Ten years ago, the most immediate barriers to an efficient design-build-test cycle were finding the proper biological parts, cloning and/or synthesizing

them, and assembling and inserting them into cells. While these barriers remain, their heights have been significantly lowered by innovations in DNA sequencing, synthesis, assembly and scaling functional assays. The combination is enabling rapid creation and screening of many variants of a design. For some applications it is now possible to screen large libraries for the proper pathway and host variations to produce a target molecule to a given level with increasing efficacy. However, many applications are complex enough that this is not an option. The initial designs must be implemented with parts that work predictably enough to produce systems with that function ABT 737 very close to specification, and safely, BEZ235 concentration so that there is minimal need for testing many variants semi-randomly. Here, the barriers concern the unpredictable operation of

biological parts in different contexts — that is, in different configurations with other parts, in different hosts and in different environments. We will start by reviewing a few key emerging complex biomedical applications that are aimed squarely beyond the bioreactor then describe systematic approaches to achieving reliable function despite variable context. While all applications can benefit from more predictable operation of synthetic biological systems in deployment environments,

few applications challenge this possibility like those in medicine. There Fossariinae have been some startling successes in using organisms as medicine. These include adoptive immunotherapy with engineered T-cells to cure certain types of cancer [3• and 4], engineered bacteria and oncolytic viruses for cancer [5 and 6], viral gene therapy for blindness [7 and 8] and hemophilia [9], and fecal transplants that harbinger designed communities for inflammation [10 and 11]. In some cases, the success of these applications might argue that there is not a need for complex design — that a combination of finding the correct natural starting points and modest modifications for our own purposes will be sufficient. However, as increasing specificity and long term reliability are needed, more sophisticated designs are being proposed. For example, Xie et al. demonstrated a multi-input RNAi logic circuit to be delivered as a gene therapy that would very specifically determine if an infected cell were a particular cancer type only then deliver a molecular therapeutic [12]. Anderson and colleagues built up several steps toward the bottom-up design of a tumor-destroying bacterium that, theoretically, would specifically invade target tumor cells after successful aggregation in the tumor necrotic region, then escape the vacuole and deliver a therapy to the cytosol or nucleus of the target cell [13, 14 and 15].