Image under UV and typhoon to observe fluorescence

Quantifying Autofluorescence

Date of experiment: July 27, 2017
Purpose: It is unsure whether strong fluorescence is partially due to autofluorescence generated from cells and/or the DNAzyme. This experiment will gather numerical data on fluorescence to determine how much autofluorescence exists in different concentrations of bacteria.

Reagents/materials:

13.8 uM DNAzyme
2x selection buffer
E. coli K12 (10^8 cells/mL) liquid culture
2 M9 plates
5 Eppendorf tubes
UV box & Typhoon imager

Protocol:

Grow K12 in liquid culture until it reaches a cell count of 10^8 cells/mL
Dilute liquid culture to 10^6 cells/mL and 10^4 cells/mL
Take 10uL of 10^8 cells/mL and place into a new eppendorf tube. Do the same with 10^6, 10^4 cells/mL
Pipette 11.45uL of selection buffer into each tube and 1.45uL DNAzyme. *** make sure to add DNAzyme last (SB may cause unintentional cleavage)
Place eppendorf tubes in an incubator for at least 30 minutes
Label 3 plates with quadrants: DNAzyme + cells, cells, DNAzyme, negative
Label each plate so it corresponds to one of the three different cell counts (10^6, 10^4, 10^2 cells)
Drop 10uL of the liquid cultures with different concentrations into the “cells” quadrant in the plate that’s labeled with its cell count
Drop 1.45uL DNAzyme separately onto its quadrant and let it spread
Remove tubes from incubator and drop 10uL from tubes onto its labeled quadrant on the plate letting it slightly spread
Image under UV and typhoon to observe fluorescence

Fluorescing Colonies

Date of experiment: July 28, 2017
Purpose:Before performing the timepoint experiment, it must be confirmed whether the DNAzyme will fluoresce when dropped onto a colony of E.coli K12

Reagents/materials:

13.8 uM DNAzyme
2x selection buffer
E. coli K12 (10^8 cells/mL) liquid culture
Delta RNAse (10^8 cells/mL) liquid culture
2 M9 plates
1 Eppendorf tube
UV box & Typhoon imager

Protocol:

Streak both bacteria onto separate plates labeled: E.coli K12 and delta RNAse
Place in incubator and let it grow for a day (~20 hours) until isolated colonies form and are noticeable
In an eppendorf tube, add 1.45uL 2x selection buffer and 1.45uL DNAzyme
Take plates out of incubator and take 1.45uL from the eppendorf to drop on top of individual colonies in the E.coli K12 plate
Repeat step 4 for the delta RNAse plate
Place both plates in the incubator and set it rest for ~20min
Remove plates from incubator and image under UV and typhoon for observations

DNAzyme Autofluorescence

Date of experiment: July 31, 2017 Purpose: As the DNAzyme shows excessive autofluorescence, it was important to see how much DNAzyme could be added to produce the least amount of autofluorescence, but the most cleavage in the presence of E. coli K12.

Reagents/materials:

13.8 uM DNAzyme
2x selection buffer
E. coli K12 (10^8 cells/mL) liquid culture
2 M9 plates
6 Eppendorf tubes
UV box
Typhoon imager

Protocol:

Dilute DNAzyme using liquid M9 media in 4 separate eppendorf tubes to obtain the following amounts: 5, 10, 20, 30, 40 pmol
Drop 20 uL onto quadrants and spread evenly using a hockey stick.
Let the DNAzyme set for a few minutes then image to observe autofluorescence
Streak bacteria onto each quadrant to grow colonies
Place in incubator overnight
Drop FDNA (positive control) and FQ (negative control) onto its quadrant and image to observe fluorescence

Bacterial Autofluorescence

Purpose: To explore the effect bacterial autofluorescence may be having on experiments, to quantify bacterial autofluorescence so as to minimize it in future experiments.

Setup:

Eppendorf tubes: (4)
E. coli K12
Bacterial Spreading Sticks (4)
M9 Plates (4)
UV Imager
Typhoon Imager

Protocol:

Make 4 dilutions for E. coli K12. This will be 10^9 cells/mL, 10^7 cells/mL, 10^6 cells/mL, and 10^5 cells/mL (4 tubes in total).
Transfer 30uL of each of these to a new separate Eppendorf tube.
Image under UV immediately and after 30 minutes of incubation.
Before the 1hr mark, plate and spread (with hockey stick) all of these bacteria separately.
Incubate plates overnight at 37 degrees Celsius upside down.
The next morning (SHOULD BE EXACTLY 16 HOURS AFTER, PLAN ACCORDINGLY), image under UV and Typhoon.

DNAzyme Autofluorescence

Date: August 21, 2017 Purpose: To quantify and minimize the amount of autofluorescence produced from the DNAzyme.

Setup:

Plates from the previous experiment can be used (the empty quadrants (8))
DNAzyme
2X SB
UV Imager
Typhoon Imager

Protocol:

After completing the bacterial experiment (2), utilize the empty quadrants (4).
Make DNAzyme dilutions: 40pmol/5uL,30pmol/5uL, 20pmol/5uL, 10pmol/5uL. = 8pmol/uL, 6pmol/uL, 4pmol/uL, 2pmol/uL
Put 5uL of these into separate Eppendorf tubes. Image under UV immediately.
Plate the 5uL in the empty quadrants of the bacterial plates. Incubate right side up for 1hr at 37 degrees Celsius and image under UV and with the Typhoon.Incubate for another hour and image again under Typhoon.

Optimal Response

Date: August 21, 2017
Purpose: To generate the optimal response for the DNAzyme to detect E. coli K12 while minimizing all autofluorescence.

Setup:

DNAzyme: pmol/5uL, pmol/5uL, pmol/5uL, pmol/5uL
E. coli K12 cells on plates from experiment (2)
E. coli K12 delta RNase cells from experiment (2)
UV Imager & Typhoon Imager

Protocol:

On the same day as the bacterial autofluorescence experiment Day 2, once results are obtained for that experiment, do this experiment.
Drop the 5uL of each DNAzyme amount on individual colonies of E. coli K12 and E. coli K12 delta RNase for each bacterial number.
Incubate the plates right side up for 1 hr, then image under UV and Typhoon Image.
Image again at 2 hrs with the Typhoon Imager.

DNAzyme Specificity

Date: August 21, 2017
Purpose: To verify the specificity of the DNAzyme solely for E. coli K12, and to show the inability to cleave against other bacterial species.

Setup:

E. coli K12 (10^8 cells/mL)
E. coli K12 delta RNase (10^8 cells/mL)
Bacillus subtilis (10^8 cells/mL)
Hafnia alvei (10^7 cells/mL)
Achromobacter xylosoxidans (10^7 cells/mL)
Serratia fonticola (10^7 cells/mL)
DNAzyme (A)
2X SB
FDNA (10uM)
M9 plates
UV Imager & Typhoon Imager

Protocol:

Grow overnight liquid cultures in M9 media of each bacterial species.
Plate 30uL of cells (10^4 cells) on each quadrant and grow for 16 hours at 37 degrees Celsius. Remember to put cell cultures back in the fridge after use!
After the 16 hours, drop the 5uL of DNAzyme on an individual colony of each bacterial species.
Incubate right side up for 1hr and UV image and Typhoon image.
Incubate for another hour and Typhoon Image.

Tracking Fluorescence Over Time

Date: August 21, 2017
Purpose: To quantify DNAzyme fluorescence over time and determine whether the addition of selection buffer impacts fluorescence results.

Setup:

E. coli K12
DNAzyme (40pmol/5uL)
2X SB
Typhoon Imager
UV Imager
M9 plates

Protocol:

Incubate E. coli cells at 37 degrees Celsius until a density of /mL
Plate 30uL of cells and let grow for 16 hours at 37 degrees Celsius.
Drop 5uL of DNAzyme (pmol) + 5uL 2xSB on a colony.
Drop 5uL of DNAzyme (pmol) + 5uL water on another separate colony.
UV image immediately. Typhoon Image Immediately.
Typhoon Image after 30 minutes, 1hr, 2hrs, 3hrs, 4hrs, and 5 hrs.
Label all results and post them here.

Experiments Redone Without Selection Buffer

Date of Experiment: September 28
Purpose: To prove the specificity of the DNAzyme and supply appropriate measurements for data analysis.

Materials:

M9 plates
E. coli K12 & E. coli K12 delta RNase
B. subtilis
A. xylosoxidans
H. alvei
S. fonticola

Protocols:

Culture all bacteria in liquid M9 media overnight
Look at the OD for bacteria and calculate cell density (around 10^8-10^9 cells/mL).
Dilute cells as necessary and plate 30uL onto a quadrant of a plate. 3 quadrants bacteria, one quadrant empty (for negative control). *made replicates to verify results
Put the plates in the incubator upside down for 16 hrs exactly
An hour before removing the plates from the incubator, prepare the DNAzyme (the optimal amount per 5uL).
Drop the 5uL of DNAzyme onto each bacterial quadrant as and one control quadrant
Incubate with the DNAzyme for 1 hour and then image with the typhoon imager.

Enzyme Kinetics & 96 Well Plate

Without Selection Buffer

Date: September 25

Materials:

96-well plate (black-clear bottom)
E. coli K12 (at least at 10^7cells/mL or 10^4cells/uL)
Liquid M9
Autoclaved distilled water
DNAzyme (RFD-EC1)

Protocol:

Grow a liquid culture of E. coli bacteria in liquid M9 overnight in a shaker incubator
Pipette 90uL of liquid M9 from A2-7 and C2-7
Dilute with liquid M9 (if necessary) to obtain a cell concentration of 10^7cells/mL or 10^4cells/uL
Transfer 100uL of cells to wells A1 and C1
Do a serial dilution and transfer 10uL across the wells (e.g. from A1 to A2, A2 to A3, etc) *once you’ve reached row 6, take out 10uL but DO NOT put into row 7
At this point, 90uL should be the volume in every well used for this experiment
Dilute using sterile distilled water (if necessary) to reach a concentration of 4uM
Using a multichannel pipette, drop 10uL of DNAzyme into the upper row (A1-7)
Scan immediately in plate reader to observe kinetics

With Selection Buffer (Replicates)

Date: October 5

Materials:

Everything else listed in previous experiment (including selection buffer)

Protocol:

Repeat all steps in previous experiment on a new plate doing triplicates to verify data and create error bars (used rows A to F)

DNAzyme Ligation Protocol

Date: Repeat performances throughout summer to replenish and refine DNAzyme stock.

Protocol

	Absorbance (OD600)	Concentration (initial)	Molecular Weight	Concentration by Weight	Concentration by Lambert-Beers
Template Short Strand	Ex:22.31A	Ex: 730 ng/uL	7884.2 g/mol	Calculated by example:90.4 uM	Calculated by example:92.6 u
DNAzyme Short Strand	Ex:21.3A	Ex:693 ng/ul	8018.2 g/mol	Calculated by example: 87.1 uM	Calculated by example: 86.4 u<

Need 5 nmol product (enough for 100 plates with 500 picomolar per plate)

10 nmol ligation because not 100% yield, therefore we need to make more than necessary.

Need 63 uL (RS28)
Template: 21.6 uL
DNAzyme: 23.1 uL to get 2 nmol
RS28: 12.8 uL

PNK Step (T4 Polynucleotide Kinase)

Description

T4 Polynucleotide Kinase enzyme catalyzes the transfer and exchange of Pi from the y position of ATP to the 5’-hydroxyl terminus of polynucleotides. This facilitates ligation because at least one of the DNA ends will now contain a 5’ phosphate, which is necessary in order for ligation to occur.

Protocol

Mix in one Eppendorf: DNAzyme: 23.1 uL 10x PNK buffer: 20 uL ATP: 4 uL PNK Enzyme: 4 uL H2O: 148.1 uL Total volume: 200 uL	Mix in another Eppendorf: RS28: 12.8 uL Template 21.6 uL H2O: 121.6 uL Total volume: 156 uL
Incubate at 37 degrees Celsius for 40 min	90 degrees Celsius for 2 min
Incubate at 90 degrees Celsius for 5 minutes	Room Temperature for 30 min

Mix both Eppendorfs together in one Eppendorf
Add 40 uL of 10x Ligation Buffer
Add 4 uL of Ligase
Centrifuge for 10 minutes
Keep at room temperature for 2 hours
Add 1000 uL of 100% EtOH
Add 40 uL of NaOAc
Centrifuge @22000 for 20 minutes, remove supernatant keeping the DNA pellet on the bottom
Keep at -20 degrees Celsius for at least 20 minutes (can leave overnight at this temperature for greater yield)
Add 500 uL of 70% EtOH
Spin in centrifuge for 10 minutes, remove the supernatant
DNA speed vacuum for 15 minutes to remove all remaining ethanol

dPAGE to purify DNA

What is a dPAGE?

A denaturing PAGE (Polyacrylamide Gel Electrophoresis) gel is a common method for separating small <500 nt nucleic acids. This gel contains high concentrations of urea as a denaturing agent. There are several methods available to visualize nucleic acids on PAGE gels, Radiolabeled nucleic acids can be visualized using film exposure or phosphor storage screens. Fluorescently modified nucleic acids can be visualized by fluorescence. Bands containing more than 300 ng of nucleic acids can be detected by UV shadow. Bands can be excised and nucleic acids extracted using Crush-Soak method.

Hazards and Safety Precautions

Lab coat, gloves are required when handling all components. Unpolymerized acrylamide is a neurotoxin, avoid contact. Use extra care when handling sharps, dispose of sharps in sharps containers.

dPAGE Gel Making

Select a set of glass plates of medium length - a set is composed of a thinner, rectangular glass plate and a thicker notched glass plate.
Select two spacers of appropriate length for the glass plates. For this case, use thick 1.5 mm spacers to cast a thick gel, the most common type used for purification due to more voluminous wells. Choose a corresponding comb of the right thickness and with wide enough teeth.
Clean glass plates with dH2O and then with 100% ethanol drying plates with paper towel. Place thinner glass plate on bottom then lay two spaces along the length of the plate on either side right at the edge. Place notched plate on top and clasp into place with 4 binder clips.
For a thick gel of medium length used for DNA purification, mix together
- 100 mL of 100% PAGE (Polyacrylamide Gel Electrophoresis liquid)
- 100 uL TEMED (Thermo Scientific Pierce Tetramethylethylenediamine)
- 1000 uL APS (Ammonium persulfate) - make sure to put APS last as the solution will solidify extremely rapidly upon addition of APS.
Swirl solution gently to mix and then slowly and steadily pour solution onto ledge formed by bottom glass plate. Let the capillary action draw the solution between the glass plates to the other side. Continue pouring while tapping glass to avoid trapped bubbles and pour until entire glass plane is enveloped in gel.
Insert teeth into notched end where gel was poured.
Let sit for 10 to 15 minutes for polymerization to complete. Gel is now ready to be loaded and run.

dPAGE Electrophoresis Protocol

Place the cast PAGE gel into the gel running apparatus flat facing glass out and adjust top reservoir height to be flush with the notched glass plate. Using binder clips, clamp the glass plate assembly to the top reservoir along the top two sides, the goal is to form a watertight seal between the reservoir gasket and the notched glass plate so as to allow buffer to enter the wells.
On a thick thickness, medium sized gel which McMaster iGEM is making, attach a metal cooling plate to the back of the glass plate assembly with 2 binder clips.
Add the minimum volume of 1X TBE buffer to the top and bottom reservoirs necessary to fill the well and contact the gel
TBE buffer stands for Tris/Borate/EDTA, the components from which it is made, and is a buffer solution used in protocols involving electrophoresis of nucleic acids. It keeps DNA deprotonated and soluble in water, contains ions which act as enzyme co-factors and protects nucleic acids against degradation.
Connect the top and bottom lids and connect the leads to a power supply. Adjust the voltage and current to 550 V.
Keep total current to less than 35 mA to prevent glass plates from cracking.
Let gel pre-run for 20 minutes to stabilize prior to loading with samples.
MEANWHILE, put all DNAzyme pellets (if making more than 2 nmol, segment into different Eppendorfs with each containing the reagents for 2 nmol of DNAzyme yield) into 1 Eppendorf
1. Add 35 uL of dH2O to Eppendorf 1, then vortex to mix. Transfer all of mixed solution to Eppendorf 2. Vortex Eppendorf 2 and transfer all of mixed total to Eppendorf 3, repeat until last Eppendorf holds all the liquid
2. Repeat 6.a again on the first Eppendorf to the last in order to wash and remove remaining traces of DNA from other Eppendorfs. Dispose of all now empty Eppendorfs à all DNA material is in the last Eppendorf now
3. Add 75 uL of loading buffer to the DNA material and spin & vortex
4. Loading buffer may crystallize so (beforehand) put on hot plate @90 degrees Celsius for 1 minute, vortex before transferring to DNAzyme solution44
Transfer ~140 uL of LB + DNAzyme into gel of the dPAGE machine using pipette and long thin “capillary” pipette tips
Just prior to loading samples, flush wells with a syringe filled with TBE buffer to remove diffused urea.
ALWAYS unplug power supply before loading and NEVER remove lids when power is running. Replug power supply after loading to let gel run.
Cover gel glass panes with a tin foil layer to protect DNA from UV degradation from light while running.
Let run for approximately 1 hour until the top yellow band visible to the naked eye reaches halfway point on the gel.

dPAGE Excision for DNA extraction

With the notched plate facing up, remove the two spacers along the edges. With a thin spatula, carefully separate the top two glass plates, the gel will stay attached to one of the glass plates. Be careful not to rip the gel! With the gel facing up, lay a sheet of plastic wrap over the gel minimizing wrinkles and bubbles.
Grasping the plastic wrap and the glass plate underneath, flip the entire assembly over so the glass plate is facing up.
Lift one end of the glass plate slightly when carefully separating the gel from the glass plate with a thin spatula, proceed until the gel has completely separated from the glass plate.
Apply a second sheet of plastic film to the gel.
Trim the excess film.
Place a blade under the gel to provide sterile surface after wrapping both ends of gel with cellophane
Unclip bottom 2 clips from the gel electrophoresis machine, let the buffer drip down, then lay the glass pane horizontal
Take out teeth and black side stripes on either side, peel off one side of glass pane and cover that side with cellophane.
Flip over and do the same for the other side so the gel is fully wrapped in cellophane
Wrap the ends and edges of excess cellophane
Image gel using UV machine (for fluorescence) as well as a handheld UV 260 for DNA
Identify the band holding the “pure” DNAzyme under UV light. Try to minimize amount of time DNAzyme spends under UV light by quickly drawing a rectangle around the glowing top yellow band (there should be 2 bands) and excising outside of the UV light room. Too much UV light exposure can cause DNAzyme to mutate, impacting specificity.
Draw outline of the top yellow band during UV lighting to later excise and cut away using microblades
Cut using microblades through the cellophane, along the lines of the outline drawn earlier
Cut box into 3 even sections
Carefully peel back cellophane and deposit each gel square into an Eppendorf, folding it if need be
Crush gel square into a paste within each Eppendorf with a pipette tip or metal spatula. Ensure no paste gets stuck within the tip or lost from the spatula and Eppendorf – wasted DNA

Crush and soak DNA Extraction from Gel

Elute with elution buffer (600 uL) for 20 minutes, spin and shake with shake machine
Take out of shaker and spin in mini-centrifuge until there is a visible divide between gel and supernatant liquid
Take out supernatant and put together in a different Eppendorf. Label as Supernatant 1 (IMPORTANT, THIS HOLDS DNAZYMES EXTRACTED FROM THE GEL)
Add 400 uL of elution buffer again to each gel only Eppendorf, shake again for 20 minutes and elute again by repeating steps 8.a to 8.b. Store in one Eppendorf if possible.
Now aliquot the supernatant Eppendorf into more Eppendorfs so that the optimal volume for each Eppendorf holds 400 uL of supernatant. If volume in each Eppendorf is not 400 uL, aliquot around 200 uL of volume into the remaining Eppendorf so that adding 1000 uL of ethanol will not overflow 1.5 mL Eppendorf volume capacities.
Add 10%* of the total volume of NaOAc to the eluted solution and then add 1000 uL of 100% ethanol add end. (Ex: so add 40 uL of NaOAc to 400 uL of elution buffer)
Freeze the solution tubes in -20 degrees Celsius freezer, put the tubes in the cold room with tin foil on top to block off light interfering with DNAzyme fluorescence for a minimum of 20 minutes (longer periods of time such as overnight may result in better yield).
Centrifuge @15000 RPM for 20 min. You should see clear-ish supernatant and at the bottom, orangey specks of solid DNAzyme pellet. These orange specks are now crucial to holding your DNAzyme… please do not lose!
Remove supernatant and to the pellet remaining in Eppendorf, add 500 uL of 70% ethanol. BE CAREFUL NOT TO REMOVE ANY OF THE PELLET ALONG WITH THE SUPERNATENT. CENTRIFUGE AGAIN IF PELLET GETS MIXED UP IN THE SUPERNATENT.
Centrifuge again for 10 min then remove supernatant, put pellet tube open in speed vacuum for 15 minutes for ethanol precipitation.
After ethanol precipitation, should have a dry visible pellet at the end of the Eppendorf tube.
Add 100 uL of H2O into each tube (and following the actions of 2.a and 2.b, one after one wash again – 50 uL wash then 50 uL H2O to wash again) to put all DNAzyme solutions into one Eppendorf
End should have 1 Eppendorf tube with ~90 uL of orangey fluid (DNAzymes)
Keep cool

Quantifying DNA

Use a spectrophotometer to meeasure the concentration and absorbance.

Concentration can be converted from ng/uL to uM by calculating with the molecular weight.
Concentration can be calculated from the absorbance value (OD600) through the Lambert Beers Equation (c=A/ɛ *ʃ)
- C = concentration to be found
- A = absorbance in terms of Abs600
- ɛ = Extinction co-efficient
- ʃ = length of light path (1 cm)

References

Osterholm, M. T. (2011). Foodborne Disease in 2011 — The Rest of the Story. New England Journal of Medicine, 364(10), 889–891. https://doi.org/10.1056/NEJMp1010907
Borzio, M., Salerno, F., Piantoni, L., Cazzaniga, M., Angeli, P., Bissoli, F., … Sangiovanni, A. (2001). Bacterial infection in patients with advanced cirrhosis: a multicentre prospective study. Digestive and Liver Disease, 33(1), 41–48. https://doi.org/10.1016/S1590-8658(01)80134-1
Velusamy, V., Arshak, K., Korostynska, O., Oliwa, K., & Adley, C. (2010). An overview of foodborne pathogen detection: In the perspective of biosensors. Biotechnology Advances, 28(2), 232–254. https://doi.org/10.1016/j.biotechadv.2009.12.004
Ali, M. M., Aguirre, S. D., Lazim, H., & Li, Y. (2011). Fluorogenic DNAzyme Probes as Bacterial Indicators. Angewandte Chemie International Edition, 50(16), 3751–3754. https://doi.org/10.1002/anie.201100477
Aguirre, S. D., Ali, M. M., Kanda, P., & Li, Y. (2012). Detection of Bacteria Using Fluorogenic DNAzymes. Journal of Visualized Experiments, (63). https://doi.org/10.3791/3961

Team:McMasterU/DNAzyme

Introduction

Timeline of Experiments

Fluorophore

Medium Optimization: M9 vs LB (Luria Bertani)

Testing DNA Specificity

Fluorescence Detecting Wavelength

Bacterial Autofluorescence

DNAzyme Autofluorescence

Optimal Response

DNAzyme Specificity - 2X Selection Buffer Added

Tracking Fluorescence Over Time

Specificity Test - Without Selection Buffer

Protocols

Testing Fluorescence of Bacterial Plates

Fluorophore Concentrations

Optimizing Concentrations & Cleavage

Materials/Reagents:

Protocol:

M9 vs LB Autofluorescence via Agar Thickness

Cell Detection Using DNAzyme in M9

Materials & Reagents:

Protocol:

Differing [NaOH] for Positive Control

Reagents:

Protocol:

Testing DNAzyme Sensitivity

Setup:

DNAzyme Specificity

Reagents:

Protocol:

Bacterial Mixture

Materials/Reagents:

Protocol:

Fluorescence detecting wavelength

Reagents/Materials:

Protocol:

Optimal DNAzyme Concentration

Reagents/materials:

Protocol:

Quantifying Autofluorescence

Reagents/materials:

Protocol:

Fluorescing Colonies

Reagents/materials:

Protocol:

DNAzyme Autofluorescence

Reagents/materials:

Protocol:

Bacterial Autofluorescence

Setup:

Protocol:

DNAzyme Autofluorescence

Setup:

Protocol:

Optimal Response

Setup:

Protocol:

DNAzyme Specificity

Setup:

Protocol:

Tracking Fluorescence Over Time

Setup:

Protocol:

Experiments Redone Without Selection Buffer

Materials:

Protocols:

Enzyme Kinetics & 96 Well Plate

Without Selection Buffer

Materials:

Protocol:

With Selection Buffer (Replicates)

Materials:

Protocol:

DNAzyme Ligation Protocol

Protocol

PNK Step (T4 Polynucleotide Kinase)

Description

Protocol

dPAGE to purify DNA