Aptamer Sensing System

Finalised protocol to test for the presence of λ-cyhalothrin (in lab) within a sample (suspended in acetone).
Protocol can be scaled up (e.g., 2x).

Materials

• PDDA (Polydiallyldimethylammonium chloride)
• 30 nm Gold Nanoparticles (AuNP)
• 500 mM MOPS Buffer
• 10 µM Aptamer of choice (suspended in TE 1X Buffer)
• Pesticide sample (suspended in acetone)

Procedure

Add the following into a microtube, in the following order and ensuring it is fully mixed:

1. 40 µL PDDA (Polydiallyldimethylammonium chloride)
2. 10 µL 500 mM MOPS Buffer
3. 15 µL 10 µM Aptamer of choice (suspended in TE 1X Buffer)
4. 30 µL Pesticide sample (suspended in acetone)
5. 60 µL 30 nm Gold Nanoparticles (AuNP)

Incubate microtube at 30°C for 10 minutes.

6. Observe any colour change; red = pesticide absence, blue = pesticide presence.
7. Dilute microtube contents with MOPS buffer (in order to create a 10x dilution).
8. Test diluted microtube contents within a spectrophotometer; at both 528 nm and 650 nm.
9. Calculate the absorbance ratio (650 nm/528 nm) - the higher the ratio, the higher the level of aggregation.

TE 1x Buffer

Materials

• 1M Tris-Cl (pH 8)
• 0.5M EDTA (pH 8)
• Deionised water

Combine 1 mL of 1M Tris-Cl with 0.2 mL 0.5M EDTA; lastly combine with 98.8 mL deionised water.

500 mM MOPS Buffer

Protocol to make 250 mL of 500 mM MOPS buffer.

Materials

• MOPS powder
• Deionised water
• 5 µM NaOH

Procedure

1. Combine 26g of MOPS powder with approximately 200 mL of deionised water.
2. Continually add NaOH to the solution, until a pH of 7 is attained.
3. Make any final adjustments to reach the 250 mL final volume using deionised water.

Chemically Competent E. coli

Filter sterilise and store at 4°C. Centrifuge at 3400 g, for 10 minutes. Liquid nitrogen can be substituted with dry ice + 100% EtOH bath, or omitted entirely for slightly less competent cells.

Procedure

1. Inoculate 10 mL of LB medium with a small portion of cell scraped from the surface of a frozen glycerol stock.
2. Incubate overnight with agitation at 37°C.
3. Dilute culture to an OD600 of 0.05 in 200 mL and further incubated until an OD600 of 0.4-0.6.
4. Aliquots of 35 mL were centrifuged and combined pellets resuspended in 60 mL ice-cold 100 mM CaCl2/10% glycerol solution.
5. Centrifuge cells again and combined pellets resuspended in 8 mL ice-cold 100 mM CaCl2/10% glycerol solution
6. Incubate on ice for 25 minutes. Aliquots of the cells were snap frozen in liquid nitrogen and stored at -80°C.

Golden Gate Assembly (pTYK-001 Level 1 Assembly)

Materials

• 28.5 ng/µL Backbone pYTK-001
• 20 ng/µL Insert Pro-RBS-Pyre1-Term
• 10X T4 Ligase Buffer (Invitrogen)
• T4 Ligase (Invitrogen)
• BsmBI
• Nuclease-free water

Procedure

1. Combine: 3.51 µL pYTK-001, 2.5 µL insert, 1 µL T4 ligase buffer, 0.5 µL T4 ligase, 0.5 µL BSMBI and 1.99 µL nuclease-free water.
2. Program and run the PCR machine for: (37°C for 10 min, 16°C for 10 min) x10, 50°C for 5 min, 65°C for 20 min, 4°C forever.
3. Transform 2 µL into E. coli competent cells and select on LB chloramphenicol (25 µg/ml) for white colonies.

iGEM Transformation Protocol

Preparation

1. Resuspend DNA in selected wells in the Distribution Kit with 10 uL dH2O. Pipette up and down several times, let sit for a few minutes. Resuspension will be red and from cresol red dye.
2. Label 1.5 mL tubes with part name or well location. Fill lab ice bucket and pre-chill 1.5 mL tubes (one tube for each transformation, including your control) in a floating form tube rack.
3. Thaw the cells on ice. This may take 10-15min for a 260 uL stock. a. Dispose off unused competent cells. Do not refreeze thawed cells, as it will drastically reduce transformation efficiency.

Protocol

1. Pipette 50 uL of competent cells into 1.5 mL tube per transformation. Tubes should be labelled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don't forget a 1.5 mL tube for your control.
2. Pipette 2 uL of resuspended DNA into 1.5 mL tube. Pipette from well into appropriately-labelled tubes. Gently pipette up and down a few times. Keep all tubes on ice.
3. Pipette 2 uL of control DNA into 2 mL tube. Gently pipette up and down a few ties. Keep all tubes on ice.
4. Close 1.5 mL tubes and incubate on ice for 30 minutes. Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
5. Heat shock tubes at 42°C for 45 seconds. 1.5 mL tubes should be in a floating foam rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
6. Incubate on ice for 5 minutes.
7. Pipette 950 uL of SOC media to each transformation. SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
8. Incubate at 37°C for 1 hour, shaking at 200-300 rpm.
9. Pipette 100 uL of each transformation into Petri dishes. Spread with sterilised spreaders or glass beads immediately. This helps ensure that you will be able to pick out a single colony. Spin down cells at 6800G for 3 minutes and discard 800 uL of the supernatant. Resuspend the cells in the remaining 100 uL, and pipette each transformation onto petri dishes. Spread plate with sterilised spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
10. Incubate transformation overnight (14-18 hours) at 37°C. Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow. (/li)
11. Pick single colonies: Pick single colonies from transformation. Do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
12. Count colonies for control transformation. Count colonies on the 100 uL control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA.