Fig 1.
The Gibson Assembly method and Picky thermodynamic junction analysis.
(a) The Gibson Assembly reagent includes three enzymes. The 5’ exonuclease erodes the 5’ ends on each dsDNA fragment, exposing single-stranded 3’ overhangs. The overhangs anneal to each other according to their compatible base-pairing. The DNA polymerase repairs gaps and the DNA ligase covalently binds the fragments to create a full-length product. (b) To design an optimal fragment set for gene assembly, the target gene is first analyzed using the Picky software to identify all its thermodynamically unique junction regions. Next, a separate Perl program takes these junction coordinates as well as some user specified design parameters such as acceptable minimum and maximum fragment lengths and the optimization goal for lower cost or fewer fragment count to finalize the optimal fragment set.
Table 1.
Assembly dsDNA fragments designed for Green fluorescent protein gene (GFP), Kanamycin resistance gene (KanR) and Tetracycline resistance gene (TetR).
Table 2.
Gibson Assembly 5’ exonuclease erosion length under different temperature and reaction time.
Table 3.
Final dsDNA fragment concentration (conc) in successful assemblies.
Fig 2.
Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix.
The agarose gel is stained with ethidium bromide.
Table 4.
Assembly quality assessment for the three assemblies.
Fig 3.
Assembly efficiency and reaction count under different conditions.
(a) The assembly efficiency and fragment count to assemble a 2000 bp gene using different fragment sizes. (b) The assembly reactions required to assemble sequences up to a million bps from 200 bp fragments under different Gibson Assembly capacities up to 30 fragments at once. In both figures the junction length between fragments is fixed at 20 bps and it is assumed that any sequence to assemble can be evenly divided by the fragments.
Fig 4.
Difference in DNA polymerase behaviors.
The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.