Table of Contents

• Transformation Protocol
• Colony PCR: Sapphire
• Loop Assembly: Golden Gate
• PCR Purification
• Glycerol Stock
• PCR: Primestar
• Plasmid Isolation
• Cloning for L0: Gibson Assembly
• Sequencing Preparation

Transformation Protocol

Plasmid or DNA transformation introduces recombinant DNA into host cells. In this project, E. coli and Alteromonas macleodii were transformed depending on the cloning step.

Ingredients (vary by method)

• Gibson Assembly: 15 μL Competent Cells, 3 μL Target Insert Volume
• Golden Gate: 12 μL Competent Cells, 0.5 μL Target Insert Volume
• 270 μL SOC Medium

Instructions

1. Thaw competent cells on ice for 5 minutes. Allow selective plates to reach room temperature.
2. Mix cells with DNA in an Eppendorf tube.
3. Hold on ice for 5–10 minutes.
4. Heat shock at 42°C for 30 seconds.
5. Return tubes to ice for 5 minutes.
6. Add 270 μL SOC medium and incubate at 37°C for 1 hour.
7. Plate 50 μL onto selective agar and incubate overnight at 37°C.

Colony PCR: Sapphire

Colony PCR confirms whether a colony contains the desired DNA insert after transformation.

Reaction Mix

• 12 μL Sapphire Mix
• 11.5 μL H₂O
• 1 μL 10 μM Primer Mix
• 0.5 μL Template DNA (heat-lysed cells if needed)

Instructions

1. Pipette all components into an Eppendorf tube.
2. Swirl a pipette tip with cells directly into the reaction mix.
3. Vortex to homogenize the mixture.

Loop Assembly: Golden Gate (BsaI)

Golden Gate assembly uses Type IIS restriction enzymes and ligase to assemble DNA fragments in one step. This method was used for constructing L1 plasmids with BsaI.

Reaction Mix (per reaction)

• 3 μL HPLC-grade H₂O
• 1 μL T4 DNA Ligase Buffer (10×)
• 0.5 μL Purified BSA (1 mg/mL)
• 0.25 μL T4 DNA Ligase (400 U/μL)
• 0.25 μL BsaI (10 U/μL)

Instructions

1. Combine components in an Eppendorf tube; scale volumes as needed.
2. Add 1 μL of each L0 part (adjust volume to total 5 μL with water).
3. Mix with 5 μL of the BsaI mix.
4. Thermocycle:
   • 37°C for 3 min
   • 16°C for 4 min (repeat 25×)
   • 50°C for 5 min
   • 80°C for 10 min

Loop Assembly / Golden Gate (SapI, L2)

Used for constructing Level 2 (L2) plasmids using SapI restriction sites.

Reaction Mix (per reaction)

• 3.5 μL HPLC-grade H₂O
• 0.5 μL T4 DNA Ligase Buffer (10×)
• 0.5 μL CutSmart Buffer (10×)
• 0.25 μL T4 DNA Ligase (400 U/μL)
• 0.25 μL SapI (10 U/μL)

Thermocycler Program

• 37°C for 3 min
• 16°C for 4 min (repeat 25×)
• 50°C for 5 min
• 80°C for 10 min

PCR Purification

Removes primers, dNTPs, enzymes, and salts to yield purified DNA for downstream use.

Ingredients

• 100% Ethanol
• DNA Wash Buffer
• DNA Binding Buffer
• DNA Elution Buffer or water

Instructions

1. Add 2–7× volumes of Binding Buffer to DNA sample (depending on type).
2. Transfer to spin column; centrifuge 30 s and discard flow-through.
3. Wash twice with 200 μL Wash Buffer.
4. Elute with ≥25 μL Elution Buffer; centrifuge 30 s to recover purified DNA.

Glycerol Stock

Glycerol stocks are used for long-term bacterial preservation at -80°C.

Ingredients

• 500 μL 30% Glycerol Solution
• 500 μL Target Bacterial Culture (broth)

Instructions

1. Combine equal volumes of bacterial culture and glycerol solution.
2. Mix gently and store at -80°C.

Plasmid Isolation

Extracts plasmid DNA from bacterial cultures for sequencing and cloning.

Instructions

1. Resuspend pellet in 250 μL Buffer P1.
2. Add 250 μL Buffer P2 and mix by inversion.
3. Add 350 μL Buffer N3 and invert to mix thoroughly.
4. Centrifuge 10 min at 13,000 rpm; collect supernatant.
5. Apply to QIAprep Spin Column and centrifuge 30–60 s.
6. Wash with 750 μL Buffer PE and centrifuge again.
7. Elute DNA with 50 μL Buffer EB; incubate 1 min and centrifuge 30 s.

Sequencing Preparation

Samples were prepared and sent to Eton Bioscience for sequencing to confirm the accuracy of plasmid constructs and rule out mutations compared to digital designs.