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Every hard-core biologist knows designing the perfect construct can be a complex puzzle to solve. This challenge, if successful, can be extremely satisfying but can also drive you crazy for weeks. Luckily, Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute designed a new easy-to-use cloning method. Better yet, this system allows for joining multiple DNA fragments in one single step.
This means, if their theory meets practice, every cloning-dummy can make the most complex construct a reality! If this promise does not awaken your interest you can stop reading here…. But if this sounds like paradise to you, then read further on how this technique works! In three simple steps you will end up with the ideal construct to perform the experiment that will finally award your career with a Nature or Cell paper!
Step 1 Preparation
The first step is to order the Gibson Assembly Cloning kit, which basically includes three different enzymes in one single buffer:
(i) exonuclease to create single-stranded 3’ overhangs that facilitate the annealing of fragments sharing complementarity at the overlap region,
(ii) DNA polymerase to fill in gaps within each annealed fragment and finally
(iii) DNA ligase to seal nicks in the assembled DNA.
Next to this kit, you’ll also need a high-fidelity DNA polymerase and Luria-Bertani (LB) agar plates with the appropriate antibiotic (vector-based).
Step 2 Primer design
The second step is to design the primers with overlapping sequences between the adjacent DNA fragments for their assembly into a cloning vector. There a several primer design programs available on the web, which give you a list of the required primers, their melting temperatures, recommended annealing temperatures, as well as the final sequence of your assembled construct and a summary of the design. This is probably the most time-consuming step in this protocol!
Step 3 Construction
Now, take out your lab coats and gloves and let’s get started!
With the primer sets you can amplify the desired inserts in a normal PCR, while a linearized vector can be prepared by either PCR amplification using a high-fidelity DNA polymerase or (if possible) by restriction digestion. If using restriction digestion, make sure that any undigested vector is removed in order to avoid high numbers of false-positive colonies.
Now, check the concentration of each fragment by using gel electrophoresis or a NanoDrop instrument. The concentration of assembly fragments should be at least 2-3 times higher than the concentration of vector. For assembly of multiple fragments into a vector, an equimolar ratio of fragments is recommended.
Finally, all you have to do is add inserts and linearized vector to the Gibson Assembly Master Mix and wait patiently for 15-60 minutes while it incubates at 50°C. The time it takes depends on the number of fragments being assembled. This mix can be transformed into NEB 5-alpha Competent E. coli (provided with the kit) or used directly in other applications.
Some final thoughts!
- This kit is suitable for large constructs: It has been used to clone a 15 kb DNA fragment into a 5.4 kb plasmid in E. coli, totalling up to 20.4 kb in length! (Note: Special competent cells are required for constructs larger than 15 kb).
- The number of DNA segments that can be assembled in one reaction is dependent on the length and sequence of the fragments. This method has been used to efficiently assemble up to 12 inserts (0.4 kb) into a vector at once. However, it’s recommended to use five or less inserts into a vector in one reaction in order to ensure a clone with the correct insert. A strategy involving sequential assembly can be used if all of the fragments cannot be assembled in a single reaction.