Inexpensive and easy method for 6 fragment Golden Gate Assembly of a modular S/MARs mammalian expression vector and its variants

Background 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. This study aims to further simplify that method and make it more suitable for small labs and students.

Methods Prior to amplification of the parental plasmids used in building the modules were domesticated using a variation of SDM (Site Directed Mutagenesis) called SPRIP. After careful design and amplification of the desired modules, using a high fidelity polymerase, amplified PCR fragments that enter the one-step-one-pot reaction were 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 fragments were designed to posses unique overhangs using NEB Golden Gate assembly tool and Snapgene, amplification of modules was performed using a Q5 high fidelity polymerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) was performed in one tube per assembly using Zymo DNA Clean and Concentrator-5, assembled using BsaI and T4 ligase, DpnI digestion performed for eliminating the background plasmids that remain after the PCR reaction and the resulting assembled product was transformed into competent E.coli cells. Transformants were screened using diagnostic digest and reporter gene expression in both bacteria and HEK293 cells was evaluated using fluorescent light.

Results Herein presented is a simple and inexpensive alternate protocol to build modular plasmids using the Golden Gate Assembly method. Four S/MARs mammalian expression vectors were designed and constructed using 6 modules previously amplified by PCR and stored in the appropriate buffer to eliminate exo- and endonuclease activity and to protect the DNA from freeze thaw cycles. The existing modules were interchangeable and new modules were easily amplified and stored for use when needed. The mammalian expression vectors constructed showed the desired restriction pattern and GFP expression in bacteria and in mammalian cells

Conclusions Provided the design steps are respected and the fragments are stored and labeled appropriately, multiple plasmid variants and combinations of the pre-designed modules can be assembled in one day, easier and using less resources than the established protocols, with good efficiency. The simplicity of the design and the affordability of the method could make modular cloning of plasmid constructs more accessible to small labs and students.