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Closing the Circuit on Cloning Complex Constructs
Patrick C. H. Lo, Ph.D.
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And in a further ironic twist that illustrates the fickle nature of scientific success, Torella's fatty acid biosynthesis project, for which the new assembly method was developed, didn't work out. “At the end, we had this approach that now everyone in the lab was using because it was clearly valuable and saving people time, but ironically the original application we had in mind hadn't come through; this is why my friend managed to publish his paper using our method before I got anything out the door.”

Future prospects

Weiss plans to find better insulator sequences, given that the present ones were not as perfect as they hoped, even after multiple cycles of improvement. He also plans to automate the method, for example using liquid handling robots, which he believes really represents an exciting direction for assembly.

In contrast, Torella and the Silver lab are not pursuing automation for their technique, not being convinced that robotics helps to save that much time for their projects based on their and other labs’ experiences. “I think whether you're working on a robot or at a lab bench as an individual, this kind of approach is going to speed up whatever pathway you're trying to design,” noted Torella.

Torella believes the major challenge will be trying to understand how to use the methodology in an intelligent way, instead of brute-force empirical testing of all possible combinations of a genetic circuit. “Now that this method exists, we're no longer held back by the construction of a 5-gene or a 10-gene circuit. …In my mind, the important point in the future probably won't be improving the assembly methodology so much as it will be improving our understanding of the systems we're trying to put together adequately so we don't have to assemble 55 constructs (for example) every time we want to build a biosynthetic pathway.”

Torella's method currently is being used in the Silver lab for building optimized alkane biosynthetic pathways in E. coli, building logic gates in stem cells for therapeutic applications, and for Torella's current project of building a “bionic leaf.” The latter is a collaboration with Dan Nocera's lab in the Dept. of Chemistry at Harvard, where they “are interfacing autotrophic bacteria with a really efficient solar water-splitting catalyst to try to make a hybrid inorganic microbial photosynthetic system.” With that system mostly working, they're now using the multi-gene assembly method to engineer a new bacterium, Ralstonia eutropha, to produce biofuel and sugar within the system. Such a fascinating and potentially high-impact synthetic biological application clearly illustrates the potential of the method developed by the Weiss and Silver labs.

1.) Gibson, D.G., L. Young, R.Y. Chuang, J.C. Venter, C.A. Hutchison, and H.O. Smith. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6:U343-U341.

2.) Guye, P., Y. Li, L. Wroblewska, X. Duportet, and R. Weiss. 2013. Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res. 41:e156.

3.) Torella, J.P., C.R. Boehm, F. Lienert, J.H. Chen, J.C. Way, and P.A. Silver. 2013. 2013. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res. [Epub ahead of print].

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