Http://2013.igem.org/Http://2013.igem.org/Team:TU-Delft/LabWork/Protocols

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Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.

1. Transforming Parts from Distribution kit:

Requirements:

1. Distilled water 2. Pipettes 3. Ice 4. Competent cells 5. SOC media 6. Agar plates with Antibiotics 7. Timer 8. Water bath

Prodecure:

1. Mark the location on distribution plate by looking at the right row and column as mentioned. Punch the plate with a pipette and add 10 µL of distilled water.
2. Resuspend the Dry DNA on the distribution plate with distilled water.
3. Inoculate the competent cells with the desired DNA from the distribution plate as described in step 2. Add 1 µL of the resuspended DNA to the competent cells. Use 2 mL tubes and keep the cells on ice at all the times for a more efficient transformation.
4. Incubate the transformation on ice for 5 min.
5. Heat shock the transformation the water bath at 42⁰C for 30 sec.
6. Incubate on ice for 2 min.
7. Add 200 µL SOC Media to the transformation.
8. Incubate the transformation at 37⁰C for 1 hour.
9. Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.
10. Incubate the agar plate overnight (14-16 hours) at 37⁰C.

Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid.

2. Growing the Single Colonies from the Agar Plates:

Requirements:

1. LB Broth 2. Pipettes 3. Antibiotics 4. Sterile flasks

Prodecure:

1. Prepare flask with LB Broth in it. Add the appropriate antibiotic needed.
2. Select the single colony using the pipette tip from the Agar plate, which contains the bacterial cells.
3. Inoculate the LB broth with the bacterial cells.
3. Grow the culture for 14-16 hours.

Next day prepare glycerol stocks and carry out a miniprep protocol to extract the desired plasmid from the bacterial cells.

3. Making glycerol stocks:

Requirements:

1. Glycerol 2. Pipettes 3. 1.5 mL Tubes 4. Culture Sample

Prodecure:

1. Label the tubes and add 180μL of glycerol.
2. Pipette out 1000 μL of the culture.
3. Add the culture to the tubes and store in -80°C

The glycerol stock can be used whenever required, by just adding 0.5 mL of stock into 5 mL of freshly prepared media.

4. Miniprep Protocol:

We use Qiagen Spin Miniprep Kits for doing small batches of minipreps. This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Note: All protocol steps should be carried out at room temperature.

Requirements :

1. Qiagen Miniprep Kit
2. Cell Culture
3. 1.6 ml Microcentrifuge tubes (2 per a miniprep)
4. TE (1:10)

Prodecure:

1. Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container.
2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet. Important: Ensure that RNase A has been added to Buffer P1.
3. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. 4. Add 350 μl Buffer N3 and invert the tube immediately and gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.
6. Apply the supernatants from step 5 to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for 30–60 s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 1 min.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

This plasmid DNA is stored in -20°C and is used to carry out restriction digestion when needed.

5. Restriction digestion:

Requirements:

1. Plasmid DNA 2. Restriction Enzymes 3. 1.5 mL Tubes 4. NE Buffer 2 5. BSA 6. Distilled water

Prodecure:

1. Add 25 μL of plasmid DNA and suitable amount of Distilled water to make up total volume to 50 μL. The amount of plasmid DNA to take depends upon the concentration of the DNA solution.
2. Add 5 μL NE Buffer 2 and 0.5 μL of BSA.
3. There should be a total volume of 50 μL. Mix well and spin down briefly.
4. Incubate the restriction digest at 37°C for 30 min, and then 80°C for 20 min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid.

Run a portion of the digest on a gel (8ul, 100ng)to check that both plasmid backbone and part length are accurate. The next step would be to carry out ligation of the digested pieces of DNA.

6. Ligation:

Requirements:

1. Cut Insert DNA 2. Cut Vector DNA 3. Ligase buffer 4. T4 DNA ligase 5. Distilled water

Prodecure:

1. Calculate the amount of vector DNA and insert DNA to add based on the ratio of the DNA and vector(3:1),if insert DNA size is 5 times smaller than vector DNA or 1:1 ratio if both sizes are same.
2. Add suitable amount of distilled water to make up total volume to 50 μL.
3. Add 1 μL T4 DNA ligase buffer.
4. Add 1 μL T4 DNA ligase.
5. Ligate at 16°C overnight or leave it at room temperature for 4 hours.

The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.

7. Gel Extraction Procedure:

Requirements:

1. Scalpel 2. Pipettes 3. Microcentrifuge tubes 4. QIA quick column and collection tubes 5. Buffer PE 6. Buffer QG 7. Sterile MilliQ

Prodecure:

1. Excise the DNA fragment from the agarose gel with a clean scalpel.
2. Weigh the gel slice in a tube. Add 3 volumes of Buffer QG to 1 volume of the gel.
3. Incubate at 50 ⁰C for 10 mins (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 mins during incubation.
4. After the gel slice has dissolved completely check that the colour of the mixture is yellow.
5. Place a QIA quick spin column in 2mL collection tube and centrifuge for 1 min.
6. Discard the flow through and place QIA quick column back in same collection tube.
7. To wash, add 0.75 mL of Buffer PE to the QIA quick column and centrifuge for 1 min at 13000 rpm.
8. Discard the flow through and centrifuge for an additional 1 min at 13000 rpm to remove the remaining ethanol.
9. Place the column on a clean microcentrifuge tube.
10. Add 40-50 µL of milliQ sterile H2O.
11. Incubate in oven for 2 mins and then centrifuge again for 1 min.
12. Measure on Nanodrop for the concentration of the DNA.

8. PCR Purification:

Requirements:

1. Buffer PB 2. Pipettes 3. Buffer PE 4. Microcentrifuge tubes 5. QIA quick spin columns

Prodecure:

1. Add 5 volumes of Buffer PB to 1 volume of the sample.
2. Transfer the mixture to the QIA quick spin columns and centrifuge the column for 1 min at 13000 rpm.
3. Discard the flow through and place the spin column in the same collection tube.
4. To wash, add 0.75 mL of Buffer PE to the column, and centrifuge for 1 min. Discard the flow through and place the column back in the same collection tube.
5. Centrifuge the empty column for an additional 1 min to remove the remaining Buffer PE from the column.
6. Now place the column in a fresh microcentrifuge tube. Add 50 µL of sterile milliQ water.
7. Place the column in oven for 2 mins, then centrifuge again for 1 min at 13000 rpm.
8. Measure the sample on Nanodrop to get the concentration.