Today it is commonplace to design and construct single electronic components with billions of transistors. These are complex systems, difficult (but possible) to design, test, and fabricate. Remarkably, simple living systems can be assembled from a similar number of atoms, most of them in water molecules. Key ideas of intentional simplification, effective design tools, and stratified designs will help us understand, engineer, and build these simple organisms.

In this talk I will present the current status of our attempts at full understanding and complexity reduction of one of the simplest living systems, the free-living bacterial species Mesoplasma florum. Our recent experiments using transposon gene knockouts identified 354 of 683 annotated genes as inessential in laboratory culture when inactivated individually. While a functional redesigned genome will certainly not remove all of those genes, this suggests that roughly half the genome can be removed in an intentional redesign.

I will discuss our recent knockout results and methodology, and our future plans for:

• Genome re-engineering using targeted knock-in/knock-out double recombination
• Re-sequencing of additional strains of Mesoplasma florum and close relatives
• Whole cell metabolic models
• Creation of plug-and-play metabolic modules for the simplified organism
• Inherent and engineered biosafety control mechanisms

Engineering biological systems requires a fundamentally different viewpoint from the science of biology. Key engineering principles of modularity, simplicity, separation of concerns, abstraction, flexibility, hierarchical design, isolation, and standardization are of critical importance. The essence of engineering is the ability to imagine, design, model, build, and characterize novel systems to achieve specific goals. Current tools and components for these tasks are primitive. Our approach is to create standard biological parts, assembly techniques, and measurement techniques. The MIT registry of standard biological parts, in collaboration with the Endy Lab, is a growing collection of DNA snippets containing transcriptional promoters, terminators, protein coding sequences, and specialized components in characterized, documented, and assembly-ready form (parts.syntheticbiology.org). Using these parts, we design, build, and test functional biological systems. To function, these components must be incorporated into a working biological system, a living cell. For most of our current research, this cell is the E. coli K-12 bacterium. With four thousand genes, this cell is by far the most complex portion of the system. Another laboratory effort is a long range project to engineer a simpler chassis and power supply for our systems. We have chosen the simple bacterium Mesoplasma florum as a starting point for this process on the basis of safety, fast growth, and small genome size. We have sequenced the organism in collaboration with the Whitehead Institute, annotated the sequence, and are now working on the reduction and standardization of its genome to perhaps 400 genes, creating a simple, manageable, understandable basis for engineering life.