Team:McMasterU/CDifficile

• HOME
• WET LAB
  • Project Description
  • DNAzyme Plate Experiment
  • C. Difficile Project
  • InterLab
  • Results
• DRY LAB
  • Image Processing
  • Enzyme Model
  • SELEX Optimization with Machine Learning
• HUMAN PRACTICES
  • Silver Medal
  • Integrated Human Practices
  • Public Engagement
  • Collaboration
• TEAM
  • Members
  • Sponsors
  • Attributions

Introduction

We are planning to generate a cis-acting DNAzyme using in vitro selection for the detection of pathogenic strains of C. difficile. This DNAzyme will be enriched from a combinatorial sequence library of around 10^15 single-stranded DNA with central random sequences. The DNAzyme will be selected using the 61-aa (NAP1) and 65-aa (NAP7) truncated variants of C. difficile TcdC (expressed in E. coli) as the target. This target was chosen because of its low identify ratio between wild type and pathogenic strains of C. difficile. An oligonucleotide containing a fluorophore-riboadenosine-quencher motif will be ligated to the random single-stranded DNA library. DNAzymes will be selected from the library based on their ability to cleave the riboadenosine in the presence of the TcdC variants specified above. Negative selection of the library will be performed sequentially in the presence of E. coli, wild type TcdC, and B. subtilis CEM. We have cloned NAP1, NAP7, and wild type TcdC into pET15 plasmids and are currently working on expressing the genes.

---

Plasmid Design

Green light inducible plasmid

Red light inducible plasmid

Chromophore producing plasmid

Project Design

In-Vitro Selection

Genetic evolution by natural selection has guided life as we know it through billions of years of Earth’s harshest environments, giving rise to millions of diverse and steadfast lifeforms that we see around us today. In the lab, we can mimic this grandiose process to our advantage on a microscale using a technique called in-vitro selection¹. By following Darwin’s principles of natural selection in a controlled environment, it is possible to artificially evolve large groups of DNA molecules from randomness and select for those with useful functions. This is done by exposing large, “random libraries” of short DNA strands to other molecules of interest, and removing the species in the pool that don’t react to them in the way we desire. These reactions are observed on the macroscale using the tried and true technique of polyacrylamide gel electrophoresis on the radio-labelled, denatured (untied and linearized) DNA libraries after exposure. In the end, an assortment of DNA molecules comprising of more than a quintillion random nucleotide sequences is reduced to a handful of highly specific, functional product species.

If selected successfully to process a substrate on que, these nucleic acids are called DNAzymes. We work with DNAzymes that have been artificially selected to serve as a detection platform for E. coli, one of the most infamous antimicrobial drug-resistant strains of bacteria in the world^2,3. Our DNAzymes react in the presence of a cocktail of molecules radiated specifically from the extracellular matrix of the E. coli bacterium. The ensuing process results in the cleavage of an RNA unit bridging a quencher-fluorophore DNA complex, resulting in the emission of a signature green glow. In essence, we’ve used one of nature’s oldest solutions to generate something innovative, creative, and potentially revolutionary to the field of medical point-of-care testing.

Compiled Protocols for TcdC Plasmids Design

I. C. Difficile gDNA Extraction Using Phenolchloroform

Materials

Resuspension Buffer

• 10 mM Tris-HCl, pH 8.0
• 20% (w/v) Sucrose

Lysis Buffer

• 10 mM Tris-HCl, pH 8.0
• 1mM EDTA
• 1% (w/v) SDS

RNase A (10mg/mL)

• 10 mg bovine pancreating RNase A
• 10 mM Tris, pH 8.0
• 500 mM NaCl
• 850 uL water
• Heated to 95 C until use

Protocol

1. 10 mL of overnight culture was pelleted at 5000 rpm for 5 minutes
2. Pellets were resuspended in 1 mL of resuspension buffer.
3. 9 mL of lysis buffer was added to resuspended pellet.
4. Cell suspension was incubated at 37 C for 1 hour, shaking at 260 rpm.
5. Crude cell lysate was extracted with an equal volume of phenol chloroform.
6. Aqueous phase was recovered to a new tube and 25 ug RNase A was added, crude lysate was incubated at 37 C for 20 minutes.
7. Crude lysate was extracted with equal volume of phenolchloroform, aqueous phase removed to a new tube.
8. DNA was precipitated using ethanol precipitation. (see Other Protocols)
9. DNA was dissolved in 1.5 mL nuclease free water

II. PCR Amplification of BAA-1870, BAA-1875, and WT TcdC

Protocol

1. Primers were diluted to 10 uM
2. dNTP was diluted to 10 mM
3. DNA samples were diluted to 125 ng/uL
4. The following master mix solution was prepared

Reagent	1x(uL)
Forward Primer	2.5
Reverse Primer	2.5
dNTP	1
Phusion Buffer	10
Phusion High Fidelity Polymerase	0.5
Nuclease-free water	31.5

5. 2 uL of DNA was added to 48 uL of the master mix
6. PCR reaction was run in a thermocycler using the following conditions for 35 cycles:

Step	Temperature (C)	Time
Initial Denaturing	98	10 minutes
Denaturing	98	30 seconds
Annealing	50	20 seconds
Elongation	72	20 seconds
Final elongation	72	10 minutes
Hold	4	Indefinite

7. The PCR reactions were run on a 1% agarose gel and purified using gel extraction (see Other Protocols)

Results:

Figure 1: PCR Amplification of NAP1, NAP7, and WT TcdC
III: Digestion of TcdC and pET15b

1. The following reaction was set up:

   Component	50 uL Reaction
   DNA	1 ug
   FastDigest Buffer	5 uL
   Ndel	1.0 uL
   BamHI	1.0 uL
   Nuclease Free Water	To 50 uL

2. Reactions were incubated at 37 Celsius for one hour
3. Digestion reaction were run on a 1% agarose gel and purified using gel extraction (see Other Protocols)

Digestion of TcdC and pET15b

Protocol

1. The following reaction was set up:

Component	50 uL Reaction
DNA	1 ug
FastDigest Buffer	5 uL
Ndel	1.0 uL
BamHI	1.0 uL
Nuclease free water	To 50 uL

2. Reactions were incubated at 37 degrees Celsius for one hour
3. Digestion reaction were run on a 1% agarose gel and purified using gel extraction (see Other Protocols)

Ligation of TcdC to pET15b

Protocol

1. The following reaction was set up:

Component	20 uL Reaction
10X T4 Ligase Buffer	2 uL
Vector DNA	70.54 ng (0.02 pmol)
Insert DNA	25.81 ng (0.06 pmol)
T4 DNA Ligase	1 uL
Nuclease-free water	To 20 uL

2. The ligation reactions were incubated at 16 degrees Celsius overnight.

V. Transformation Using Chemically Competent Cells

Protocol

1. Invitrogen DH5 alpha cells were thawed on ice for 10 minutes, 50 uL of cells were pipetted into transformation tube
2. 5 uL of ligation reaction was added into cell mixture
3. Mixture was incubated on ice for 30 minutes
4. Mixture was heat shocked at 42 C for 30 seconds
5. Mixture was transferred to ice for 5 minutes.
6. 950 uL of LB broth was added to mixture
7. Mixture was incubated at 37 C for 60 minutes, shaking at 250 rpm
8. Selection plates containing 50 ug/mL of ampicillin were warmed to 37 C
9. Cells were pelleted at 4000 g for 1 minute
10. 900 uL of LB broth was removed
11. Cells were plated onto selection plates and incubated for 16 hours at 37 C
12. Colonies were picked and grown in 5 mL of LB broth for 16-18 hours

VI. Miniprep Plasmids

Promega Miniprep Kit was used.

1. 1.5 mL of liquid culture was centrifuged at maximum speed for 30 seconds. Supernatant was discarded
2. An additional 1.5 mL of bacterial culture was added to the same tube and step 1 was repeated.
3. 600 uL of nuclease-free water was used to resuspend the cells
4. 100 uL of cell lysis buffer was added and mixed by inverting the tube 6 times
5. 350 uL of cold neutralization solution was added
6. Mixture was centrifuged at maximum speed for 3 minutes
7. Supernatant (~900 uL) was transferred to a minicolumn without disturbing the cell debris pellet and centrifuged at maximum
8. speed for 15 seconds. Supernatant was discarded.
9. 200 uL of endotoxin removal wash was added to the minicolumn. Minicolumn was centrifuged at maximum speed for 15 seconds
10. 400 uL of column wash solution was added to the minicolumn and centrifuged for 30 seconds
11. Minicolumn was transferred to a clean 1.5 mL Eppendorf tube and 30 uL of elution buffer was added
12. After 1 minute, tubes were centrifuged for 15 seconds to elute the plasmid DNA.

Other Protocols

Ethanol Precipitation

1. Add 0.1 volumes of 3M NaOAc, 2.5 volumes of ice cold 100% ethanol, and mix
2. Precipitate at -20 C for at least one hour
3. Centrifuge at 15000 rpm at 4 C for 30 minutes
4. Wash pellet twice with 0.5 mL of ice cold 75% ethanol, spinning at 4 C for 10 minutes each time
5. Remove ethanol and dry the pellet in a Speed-Vac
6. Resuspend pellet in nuclease free water

Agarose Gel Electrophoresis

1. Dilute 50X TAE to 1X
2. Add 1% mass of agarose to TAE
3. Microwave until agarose has dissolved
4. Cool to around 50 C, then add SYBR Safe (1 uL for every 10 mL of TAE)
5. Pour gel into casting tray with a comb
6. Once gel has solidified, place the gel into a gel electrophoresis machine and add 1X TAE until the gel is fully covered
7. Load 5 uL of 1kb DNA ladder into the first well and DNA samples into subsequent wells
8. Run the gel for about 45 minutes

DNA Extraction from Agarose Gels

Nucleospin Gel and PCR Clean-up Kit was used

1. Use a clean razor blade to excise DNA fragment from gel
2. For every 100 mg of gel, add 200 uL of Buffer NTI (gel solubilization solution)
3. Place a binding column into a collection tube and add 700 uL of sample. Centrifuge for 30 seconds at 11,000 g. Discard flow through. Repeat if necessary
4. Add 700 uL of Buffer NT3 (wash buffer). Centrifuge for 30 seconds at 11,000 g. Discard flow-through. Repeat this step to minimize chaotropic salt carry-over.
5. Centrifuge for 1 minute at 11,000 g to remove Buffer NT3 completely. To remove residual ethanol, incubate the columns for 5 minutes at 70 C prior to elution
6. Place the column into a new 1.5 mL Eppendorf tube and add 30 uL of nuclease-free water. Incubate for 1 minute before centrifuging for 1 minute at 11,000 g.

Parts

<groupparts>iGEM17 McMasterU</groupparts>

[1] Wilson, D. S. & Szostak, J. W. In Vitro Selection of Functional Nucleic Acids. Annual Review of Biochemistry 68, 611–647 (1999).

[2] Aguirre, S., Ali, M., Salena, B. & Li, Y. A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection. Biomolecules 3, 563–577 (2013).

[3] Antimicrobial resistance: global report on surveillance. (World Health Organization, 2014).

Follow Us

• 
• 
• 
• 

© 2017 mGEM