Haynes:TypeIIS Assembly

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Type IIS Assembly

by Karmella Haynes, 2013

Principle: The familiar "BioBrick cloning" enzymes (i.e., EcoRI, NotI, XbaI, SpeI, PstI) are Type II restriction enzymes, which cut the sequences that they specifically bind to. The Type IIS Assembly method uses a Type IIS restriction enzyme, which binds at a specific sequence and cuts at a non-specific location exactly five base pairs away. As a result, the enzyme cleaves away its own binding site and leaves behind the most useful feature of assembly, sticky overhangs. When designed properly, Type IIS sites can be used to perform seamless assembly of parts. As an added convenience, this protocol allows cutting and ligation to occur in a single tube, as a single reaction. Thus, gel purification steps can be eliminated.

This protocol uses the Type IIS restriction enzyme BsmBI (CGTCTCnnnnn/).

Use PCR to prepare the parts

• Multiple parts can be assembled in one step.
• Parts and the destination vector should be amplified by PCR.
• Make sure that none of the parts/ vector have any BsmBI sites!

	First, map out your assembly. In this example, three parts, A, B, and C will be assembled and inserted into a Vector.
	Design a pair of primers to add BsmBI sites to the ends of a vector backbone. The "cacacca" before BsmBI is used to help restriction enzyme positioning. The "a" after BsmBI is a spacer that is required to generate a correct 4-base sticky end. Vector Primers Forward Primer: 5'-cacaccaCGTCTCa + 15 bp of "vector right" top strand Reverse Primer: 5'-cacaccaCGTCTCa + 15 bp of "vector left", bottom strand pSB1A3 Vector Primers - already available in the Haynes lab freezer Forward Primer gg0001: 5'-cacaccaCGTCTCaactagtagcggccgct Reverse Primer gg0002: 5'-cacaccaCGTCTCatctagatgcggccgcg
	Part A Primers Forward Primer: 5'-cacaccaCGTCTCa + 4 bp of "vector left" top strand + 15 bp of Part A top strand Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part B, reverse complement (bottom strand) + last 15 bp of Part A, reverse complement (bottom strand) Note: For insertion into pSB1A3, "last 4 bp of vector left" = TAGA
image	Part B Primers Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part B (top strand) Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part C, reverse complement (bottom strand) + last 15 bp of Part B, reverse complement (bottom strand)
image	Part C Primers Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part C (top strand) Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of "vector right", reverse complement (bottom strand) + last 15 bp of Part C, reverse complement (bottom strand) Note: For insertion into pSB1A3, "first 4 bp of vector right, reverse complement" = TAGT
image	Purify the PCR products using a Zymo clean and Concentrator kit.

Digestion/ Ligation Reaction

1. Dilute the purified PCR product to 20 fmol/μL

• Measure ng/μL of the purified sample.
• Use the following formula to calculate the volume of purified DNA (x) you will need to dilute in a final volume of 20 μL
  • Formula: x = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL

2. Golden Gate Reaction

Reagent	Vol.
20 fmol of each DNA part	up to 8.0
10x T4 ligase buffer (Promega)	1.0
T4 ligase (NEB)	0.25
BsmBI	0.5
dH2O	0.25
	10.0 μL

Thermal cycling

• [45°C, 2 min.; 16°C 5 min.] x25
• 60°C, 10 min.
• 80°C, 20 min.
• 4°C, ∞

3. Transformation

• Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
• Incubate on ice for 2 min., heat shock at 42°C for exactly 90 sec., immediately place on ice.
• Add 800 μL sterile SOC medium.
• Grow with shaking at 37°C for 30 min.
• Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp.
• Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic.
• Plate cells on pre-warmed LB agar + antibiotic. Grow overnight at 37°C.
  • Quick-transormation (e.g., DH5α-Turbo) is not recommended