ICSynBio:Protocols

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Promoter Engineering

• Segment-specific mutagenesis: Extensive mutagenesis of a lac promoter/operator element
• QuikChange Mutagenesis
• Site Directed Mutagenesis

Oligoannealing

• Promoters need to be transformed from single strand to double strand through oligoannealing

RBS Engineering

• Custom RNA Oligos

Plasmid Construction

• Restriction Enzyme Protocol

Plasmid Insertion

• Blunt End Cloning - Transformation of designed parts to become chemically competent E.Coli cells
• Bacterial Transformation

Miniprep

Isolation of plasmid DNA from bacteria

1. Inoculate 5 ml 2x YT medium with the bacterial strain and incubate the culture for 12-16 hours at 37°. Avoid overgrowth, which may lead to reduced plasmid yields.
2. Pellet 5ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature.
3. Completely resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that no clumps of cells remain.
4. Add 250μl Buffer P2 and mix by inverting the tube 10-12 times until the solution turns completely blue. Incubate for 5 min. do not allow lysis to proceed for more than 5 min.
5. Add 350μl Buffer N3 and mix immediately and thoroughly by inverting the tube 10-12 times until the solution turns colourless.
6. Centrifuge for 10 min.
7. Apply the supernatant from step 6 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through.
8. Wash QIAprep spin column by adding 500μl Buffer PB. Centrifuge again as in step 7.
9. Wash the QIAprep spin column by adding 750 μl Buffer PE. Centrifuge as in step 7.
10. Centrifuge for 1 min to remove residual wash buffer.
11. Place the QIAprep spin column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 60 μl Buffer EB (10mM Tris-Cl, pH 8.5) to centre of the QIAprep spin column, let it stand for 1 min and centrifuge for 1 min.

Glycerol Stock

Storing the bacteria

1. Add 500μl of glycerol to a centrifuge tube.
2. Add 500μl of the bacteria solution to the same tube.
3. Centrifuge for 5 minutes at 6700rpm.
4. Remove supernatant and freeze.

Imaging the Bacteria

Agarose Pad Imaging

Flow Cytometry

• Flow Cytometer Calibration

MoClo

• Assembly of Toggle Switch and insertion into Plasmid

Pouring Agar Plates

(Taken from https://www.addgene.org/protocols/pouring-lb-agar-plates/)

• Equipment needed: Autoclave, Water bath, Pipetman
• Reagents needed:
• 1 L Sterile H2O
• Sterile plates (60 mm x 15 mm)
• Autoclavable flask
• Sterile pipettes
• Ice bucket to hold antibiotic
• Ampicillin and Kanamycin

1. Measure 37g of pre-mixed LB-agar powder per L of molten agar needed

2. Transfer the LB-agar powder into autoclavable flask.

3. Transfer the sterile water into the bottle and swirl to form a medium/agar colloid.

4. Cover the opening of the bottle with aluminium foil and tape the bottle with autoclave tape.

5. Label the bottle with your initials, the date, and the bottle contents.

6. Place the gel mix in the autoclave and run on 121 ℃ under 20 psi for at least 30 min.

7. Spray down the bench with a 70% ethanol solution

8. Label the plates with the date, medium, and antibiotic.

9. Create a 1000xstock solution of antibiotic

10. Retrieve your molten agar mix from the autoclave and partially submerge it in a 60 ℃ water bath.

11. Light the flame at the plate pouring station and dilute antibiotic into molten gel mix using sterile technique. Swirl the bottle to ensure even distribution of the antibiotic

12. Open one plate at a time next to the flame and begin pouring.

13. Leave your plates out on the bench to solidify.

Plate Streaking Protocol

(Taken from https://www.addgene.org/protocols/streak-plate/)

• Equipment needed: Wire loop, Bunsen burner, Incubator, Marker
• Reagents needed: LB agar plate (with ampicillin), Bacterial stab

Protocol:

1. Spray workspace with 70% ethanol. Maintain sterility by working near a flame or Bunsen burner.

2. Obtain the appropriate bacterial stab or glycerol stock, and LB agar plate

3. Using a sterile loop, touch the bacteria growing within the punctured area of the stab culture or the top of the glycerol stock.

4. Gently spread the bacteria over a section of the plate

5. Using a fresh sterilized loop, drag through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.

6. Using a third sterile loop, drag through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.

7. Incubate plate with newly plated bacteria overnight (12-18 hours) at 37 °C.

Miniprep Protocol: (Taken from QIAGEN Plasmid Purification Handbook)

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm).

2. Dilute the starter culture 1/500 to 1/1000 into 3 ml selective LB medium. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).

3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.

4. Resuspend the bacterial pellet in 0.3 ml of Buffer P1.

5. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.

6. Add 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4– 6 times, and incubate on ice for 5 min.

7. Centrifuge at maximum speed in a microcentrifuge for 10 min. Remove supernatant containing plasmid DNA promptly. Optional: Remove a 50 µl sample from the cleared lysate and save it for an analytical gel (sample 1).

8. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow.

9. Apply the supernatant from step 7 to the QIAGEN-tip 20 and allow it to enter the resin by gravity flow. Optional: Remove a 50 µl sample of the flow-through and save for an analytical gel (sample 2).

10. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC. Optional: Remove a 220 µl sample of the combined wash fractions and save for an analytical gel (sample 3).

11. Elute DNA with 0.8 ml Buffer QF. Collect the eluate in a 1.5 ml or 2 ml microcentrifuge tubes (not supplied). Optional: Remove a 45 µl sample of the eluate and save for an analytical gel (sample 4).

12. Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of roomtemperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g rpm for 30 min in a microcentrifuge. Carefully decant the supernatant.

13. Wash DNA pellet with 1 ml of 70% ethanol and centrifuge at 15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet.

14. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris·Cl, pH 8.5)

Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus)

1. Set-up the following reaction in a microcentrifuge tube on ice:
   1. DNA
   2. up to 300 pmol of 5´ termini
   3. T4 PNK Reaction Buffer (10X)
   4. 5 µl
   5. ATP (10 mM)
   6. 5 µl
   7. T4 PNK
   8. 1 µl (10 units)
   9. Nuclease-free Water
   10. up to 50 µl
2. Incubate at 37°C for 30 minutes.
3. Heat inactivate by incubating at 65°C for 20 minutes.

Annealing Oligonucleotides

(Taken from https://www.sigmaaldrich.com/technical-documents/protocols/biology/annealingoligos.html)

Equipment: Heat block or Thermocycler

Supplies: 2 mL centrifuge tubes, Pipette tips, Milli-Q® H2O, EDTA, NaCl, Trizma®, Two singlestranded oligonucleotides with complementary sequences

Protocol:

• Dissolution: Dissolve each oligonucleotide in a volume of Annealing Buffer (Concentration of

each oligonucleotide needs to be 2X the desired concentration of the duplex oligonucleotide.

• Annealing: Mix Heat Block, equal volumes of the equimolar oligonucleotides in a microtube.

Incubate the microtube at 95 °C for 5 min. Allow the microtube to slowly cool to room temperature (should take <60 min).

• Thermocycler: Mix equal volumes of the equimolar oligonucleotides in a PCR tube.

1. Use the following thermal profile
   1. Heat to 95 °C and maintain the temperature for 2 min.
   2. Cool to 25 °C over 45 min
   3. Cool to 4 °C for temporary storage.
2. Centrifuge the PCR tube briefly to draw all moisture away from the lid.

Golden Gate Assembly Protocol

• The following components were added to a 0.2 mL tube: 10−60 fmol of each DNA component,

equimolar 10−50 U of BsaI , 5−50 U of T4 DNA ligase , 1× T4 DNA ligase buffer, and deionized water to at total volume of 10−60 μL.

• Reactions were performed using the following parameters: 15−40 cycles (37°C 1.5−3 min, 16 °C

3−5 min), followed by 50 °C for 5 min and 80°C for 10 min and were then held at 4 or −20°C until they were transformed.

Gibson’s Assembly Protocol

• The following components were added to a 20μl tube on ice: 0.2-1pmols of each DNA component,

10 μL Gibson Assembly Master Mix, 10μl deionised water. This was then incubated in a thermocycler at 50C for 60 minutes. Then, the samples were transferred and rested on ice to prepare for transformation.

Heat shock protocol

1. Mix 50 μL of 5X KCM into 200 μL of comp cell prep, thawed on ice
2. Add 50 – 100 μL of comp cell-KCM cocktail to DNA
3. 10min on ice (4°C)
4. 1 min 42°C
5. 1min on ice (4°C)
6. 37°C recovery for 15-60min

PCR

Taken from (https://www.addgene.org/protocols/gel-electrophoresis/)

Reagents:

• TAE: Tris-base: 242 g, Acetate (100% acetic acid) 57.1 ml, 100 ml 0.5M sodium EDTA, water
• Agarose
• Invitrogen SYBR Safe DNA Gel Stain

Pouring a Standard 1% Agarose Gel:

• Mix 1 g of agarose powder with 100 mL 1xTAE in a microwavable flask.
• Microwave for 1-3 min until the agarose is completely dissolved (
• Let agarose solution cool down to about 50 °C
• Add Invitrogen SYBR Safe DNA Gel Stain
• Pour the agarose into a gel tray with the well comb in place.

Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins.

Gel electrophoresis

Taken from (https://www.addgene.org/protocols/gel-electrophoresis/)

• Add loading buffer (HC or GF) to each of your DNA samples
• Once solidified, place the agarose gel into the gel box (electrophoresis unit).
• Fill gel box with 1xTAE (or TBE) until the gel is covered.
• Carefully load a molecular weight ladder into the first lane of the gel, and load samples into the

additional wells of the gel.

• Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.
• Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the

gel from the gel box.

• Visualize DNA fragments using UV light.