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Revision as of 19:34, 18 July 2017
Project Description
Gibson assembly is a useful molecular biology technique published in 2009 by Dr. Daniel Gibson and his colleagues. This technique allows for the possibility of assembling up to 15 different DNA fragments in a single in vitro reaction, which allows for more efficient use of time and resources when compared with conventional cloning techniques.
Escherichia coli is a model organism that has been developed into a useful tool for researchers to express genes of interest due to their ease of genetic manipulation and high replication rate. We believe that we can use engineered E. coli to overcome the high cost of using Gibson assembly method. Theoretically, two of the three genes required to encode for the proteins required in Gibson Assembly could be inserted into a duet vector plasmid and the third into a single plasmid vector to be transformed into E. coli. These E. coli strains harboring the Gibson Assembly machinery will then naturally replicate and the plasmids that contain the genes for the 5'-exonuclease, DNA polymerase, and DNA ligase will be expressed. Afterwards, cell lysate can be mixed with a plasmid backbone and DNA fragments of interest. In theory, this should produce the same combination effect as the traditional Gibson assembly method.
Estimates for the current cost of running a Gibson assembly method is $185 for ten reactions. The method we are developing will be significantly cheaper than the method now, as we will only need the cost of making an LB media (which ranges from $0.03 to $0.30 depending on the method of acquiring the LB media).
Reference
- Pierucci, O. (1972). Chromosome replication and cell division in Escherichia coli at various temperatures of growth.Journal of bacteriology. 109(2), 848-854.
- Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods. 6 (5): 343–345.
