Efficient cloning techniques are a requirement for synthetic biology. This study provides a simplified cloning method based on Golden Gate Assembly that can be used for rapid vector construction. The building of multiple expression vectors with customizable modules is achieved in a timely manner with minimal hands-on time by removing unnecessary steps in the workflow. The authors constructed a total of 21 mammalian episomal expression vectors and conducted a fluorescence expression comparison for different regulatory region combinations post-transfection in HEK293T and HEPG2 cells. Screening revealed that using the EF-1α promoter in combination with the bovine growth hormone polyadenylation sequence seemed to perform best in the types of cells tested compared with other variants.
A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. Here described is a simplified and inexpensive Golden Gate Cloning Protocol in which the amplified PCR fragments that enter the one-step-one-pot reaction are stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The protocol inludes the design step, in which fragments are designed to posses unique overhangs, amplification of modules using a high fidelity polimerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) in one tube, assembly, DpnI digestion for eliminating the background plasmids that remain after the PCR reaction and transformation of the resulting assembly reaction into competent E.coli cells. The protocol eliminates the need for vectors and inserts. The plasmid is build using only PCR products or fragments, with one of them containing the bacterial origin of replication and the antibiotic resitance gene for selection. Multiple modular plasmids can be constructed in just one day with minimal hands-on time.
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