Summary for the Conference

• Synthesize genomes of several species completely.

• Synthetic biology in industrial development.

• Bio-systems and quantitive analysis.

• BioBricks

• The ethical issues, biological security and political issues.

The reports in Synthetic Biology 4.0 Conference can be roughly divided into several parts:

Content

• Building Genomes

• Setting up bio-systems

Building Genomes

• A key aim of synthetic genomic research is to be able to design and build artificial whole genomes.

• Some progress have been achieved including synthetic Mycoplasma genitalium JCVI 1.0 genome (J. Craig Venter Institute), genomes building in the Bacillus subtilis genome vector and so on.

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

J. Craig Venter Institute

Building Genomes

• Mycoplasma genitalium is a bacterium with the smallest genome of any independently replicating cell that has been grown in pure culture.

• The M. genitalium genome sequence was partitioned into 101 cassettes of approximately 5 to 7 kb in length that were individually synthesized, verified by sequencing, and then joined together in stages.

Assembly of synthetic cassettes by in vitro recombination

• The essential steps of the reaction are (i) the overlapping DNA molecules are digested with a 3′ exonuclease to expose the overlaps, (ii) the complementary overlaps are annealed, and (iii) the joints are repaired.

Fig 1. Diagram of steps in vitro recombination reaction

Fig 2. BAC vector is prepared for the assembly reaction by PCR amplification

Fig 3. The desired assembly is circular DNA containing the four cassettes and the BAC DNA

Fig 4. Repair of annealed junctions containing nonhomologous 3′ and 5′ Not I sequences

Assembly by in vivo recombination in yeast

• Larger assemblies were simply not stable in E. coli.

• Using S. cerevisiae as a cloning host. Yeast will support at least 2 Mb of DNA in a linear centromeric yeast artificial chromosome (YAC) and has been used to clone sequences that are unstable in E. coli.

Fig 5. Yeast TAR cloning of the complete synthetic genome.

Recovery of the synthetic M. genitalium genome from yeast and confirmation of its sequence

• They used a strategy of total DNA isolation in agarose, selective restriction digestion of yeast host chromosomes, and electrophoretic separation of these linear fragments from the large, relatively electrophoretically immobile circular molecules.

Setting Up Bio-systems

Pamela SILVER

Department of System Biology

Harvard Medical School

Can we build biological systems in mammalian cells with predictable

properties?

• The field of synthetic biology aims to design biological systems to perform tasks to better understand analogous natural systems and for direct applications in research and medicine.

• Much effort in synthetic biology has correctly been placed in the logical design of systems in prokaryotes. However, can we move to a predictable biological design in eukaryotes-in particular in human cells.

Synthetic transcriptional activators in yeast

• In this study, will describe the rational design and construction of a high fidelity, modular memory device in yeast based on transcriptionally controlled autoregulatory positive feedback.

Fig 6. Schematic diagram of an activator cascade composed of an activator gene and a reporter gene

Fig 7. DIC and fluorescence images of live cells

Fig 8. Time-lapsed RFP and YFP fluorescence images of cells

Fig 9. Schematic diagram of the sensor and autofeedback genes of the memory device

Creating a cellular memory device

Fig 10. DIC and fluorescence images of live cells

Fig 11. DIC and fluorescence images of cells

Thank you!