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/).
Prepare the parts with PCR
- 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!
|image||First, map out your assembly. In this example, three parts, A, B, and C will be assembled and inserted into a Vector.|
|image|| Design a pair of primers to add BsmBI sites to each end. "cacacca" is used to help restriction enzyme positioning. The "a" is a spacer that is required to generate a correct 4-base sticky end. |
pSB1A3 Primers - already available in the Haynes lab freezer
|image|| Part A Primers
Note: For insertion into pSB1A3, "last 4 bp of vector left" = TAGA
|image|| Part B Primers
|image|| Part C Primers
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
|20 fmol of each DNA part||up to 8.0|
|10x T4 ligase buffer (Promega)||1.0|
|T4 ligase (NEB)||0.25|
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 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