Engineering Genes Vectors Constructs and Clones

The demand for high quality biotherapeutic proteins has never been greater. To meet this goal of producing functional proteins at even higher levels requires engineering (science) and insight (art). Thus, efficient biotherapeutic protein expression is both science and art. Cambridge Healthtech Institute’s Third Annual Engineering Genes, Vectors, Constructs and Clones continues the tradition of applying effective engineering strategies for protein discovery research that leads to functional biotherapeutic products. Learn from seasoned savvy researchers as they share their real-world experiences, applications, and results.

Day 1 | Day 2 | Download Brochure

MONDAY, JANUARY 9

7:30 am Conference Registration and Morning Coffee

8:55 Chairperson's Opening Remarks
Armelle Gaussin, Ph.D., Selexis

» KEYNOTE PRESENTATION

9:10 Enhanced Protein Secretion via Translation Engineering

Kelvin Lee Kelvin H. Lee, Ph.D., Professor, Chemical Engineering, Director, DE Biotechnology Institute, University of Delaware

Several strategies have been previously proposed to improve recombinant protein production. Using a systems biology approach to study supersecreters, we observed interesting expression changes in genes and proteins related to translation. Based on these observations, we developed a strategy to significantly increase protein secretion using translational engineering.

9:50 j5 and DeviceEditor: DNA Assembly Design Automation

Nathan J. Hillson, Ph.D., Director, Synthetic Biology, Joint BioEnergy Institute

We have developed two on-line software tools, j5 and DeviceEditor, that automate the design of sequence agnostic, scar-less, multi-part assembly methodologies and translates them to robotics-driven protocols. Given a target library to construct, the software provides automated oligo, direct synthesis, and cost-optimal assembly process design, and integrates with liquid-handling robotic platforms to set up the PCR and multi-part assembly reactions. This work reduces the time, effort and cost of large scale cloning and assembly tasks, as well as enables research scales otherwise unfeasible without the assistance of computer-aided design tools and robotics.

10:20 Networking Coffee Break

10:45 Reliable and Precise Gene Expression via Active Translational Coupling

Vivek K. Mutalik, Ph.D., Team Lead, BioFAB

We have developed a method that allows for reliable functional coupling of prokaryotic transcription and translation control elements with user-specific coding sequences for genes of interest. We have engineered a collection of standard biological parts that implements the method across a range of transcription and translation levels, and with native and also a phage RNA polymerase. For the genes tested our system realizes a > 90% chance to express a gene to within a two-fold protein concentration target window.

11:15 Efficient Optimization of Protein Expression Using High-Throughput Gene Synthesis and Screens

Jingdong Tian, Ph.D., Assistant Professor, Biomedical Engineering, Duke University

A parallel on-chip gene synthesis technology is developed. Used with high-throughput expression screens, it enables efficient and reliable optimization of protein expression in host cells.

11:45 Rapid Expression, Engineering and Scale-Up of Inaccessible Proteins in a Cell-Free System

Sonia Pollitt, Ph.D., Director, Protein Sciences, Sutro Biopharma

Advances in E. coli based cell-free expression technology that enable rapid high-yield production of complex proteins with multiple disulfide bonds make it an ideal platform for screening and selection of protein variants. Here we illustrate the use of this technology to enable engineering of IgGs and Diphtheria toxin fusion proteins. In addition, we show how use of a cell-banked E. coli extract allows rapid scale-up to generate material for IND enabling studies and transitions smoothly to GMP production of clinical drug product.

12:15 pm Close of Morning Session

Sponsored by

12:30 Luncheon Presentation
GENEART®Genetic Assembly Tools for Synthetic Biology Engineering
Federico Katzen, Ph.D., Senior Staff Scientist, R&D Synthetic Biology, Life Technologies
Synthetic biology applications require the precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes. We will present a set of technologies that allow the seamless, simultaneous, and highly efficient assembly of genetic material, designed for a wide size dynamic range (10s to 100,000s base pairs). The assembly can be performed either in vitro or within the living cells and the DNA fragments may or may not share homology at their ends. A novel site-directed mutagenesis strategy enhanced by homologous recombination will be also presented.

1:00 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own

2:00 Chairperson's Remarks

Ron Godiska, Ph.D., Lucigen

2:05 So Many Options, So Little Money: How to Choose the Right Vector and Screen for Functional Proteins

Bill Gillette, Ph.D., Senior Scientist, Group Leader, Protein Expression Laboratory, Advanced Technology Program, SAIC-Frederick, Inc.

Producing and purifying functional proteins is a process replete with possibilities for failure. Yet there can be method to the madness and the talk will focus on relating the options that have worked (and failed) in the context of a core service lab that provides support for work from discovery to scale-up phases in an R&D setting. Emphasis will be on project design and execution as well as the benefits and drawbacks of available expression systems, vectors and strategies.

2:35 Parallel Construct Expression Evaluation Using a
High-Throughput Process for Baculovirus-Infected Insect Cells

W. Clay Brown, Ph.D., High-Throughput Protein Lab, Center for Structural Biology, Life Sciences Institute, University of Michigan

The ligation-independent cloning (LIC) region developed at the Midwest Center for Structural Genomics was used to develop vectors compatible with producing recombinant baculovirus to extend this system into high-throughput eukaryotic expression analysis. This series of plasmids can be used for examining the effect of various fusion proteins alone and in combination with different signal peptides on soluble protein expression in the cytosol and secreted into the growth medium. Data for trials with target proteins relevant to the study of human diseases will be presented.

3:05 Re-Engineering Multicloning Sites for Function and Convenience

Hal Alper, Ph.D., Assistant Professor, Chemical Engineering, University of Texas at Austin

Multicloning sites (MCSs) in standard expression vectors are widely-used and thought to be benign, non-interacting elements. However, MCSs impose a necessary distance between promoter elements and genes of interest, resulting in additions to the 5'untranslated region which may influence downstream gene expression in eukaryotes. This talk describes the first performance-based assessment of MCSs in yeast to demonstrate that this structure-based inhibition can impact protein expression in a promoter and gene specific manner. To mitigate this inhibitory effect, a model-based approach was used to redesign superior yeast MCSs with nearly uniform expression profiles across restriction sites.

Sponsored by
3:35 Expressioneering™: Advanced Tools for High-Throughput and Pathway Protein Expression

David Mead, Ph.D., CEO, Lucigen

3:50 Networking Refreshment Break

» FEATURED PRESENTATION

4:15 Semi-Automatic Cloning and Expression of Immunoglobulin Genes from Single-Plasma Cells for
High-Throughput Generation of Monoclonal Antibodies

Masaharu Isobe, Ph.D., Professor, Molecular and Cellular Biology,
Life Sciences and Bioengineering, University of Toyama

We have established a system for high-throughput generation of monoclonal antibodies from single plasma cells. The new method is called Magnetic-beads reaction, through which arrayed hanging drops (MAGrahd) enabled us semi-automatic 5'RACE-ready cDNA synthesis from many single cells. The digestion and purification-free target-selective protocol was also developed for the construction of gene expression unit after amplification of immunoglobulin variable gene. Our new system significantly reduced the cost and the time for isolation of monoclonal antibodies so that it allows us to obtain hundreds of monoclonal antibodies within four to five days.

4:45 The Use of a Combinatorial Recombinational Cloning Platform to Simplify Construction of Expression Vectors for Wide-Ranging Areas of Proteomic and Genetic Research

Dominic Esposito, Ph.D., Interim Director, Protein Expression Laboratory; Group Leader, Clone Optimization Group, Advanced Technology Program, SAIC-Frederick, Inc.

We have developed a cloning platform for the rapid production of expression vectors from a combinatorial library of elements. Although mostly used for generic protein production in standard hosts, the platform allows for simplified and highly flexible construction of esoteric vectors for specialized purposes. Examples will be shown which utilize the platform for multiprotein complex production, protein-protein interaction studies, in vivo gene targeting, gene expression studies using miRNA, and transgenic mouse vector construction.

5:15 Vector Set for Systematic Pathway Engineering in Saccharomyces cerevisiae
Nancy A. DaSilva, Ph.D., Professor, Chemical Engineering and Materials Science, Biomedical Engineering, University of California, Irvine
A unique vectors series, designed to facilitate rapid and systematic combinatorial expression of pathway genes, was constructed for metabolic engineering in the yeast Saccharomyces cerevisiae. To allow differential expression of genes, six different promoters and six different reusable selection markers are included on 2μ- and CEN/ARS- containing plasmids. Furthermore, a fragment appropriate for integration into the genome via homologous recombinations can be readily generated using PCR, and markers can be recycled following cassette integration. We have fully characterized expression from the vectors, compared protein expression following integration at multiple different loci, and employed the vector set for a diverse group of applications.

5:45 – 7:00 Welcoming Reception in the Exhibit Hall

7:00 Close of Day

Day 1 | Day 2 | Download Brochure

Links to Companion Meetings

Pipeline 4

Choosing, Designing and Optimizing Hosts and Platforms

Overcoming Challenges, Finding Solutions


Advance Registration Deadline Extended! Register by December 2 and Save up to $200 Poster Submission Deadline is November 18 Final agenda now available!	The demand for high quality biotherapeutic proteins has never been greater. To meet this goal of producing functional proteins at even higher levels requires engineering (science) and insight (art). Thus, efficient biotherapeutic protein expression is both science and art. Cambridge Healthtech Institute’s Third Annual Engineering Genes, Vectors, Constructs and Clones continues the tradition of applying effective engineering strategies for protein discovery research that leads to functional biotherapeutic products. Learn from seasoned savvy researchers as they share their real-world experiences, applications, and results. Day 1 \| Day 2 \| Download Brochure MONDAY, JANUARY 9 7:30 am Conference Registration and Morning Coffee Targeting Genes 8:55 Chairperson's Opening Remarks Armelle Gaussin, Ph.D., Selexis » KEYNOTE PRESENTATION 9:10 Enhanced Protein Secretion via Translation Engineering Kelvin H. Lee, Ph.D., Professor, Chemical Engineering, Director, DE Biotechnology Institute, University of Delaware Several strategies have been previously proposed to improve recombinant protein production. Using a systems biology approach to study supersecreters, we observed interesting expression changes in genes and proteins related to translation. Based on these observations, we developed a strategy to significantly increase protein secretion using translational engineering. 9:50 j5 and DeviceEditor: DNA Assembly Design Automation Nathan J. Hillson, Ph.D., Director, Synthetic Biology, Joint BioEnergy Institute We have developed two on-line software tools, j5 and DeviceEditor, that automate the design of sequence agnostic, scar-less, multi-part assembly methodologies and translates them to robotics-driven protocols. Given a target library to construct, the software provides automated oligo, direct synthesis, and cost-optimal assembly process design, and integrates with liquid-handling robotic platforms to set up the PCR and multi-part assembly reactions. This work reduces the time, effort and cost of large scale cloning and assembly tasks, as well as enables research scales otherwise unfeasible without the assistance of computer-aided design tools and robotics. 10:20 Networking Coffee Break 10:45 Reliable and Precise Gene Expression via Active Translational Coupling Vivek K. Mutalik, Ph.D., Team Lead, BioFAB We have developed a method that allows for reliable functional coupling of prokaryotic transcription and translation control elements with user-specific coding sequences for genes of interest. We have engineered a collection of standard biological parts that implements the method across a range of transcription and translation levels, and with native and also a phage RNA polymerase. For the genes tested our system realizes a > 90% chance to express a gene to within a two-fold protein concentration target window. 11:15 Efficient Optimization of Protein Expression Using High-Throughput Gene Synthesis and Screens Jingdong Tian, Ph.D., Assistant Professor, Biomedical Engineering, Duke University A parallel on-chip gene synthesis technology is developed. Used with high-throughput expression screens, it enables efficient and reliable optimization of protein expression in host cells. 11:45 Rapid Expression, Engineering and Scale-Up of Inaccessible Proteins in a Cell-Free System Sonia Pollitt, Ph.D., Director, Protein Sciences, Sutro Biopharma Advances in E. coli based cell-free expression technology that enable rapid high-yield production of complex proteins with multiple disulfide bonds make it an ideal platform for screening and selection of protein variants. Here we illustrate the use of this technology to enable engineering of IgGs and Diphtheria toxin fusion proteins. In addition, we show how use of a cell-banked E. coli extract allows rapid scale-up to generate material for IND enabling studies and transitions smoothly to GMP production of clinical drug product. 12:15 pm Close of Morning Session Sponsored by 12:30 Luncheon Presentation GENEART®Genetic Assembly Tools for Synthetic Biology Engineering Federico Katzen, Ph.D., Senior Staff Scientist, R&D Synthetic Biology, Life Technologies Synthetic biology applications require the precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes. We will present a set of technologies that allow the seamless, simultaneous, and highly efficient assembly of genetic material, designed for a wide size dynamic range (10s to 100,000s base pairs). The assembly can be performed either in vitro or within the living cells and the DNA fragments may or may not share homology at their ends. A novel site-directed mutagenesis strategy enhanced by homologous recombination will be also presented. 1:00 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own Vector Construction 2:00 Chairperson's Remarks Ron Godiska, Ph.D., Lucigen 2:05 So Many Options, So Little Money: How to Choose the Right Vector and Screen for Functional Proteins Bill Gillette, Ph.D., Senior Scientist, Group Leader, Protein Expression Laboratory, Advanced Technology Program, SAIC-Frederick, Inc. Producing and purifying functional proteins is a process replete with possibilities for failure. Yet there can be method to the madness and the talk will focus on relating the options that have worked (and failed) in the context of a core service lab that provides support for work from discovery to scale-up phases in an R&D setting. Emphasis will be on project design and execution as well as the benefits and drawbacks of available expression systems, vectors and strategies. 2:35 Parallel Construct Expression Evaluation Using a High-Throughput Process for Baculovirus-Infected Insect Cells W. Clay Brown, Ph.D., High-Throughput Protein Lab, Center for Structural Biology, Life Sciences Institute, University of Michigan The ligation-independent cloning (LIC) region developed at the Midwest Center for Structural Genomics was used to develop vectors compatible with producing recombinant baculovirus to extend this system into high-throughput eukaryotic expression analysis. This series of plasmids can be used for examining the effect of various fusion proteins alone and in combination with different signal peptides on soluble protein expression in the cytosol and secreted into the growth medium. Data for trials with target proteins relevant to the study of human diseases will be presented. 3:05 Re-Engineering Multicloning Sites for Function and Convenience Hal Alper, Ph.D., Assistant Professor, Chemical Engineering, University of Texas at Austin Multicloning sites (MCSs) in standard expression vectors are widely-used and thought to be benign, non-interacting elements. However, MCSs impose a necessary distance between promoter elements and genes of interest, resulting in additions to the 5'untranslated region which may influence downstream gene expression in eukaryotes. This talk describes the first performance-based assessment of MCSs in yeast to demonstrate that this structure-based inhibition can impact protein expression in a promoter and gene specific manner. To mitigate this inhibitory effect, a model-based approach was used to redesign superior yeast MCSs with nearly uniform expression profiles across restriction sites. Sponsored by 3:35 Expressioneering™: Advanced Tools for High-Throughput and Pathway Protein Expression David Mead, Ph.D., CEO, Lucigen 3:50 Networking Refreshment Break » FEATURED PRESENTATION 4:15 Semi-Automatic Cloning and Expression of Immunoglobulin Genes from Single-Plasma Cells for High-Throughput Generation of Monoclonal Antibodies Masaharu Isobe, Ph.D., Professor, Molecular and Cellular Biology, Life Sciences and Bioengineering, University of Toyama We have established a system for high-throughput generation of monoclonal antibodies from single plasma cells. The new method is called Magnetic-beads reaction, through which arrayed hanging drops (MAGrahd) enabled us semi-automatic 5'RACE-ready cDNA synthesis from many single cells. The digestion and purification-free target-selective protocol was also developed for the construction of gene expression unit after amplification of immunoglobulin variable gene. Our new system significantly reduced the cost and the time for isolation of monoclonal antibodies so that it allows us to obtain hundreds of monoclonal antibodies within four to five days. 4:45 The Use of a Combinatorial Recombinational Cloning Platform to Simplify Construction of Expression Vectors for Wide-Ranging Areas of Proteomic and Genetic Research Dominic Esposito, Ph.D., Interim Director, Protein Expression Laboratory; Group Leader, Clone Optimization Group, Advanced Technology Program, SAIC-Frederick, Inc. We have developed a cloning platform for the rapid production of expression vectors from a combinatorial library of elements. Although mostly used for generic protein production in standard hosts, the platform allows for simplified and highly flexible construction of esoteric vectors for specialized purposes. Examples will be shown which utilize the platform for multiprotein complex production, protein-protein interaction studies, in vivo gene targeting, gene expression studies using miRNA, and transgenic mouse vector construction. 5:15 Vector Set for Systematic Pathway Engineering in Saccharomyces cerevisiae Nancy A. DaSilva, Ph.D., Professor, Chemical Engineering and Materials Science, Biomedical Engineering, University of California, Irvine A unique vectors series, designed to facilitate rapid and systematic combinatorial expression of pathway genes, was constructed for metabolic engineering in the yeast Saccharomyces cerevisiae. To allow differential expression of genes, six different promoters and six different reusable selection markers are included on 2μ- and CEN/ARS- containing plasmids. Furthermore, a fragment appropriate for integration into the genome via homologous recombinations can be readily generated using PCR, and markers can be recycled following cassette integration. We have fully characterized expression from the vectors, compared protein expression following integration at multiple different loci, and employed the vector set for a diverse group of applications. 5:45 – 7:00 Welcoming Reception in the Exhibit Hall 7:00 Close of Day Day 1 \| Day 2 \| Download Brochure Links to Companion Meetings Choosing, Designing and Optimizing Hosts and Platforms Overcoming Challenges, Finding Solutions	PepTalk DVDs are archived short course experiences. They allow the user to navigate, pause, rewind and listen to recorded sessions that have been formatted by CHI into a multimedia presentation. Click here for more info SC1: Characterization and Analysis of Visible and Sub-Visible Particulates SC3: Biosimilars SC4: Transient Protein Production in Mammalian Cells: A Short Story SC5: How to Obtain Reliable Information from DLS and Multi-Detector SEC Systems for Biophysical Characterization SC6: A Practical Guide to the Use of Affinity Tags for Protein Purification SC7: Optimized Strategies for Therapeutic Antibodies Discovery and Engineering SC8: Harnessing CHO Cells: The Work Horses of Bioproduction Access recently published whitepapers, podcasts, DVD’s and more.