BioByte Assembly

What you will need:

• Annealed Anchor
• Annealed Terminator
• Prepared BioBytes
• Magnet
• Streptavidin-Coated Magnetic Microsphere "Beads" (4mg/mL)
• Wash/Binding Buffer
• T4 DNA Ligase/Buffer
• USER™ (1 U/μL)
• Competent Cells
• LB
• Ice
• Agar Plates

Preparation:

1. Prepare X 1.5 microcentrifuge tubes, where X is the number of constructs to be made.
2. The beads are stored at 4ºC, vortex briefly to resuspend the beads and dispense 40 μL into each tube. Return the stock of beads to 4ºC immediately.
3. Apply the magnet to the side of the tube, wait until solution clears completely, aspirate supernatant.
4. Wash beads by adding 75 μL of “Wash/Binding Buffer” and resuspend by flicking.
5. Apply the magnet as before, wait until solution clears completely, aspirate supernatant.
6. Repeat steps 4 & 5 once more.

Anchor

7. Apply 20 μL of Anchor (A or B) to the bead solution
8. Incubate at room temperature for 10 minutes
9. Apply the magnet, wait until solution clears completely, aspirate supernatant.
10. Wash twice as in steps 4-6.
11. Wash once more with 75 μL 1X ligase buffer.

BioByte Addition and Construct Release

12. Add 20 μL of the first Byte with 5' end complementary to the anchor (AB Byte to A anchor and BA Byte to B anchor), resuspend beads, then add 2.3 μL 10x Ligase Buffer + 1 μL Ligase.
13 Incubate at room termpature for 20 minutes, every few minutes resuspend the beads by flicking the tube. BE GENTLE and tap the droplets back to the bottom of the tube after mixing.
14. Wash twice as in steps 4-6 with Wash Buffer and once more with 1X ligase buffer.
15. Repeat steps 12-14 for each BioByte to be added, always adding a Byte with a 5' end complementary to the 3' end of the previous end. Add AB Bytes to BA Bytes and BA Bytes to AB Bytes.

If you wish to circularize:
16. Add 20 μL of Terminator (A or B depending on previous Byte), resuspend beads, then add 2.3 μL 10x Ligase Buffer + 1 μL Ligase.
17. Incubate at room temperature for 20 minutes
18. Wash twice with Wash Buffer and once more with 1X ligase buffer.

Release from the bead:
19. Resuspend beads in 20 μL dH2O.
20. Add 2.5 μL 10x Pfu Buffer + 1 μL USER.
21. Incubate @ 37ºC 1 hr
22. Remove the beads by applying magnet, wait for solution to clear, aspirate supernatant into a fresh tube.
23. If circularizing: heat to 95ºC for 1 min, cool to RT slowly (let it sit on the bench).

Transformation

1. Add 5-10 μL circular construct to pre-chilled 1.5 mL microcentrifuge tube.
2. Chill on ice.
3. Add 75 μL or 100 μL competent cells to tube; mix gently.
4. Incubate on ice for 30 min.
5. Heat shock at 42ºC for 30-90 s.
6. Sit on ice for at least 2 min.
7. Add 400 – 800 μL LB broth to tube.
8. Incubate at 37ºC for 1 hour, with shaking.
9. Plate onto LB plates with correct antibiotics (usually 20 μL and 200 μL or rest).