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Project Description

References

Polymerase to the people. The DIY bio community has developed a number of open source and relatively inexpensive designs for laboratory equipment necessary for synthetic biology lab work, but the prices of commercial DNA polymerases essential for PCR remain a significant financial hurdle for community labs operating on shoestring budgets. Founding members of the Baltimore Underground Science Space (BUGSS) began to address this problem in the 2010 iGEM competition, developing a Biobrick for Taq polymerase synthesis. The BUGSS team has built on this earlier work by developing a new Biobrick for Pfu DNA polymerase, which offers a replication error rate approximately 6 fold lower than that of Taq polymerase. The development of this Biobrick began with the amplification and cloning of the pol gene for Pfu DNA polymerase from contaminant DNA in a commercial stock enzyme. Silent single bp substitutions were needed to remove two restriction sites incompatible with Biobrick Assembly Standard 10, and finally restriction sites SpeI and PstI were added at the 3’ end and EcoRI and XbaI at the 5’ end to fit the standard. The team is now working to develop a kit for easy purification of Pfu polymerase with the goal of reducing the cost of this essential tool.

In addition to developing cost effective Pfu, the BUGSS team has been working to cultivate a strain of E. Coli that produces metallothionein and RFP in the presence of heavy metals like lead. Adequate production of metallothionein protein can sequester lead in order to make the metal non-bioavailable. Again, the BUGSS team is building on earlier work on lead sequestration,utilizing some parts from the iGEM registry while producing and submitting new and composite parts. Sequences were found and edited for metallothionein production (BmtA), and the PbrR regulator. PpbrA promoters were connected to RFP and Bmta genes in order to regulate metallothienein and RFP production, inducing their generation in the presence of lead. In order for this new construct to work, the E. Coli's natural heavy metal resistance gene, Znta, must be deactivated. The team is utilizing a one step gene inactivation protocol using phage lambda red recombinase to recombine the gene with a kanamycin resistance insert.

Datsenko, K.A., Wanner, B.L.(2000). One step inactivation of chromosomal genes in Escherichia Coli K-12 using PCR products. PNAS. 19(12), 6640-6645.

Heo,J.H., Kim, S.W.(2013). Cloning the Pfu DNA polymerase from DNA contaminants in perparations of pfu DNA polymerase. African Journal of Microbiology, 7(9),745-750.

Liu,T., Nakashima,S., Hirose,K., Uemura,Y., Shibasaka,M., Katsuhara,M. and Kasamo,K. (2003).A metallothionein and CPx-ATPase handle heavy-metal tolerance in the filamentous cyanobacterium Oscillatoria brevis . FEBS Lett. 542 (1-3), 159-163.