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Conference Short Courses* - View Details
Sunday, January 9 - 3:00 pm - 6:00 pm
Tuesday, January 11 - 4:30 pm - 7:30 pm
Thursday, January 13 - 6:30 pm - 9:00 pm
The demand for high quality biotherapeutic proteins has never been greater, yet meeting this demand is challenging because protein expression is both an art and a science. Bottlenecks frequently arise because functional proteins are difficult to produce. This usually requires designing new cloning schemes including lengthy verification and sequence analysis of the gene or protein of interest, moving a gene from one vector to another, transfecting the vector in an alternative host, or re-characterizing the expressed protein —an inefficient, time-consuming and expensive process. This meeting continues the tradition of applying effective protein discovery research leading to functional products.
7:30 am Conference Registration and Morning Coffee
8:30 Chairperson’s Opening Remarks
8:45 Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
Daniel Gibson, Ph.D., Associate Professor, Synthetic Biology and Bioenergy Department, J. Craig Venter Institute
For the past 15 years, the genomes of many organisms have been sequenced and deposited in databases. We recently showed that it is possible to reverse this process and synthesize bacterial cells starting from digitized information. To make this happen, our group needed to learn how to sequence, synthesize, and transplant genomes. Many hurdles had to be overcome, but we are now able to combine all of these steps to produce synthetic cells in the laboratory. As a proof of concept, we designed, synthesized, and assembled the 1.08–mega–base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and transplanted it into a Mycoplasma capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. We now will extend what we have learned in this proof of concept experiment to begin designing and producing new organisms with useful properties. For example, we will use available sequencing information to create cells that can produce energy, pharmaceuticals, and industrial compounds, and sequester carbon dioxide. In addition, we have already begun working on our ultimate objective which has been to synthesize a minimal cell that has only the machinery necessary for independent life. Now that we can produce a living cell from the genome we have synthesized, we can test for the functionality of the genome. We can whittle away at the synthetic genome and repeat transplantation experiments until no more genes can be disrupted and the genome is as small as possible. This will help us to better understand the function of every gene in a cell and what DNA is required to sustain life in its simplest form. The M. mycoides genome that we assembled is over one million base pairs in length and is the largest chemically defined structure ever synthesized in the laboratory. It is almost twice as large as the synthetic Mycoplasma genitalium genome we reported on in 2008, and is more than 30 times larger than any reported DNA sequence synthesized outside of the J. Craig Venter Institute. This work and new powerful methods for constructing small and large DNA molecules starting from chemically-synthesized oligonucleotides will be discussed.
9:35 Applications with an Enhanced Unnatural Amino Acid Incorporation Platform
Travis Young, Ph.D., Postdoctoral Scholar, Biological Chemistry & Molecular Pharmacology, Harvard Medical School
We recently reported a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins though the use of a novel induction system and an optimized suppressor tRNACUA. Using this expression platform, we have discovered aaRS enzymes with unique polyspecifities which allow the incorporation of multiple unnatural amino acids with a single aaRS/tRNA pair. The pEVOL system and polyspecific aaRS enzymes will expand the applications of unnatural amino acids in E. coli.
10:05 Networking Coffee Break
10:45 Genome Surfing
Ryan T. Gill, Ph.D., Assistant Professor, Chemical and Biological Engineering, University of Colorado, Boulder
Recent advances in synthetic biology and genomics has now enabled the extension of directed evolution algorithms to the genome-scale. This algorithm includes strategies for creating relevant genetic diversity, mapping such diversity onto phenotypes of interest, and then exhaustively searching combinatorially mutational space to find optimal solutions. We will describe our efforts to develop and apply tools within this general approach to directed genome engineering.
11:15 Next-Gen Pichia pastoris Protein Expression
Anton Glieder, Ph.D., Scientific Director, Institute of Molecular Biotechnology, Graz Technical University, Austria
Pichia pastoris is broadly available and a major efficient host for heterologous protein production and especially useful for proteins which are difficult to fold or need eukaryotic posttranslational modification. Based on the Pichia pastoris CBS7435 WT genome sequence a well characterized expression platform was developed. Combining computational design with high throughput wet lab experiments new tools and concepts for protein expression with highest efficiency are now available.
11:45 Influence of Construct Design on Downstream ApplicationsSponsored by
Frank Schäfer, Associate Director, R&D, Head of DNA & Protein Sciences, QIAGEN
The basis for success in recombinant protein production and characterization is established early on in a project, e.g by the way the template is constructed and by the choice of the expression system. It will be shown how early decisions carry on through the workflow starting with rational gene design and protein production through to functional characterization and crystallization. New technologies and procedures for each of these steps will be introduced along with application data from recent successes, including studies on membrane proteins and antibody fragments.
12:15 pm Close of Morning Session
12:30 Luncheon Presentations (Sponsorship Opportunities Available) or Lunch on Your Own
2:00 Chairperson’s Remarks
2:05 Recombinant Yeast Libraries as a Tool for Engineering Novel Antibacterial Proteins
Karl E. Griswold, Ph.D., Assistant Professor, Thayer School of Engineering, Dartmouth College
The spread of drug-resistant pulmonary infections is intensifying clinical demand for next-generation antibiotics, and human lysozyme (hLYS) represents a particularly interesting biotherapeutic candidate. Wild type hLYS, however, is inactivated in the infected lung by inhibitory anionic biopolymers. The development of performance-enhanced hLYS variants will be discussed, highlighting challenges associated with high-throughput functional screens of recombinant yeast libraries.
2:35 Modulation of Plasmid Replication for Optimal Expression of Recombinant Genes
Manel Camps, Ph.D., Assistant Professor, Microbiology and Environmental Toxicology, University of California, Santa Cruz
Plasmids continue to be the main vectors used for gene amplification and expression. In the vast majority of these vectors, replication is regulated by a ColE1 plasmid origin of replication, which is sensitive to recombinant protein expression. Therefore a detailed understanding of the interplay between recombinant gene expression and regulation of plasmid replication is critical for successful stable gene expression. Here I present how protein expression affects plasmid replication, interfering with stable recombinant gene expression, as well as available strategies to address this problem.
3:05 A Molecular Biology Toolkit: Engineering, Automating and Constructing Large Clone Sets and Their Biological Applications
Jason Steel, Ph.D., Research Scientist, Center for Personalized Diagnostics, Arizona State University
Advances emerging from the genome projects have initiated a dramatic acceleration in the pace of biological research, driving towards the elucidation of the functional roles for all proteins. The Biodesign Institute at Arizona State University has a continuing project to create a sequence-verified collection of full-length cDNAs representing all coding regions for several organisms in a vector system that is protein expression-ready. Through recombination-based cloning, users routinely execute the automated transfer of thousands of genes into useful protein expression vectors overnight. These clones are available through our non-profit repository: DNASU. The DNASU plasmid repository has a collection of nearly 120,000 plasmids including complete genomic collections of several organisms, human and mouse genes from the ORFeome, protein expression plasmids and vectors from the Protein Structure Initiative, all of which are available at the freely searchable database at http://dnasu.asu.edu. We are currently developing a collection of glycan-related enzymes and continue to sequence validate protein expression plasmids from PSI:Biology centers. Applications using these clone sets include NAPPA (protein microarrays), protein-protein interactions, screening of biomarkers of cancer, autoimmunity, and infectious diseases and expression studies of mammalian, yeast and embryonic stem cells. The goal of the Center for Personalized Diagnostics is to build this resource, provide it to the community and to exploit it towards a better understanding of protein function in health and disease.
3:35 Sponsored Presentation (Opportunity Available)
3:50 Networking Refreshment Break
4:30 Use of Lentiviral Vector Promoter Libraries to Optimize Protein Expression in Mammalian Cell Lines
Dominic Esposito, Ph.D., Principal Scientist, Protein Expression Laboratory, SAIC-Frederick, Inc.
We have generated libraries of lentiviral vectors using a number of different promoters in order to explore the optimal expression context for protein production in various mammalian cell lines. Our results suggest that promoter strengths vary among different cell lines, and that optimal conditions need to be determined for each particular cell type. A recombinational cloning platform can be used to simplify the process of generating lentiviruses for this optimization process.
5:00 Variants of a Novel Bipartite Plant Viral Vector System Are Adapted for either High Level Protein Expression or Virus-Induced Gene Silencing
John Hammond, Ph.D., Research Plant Pathologist, Floral and Nursery Plants Research Unit, USDA-ARS
We have developed a plant viral vector delivery system suited to: a) high level protein expression; or b) Virus-Induced Gene Silencing, dependent upon the strength of RNA silencing suppression conferred by variants of the viral TGB1 protein. Plant infection by agroinoculation using an artificial bipartite launch system allows efficient cloning and high throughput. The bipartite launch system separates replication and movement functions of the virus, allowing libraries to be constructed directly in the multiple cloning site of the launch plasmid. Additional variants allow RNA transcription for infection of hosts (including soybean) not amenable to agroinfiltration.
5:30 A Suite of Biosafe Deleted Plant Viral Vectors for High Level Protein Expression
Christopher Kearney, Ph.D., Associate Professor, Biology, Baylor University
A series of plant viral vectors from three different viruses were created, yielding expression of GFP at 25-40% total soluble protein. Other proteins expressed included glycanases, protein insecticides, immunoglobulins and allergens at varying levels. One to four genes were eliminated from native viruses, rendering them nearly noninfectious. Co-inoculation with the siRNA-scavenging p19 gene produced high yields, effecting an on/off switch.
6:00-7:30 Welcoming Reception in the Exhibit Hall
7:30 Close of Day
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Links to Companion Meetings
Choosing, Designing, and Optimizing Hosts and Platforms
Overcoming Protein Expression Challenges with Solutions