Efficient Multi-part DNA Assembly using Type IIS Enzymes

Restriction-ligations were set up by pipetting in one tube approximately 40 fmol (∼100 ng of DNA for a 4 kb plasmid) of each DNA component (PCR product or plasmid), 10 U of the required restriction enzyme (BsaI or BpiI) and 10 U T4 DNA ligase (using high concentration ligase, 20 U/µl) in Promega ligation buffer in a final reaction volume of 20 µl. The reaction was incubated in a thermocycler for 5 hours at 37°C, 5 min at 50°C and 10 min at 80°C. The reaction mix was then added to 100 µl chemically competent DH10b cells, incubated for 15–30 min on ice and transformed by heat shock. 800 µl of liquid LB was then added to the transformation, and the cells were let to recover 45 min at 37°C. Different aliquots of the transformation were plated on LB plates containing the appropriate antibiotic. The number of colonies was counted for one or two selected plates (containing countable number of colonies), or from a section of the plates when very high number of colonies were obtained even for the lowest volume plated. The number of colonies was then extrapolated for the entire transformation.
For level 2-2 cloning, two type IIS enzymes were required, BpiI and BsaI. The same protocol was used as described above except that 10 U and 2.5 U were used for the enzymes BpiI and BsaI, respectively. To optimize efficiency of the restriction-ligation for the final construct containing 11 transcription units (cL2-13*), a variation of this protocol was used as follows. The reaction mix was set up containing 20 U ligase, 5 U BpiI and 5 U BsaI, in a total reaction volume of 20 µl. The mix was incubated in a thermocycler with the following parameters: incubation for 2 minutes at 37°C, 5 minutes at 16°C, both steps repeated 45 times, followed by incubation for 5 minutes at 50°C and 10 minutes at 80°C. The reaction mix was transformed in E. coli chemically competent cells as described above.