Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.

Alternatives for the intermediate recovery of plasmid DNA: performance, economic viability and environmental impact.

Robust cGMP manufacturing is required to produce high-quality plasmid DNA (pDNA). Three established techniques, isopropanol and ammonium sulfate (AS) precipitation (PP), tangential flow filtration (TFF) and aqueous two-phase systems (ATPS) with PEG600/AS, were tested as alternatives to recover pDNA from alkaline lysates. Yield and purity data were used to evaluate the economic and environmental impact of each option. Although pDNA yields > or = 90% were always obtained, ATPS delivered the highest HPLC purity (59%), followed by PP (48%) and TFF (18%). However, the ability of ATPS to concentrate pDNA was very poor when compared with PP or TFF. Processes were also implemented by coupling TFF with ATPS or AS-PP. Process simulations indicate that all options require large amounts of water (100-200 tons/kg pDNA) and that the ATPS process uses large amounts of mass separating agents (65 tons/kg pDNA). Estimates indicate that operating costs of the ATPS process are 2.5-fold larger when compared with the PP and TFF processes. The most significant contributions to the costs in the PP, TFF and ATPS processes came from operators (59%), consumables (75%) and raw materials (84%), respectively. The ATPS process presented the highest environmental impact, whereas the impact of the TFF process was negligible.

Growth medium selection and its economic impact on plasmid DNA production.

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 microg in 2002 to 500-5000 microg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5alpha harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 microg/ml and 6.3 microg/mg dry cell mass respectively) and the lowest was for LB (2.8 microg/ml and 3.3 microg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 microg/ml and 11.2 microg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol.

In an effort to reduce plasmid template preparation costs for large-scale genomic sequencing projects, a boiling minipreparation protocol has been developed that enables a single individual to easily prepare 768 sequence-quality templates in 8 h, without automation. The maximum throughput for one individual using one centrifuge in the manual configuration is 1920 templates in about 8 h. The most time-consuming manual steps of this method involve pipetting, which can be automated, resulting in a significant increase in throughput and about a 60% increase in yield. This method in the fully manual configuration yields sufficient double-stranded template for two sets of cycle sequencing reactions, membrane spotting for hybridization analysis and host cell transformation for the recovery of the original recombinant. The current materials cost per template using this method is less than twenty cents. The quality of the sequence generated has been evaluated by manual 35S radioactive cycle sequencing. Initial results have shown templates prepared by this method to yield greater than 300 bp of readable sequence when the radioactively labeled products were resolved on 6% modified denaturing polyacrylamide gels.