The main project goal is to develop principles and demonstrate the feasibility of a fast in vitro method that will increase the length of synthetic double-stranded DNA from its current limit of about 10 kb to 100 kb. This, in turn, will facilitate the ability to more rapidly create artificial genes and chromosomes and thus become an important tool in synthetic biology. The proposed approach involves ligations of 10 kb DNA fragments (“Building Blocks”) into larger, approximately 100 kb DNAs, which are then transferred into larger structures by transformation into yeast cells. The key element facilitating ligations relies on stretching the DNA Building Blocks by first immobilizing the DNA on micro- or nanoparticles, and then subjecting the particles and DNA to either magnetic or electric field during the ligation reaction. Major tasks include: (1) Development of hardware suitable for a feasibility study. The system will include a custom instrument which generates and control the electric and magnetic fields. We will also build a special test tube ("L-tube") for conducting ligations during which an electric and magnetic field are applied to the ligation reaction components in the new instrument. (2) Assembly a 100 kb DNA construct. Toward this goal, we will study the ligation efficiency in a special assay in which DNA fragments are fluorescently labeled-- the ligation efficiency is monitored by observing fluorescence. The DNA Building Blocks will be combined in vitro in a stepwise fashion to yield the final product. (3) Transfer of the 100 kb ligated DNA fragment into yeast by electroporation to confirm the integrity of the assembled DNA which will be established by biological selection and screening for proper biomarkers, as well as by q-PCR and DNA sequencing. The large assembled DNA fragments can be used in gene expression experiments to create novel biological products in different organisms.
Public Health Relevance Statement: NARRATIVE The purpose of this project is to design a new generation of scientific instruments and related methods that will enable the realization of synthetic biology. This will be done by developing and characterizing novel methods that allow scientists to quickly and easily assemble many synthetic permutations of genetic material found in many organisms for testing genetic constructs. Such major advancements in synthetic genomics will have wide applicability in improved crop production, alternative energy, animal husbandry, drug discovery and other areas, such as deciphering human noncoding DNA variants. It is anticipated that the methods being created will be adopted worldwide by a wide range of scientists in academia, government and private industries.
Project Terms: Academia; Adopted; Animal Husbandry; Area; Artificial Chromosomes; auxotrophy; Biological; Biological Assay; Biological Markers; Biological Products; Caliber; Cells; Chemicals; Chromosome Arm; Computer software; cost; Custom; design; Development; Devices; Dimensions; DNA; DNA Ligation; DNA sequencing; drug discovery; Drug resistance; ds-DNA; electric field; Electrodes; Electronics; Electroporation; Elements; Engineering; Equipment; experimental study; Feasibility Studies; Fluorescence; Frequencies; Gene Expression; Generations; Genetic Materials; Genetic screening method; Goals; Gold; Government; Height; Hour; Human; hydrodynamic flow; Immobilization; improved; In Vitro; Industry; instrument; interest; Label; Length; Ligation; Link; magnetic beads; magnetic field; Magnetic nanoparticles; Magnetism; Methods; Monitor; nanoparticle; novel; Oligonucleotides; Organism; particle; Performance; Phase; Physiologic pulse; plasmid DNA; Power Sources; Privatization; Production; prototype; Reaction; restriction enzyme; Robotics; Saccharomyces cerevisiae; Scientist; screening; Solid; Stretching; Structure; synthetic biology; Synthetic Genes; synthetic genomics; System; Temperature; Testing; tool; Tube; Variant; Work; Yeasts
