Fisk Lab Publication!

"Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair "

In this Genes publication, we discuss utilization of our fluorescence-based screen for codon reassignment to probe sequence context effects on the efficiency of decoding the amber stop codon as a sense codon. We perform evaluate several single stop codon containing GFP variants and several combinations thereof. The suite of measurements if performed in two related cell strains, one of which has had the release factor which reads the amber codon as a stop signal removed. The comparison of reassignment efficiencies between the 2 cell lines allowed us to quantify the contribution of release factor competition to the overall efficiency of stop codon suppression.

We were especially honored to be invited to submit this manuscript to the special issue of Genes on “Synthetic DNA and RNA Programming”

Congratulations to Dave and Meg!

Genes, 2018, vol 9, pg 546

DOI: 10.3390/genes9110546

 

Speaking Across the Pond

Nick was invited to speak about the lab’s computational model of the M13 life cycle at the First Ever UK Synthetic Biology Congress held in London on November 8 and November 9, 2018. It was a great opportunity to meet some European colleagues and establish new relationships with laboratories that are also interested in understanding and exploiting virus particles and computational modeling to create exciting bio-diagnostics and therapeutics.

 

Funding! NSF Grant Awarded, May 2018!

The National Science Foundation has awarded the Fisk Lab funding to continue our exploration of the quantitative biology of bacteriophage M13! This project is funded by the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences and the Systems and Synthetic Biology Cluster.

Building on previous work involving a simulation of M13 growth that integrates 50 years of experimental observations, the goals of this project are to evaluate the nested feedback control loops in the M13 life cycle and fill in gaps in the quantitative understanding of M13 biology. The first objective of the current project involves quantification of the poorly-understood M13 protein-protein and protein-DNA interactions that govern phage DNA replication and the system level response to modifications of the DNA replication program. The second objective involves the identification and quantification of the protein-RNA interactions that regulate the timing and extent of phage protein production. Within this aim the system level effects of modifying the protein-RNA control mechanisms will be investigated. The final objective of the project is to integrate the results of the experiments into the simulation model. This aim will focus on augmenting the model with new quantitative data from Aims 1 and 2, and evaluating the present state of understanding by comparing the results of the system level experiments to predictions. Repeated cycles of theoretical refinement and experimental testing are required to build an engineering design cycle for M13 phage. This proposal will result in a greatly improved quantitative understanding of M13 biology and an infrastructure for the design and production of highly modified particles.

 

On the road again!

The Fisk lab is thrilled to announce that we have joined the Department of Chemistry at the University of Colorado Denver. We are looking forward to everything this new opportunity offers us, from fantastic facilities to exciting opportunities to collaborate to affiliation with a university that has a medical school. Our move will happen over the summer of 2018, and we will be ready to dive in again in the fall semester.

 

Congratulations Dr. David Schwark!

Dave Schwark successfully defended his Ph.D. thesis in March 2018! Dave worked on sense codon reassignment in the Fisk lab; one of his most significant contributions was the identification and directed evolution of a variant of the M. barkeri pyrrolysine aaRS that aminoacylates tyrosine. Dave then utilized this orthogonal pair in combination with the fluorescence-based screen for the evaluation of sense codon reassignment at more than 30 codons using the M. barkeri pair. This massive data set is complementary to the evaluation of sense codon reassignment performed using the M. jannaschii orthogonal pair. Together, the 2 suites of data will allow a focused dissection of the relative quantitative importance of the factors affecting the fidelity of translation. Dave's work contributed heavily to the DNA infrastructure for the Fisk lab for future work on both genetic code engineering and non-canonical amino acids understanding protein translation.  Dave is currently identifying his next career step and finishing up a few projects in the lab. Congrats and best wishes, Dave!

 

Fisk Lab Publication!

"Mapping the Plasticity of the Escherichia coli Genetic Code with Orthogonal Pair-Directed Sense Codon Reassignment"

In our Biochemistry manuscript, we discuss 20 new, related genetic codes, in which a targeted E. coli wobble codon is reassigned to tyrosine utilizing the orthogonal tyrosine tRNA/aminoacyl tRNA synthetase pair from Methanocaldococcus jannaschii. One at a time, reassignment of each targeted sense codon to tyrosine is quantified in cells by measuring the fluorescence of GFP variants in which the essential tyrosine residue is encoded by a non-tyrosine codon. Our strategy of using orthogonal tRNA/aminoacyl tRNA synthetase pairs to precisely direct the incorporation of a single amino acid in response to individual sense and nonsense codons provides a suite of related data with which to examine the plasticity of the code.

Congratulations to Meg and Wil!

Biochemistry, 2018, vol 57, pg 2762-2774

DOI: 10.1021/acs.biochem.8b00177

 

Nick will be speaking at aaRS 2017: The IUBMB Focused Meeting on Aminoacyl tRNA Synthetases!!

Nick Fisk (PI) will be giving a talk on Thursday, November 2, 2017. Come visit Nick on the beach in Clearwater Beach, FL and enjoy some great weather and great science!!!!

 

Come see us in Vancouver at SEED: Synthetic Biology, Engineering, Evolution, and Design!!

Nick Fisk (PI) will be giving a talk on Wednesday, June 21, 2017 at 2:15 PM entitled "Orthogonal Pair-Directed, Codon Specific Sense Codon Reassignment: An Improved Tool for Evaluating the Plasticity of the E. coli Genetic Code".

Meg Schmitt will present a poster in poster session 1 on Wednesday, June 21, 2017 from 6:00-7:30 PM. You can find her poster "Dissecting the Factors That Determine the Fidelity of the Genetic Code through Sense Codon Reassignment" up in Poster Space 129 starting on Tuesday!

David Schwark will present a poster in poster session 2 on Thursday, June 22, 2017 from 6:00-7:30 PM. You can find his poster "An Engineered Pyrrolysyl tRNA Synthetase for in vivo Measurement of Sense Codon Reassignment in E. coli" up in Poster Space 130 on Thursday and Friday!

We would love to chat with you about this work, and ALL the exciting work happening right now in the Fisk Lab.  Come see us!!!!

 

Congratulations Dr. Wil Biddle!

Wil Biddle successfully defended his Ph.D. thesis in June 2017! Wil worked on sense codon reassignment in the Fisk lab and developed the simple fluorescence-based screen for sense codon reassignment. Wil's work provided a lot of valuable DNA infrastructure for the Fisk lab for future work on both genetic code engineering and non-canonical amino acids and understanding protein translation.  Wil is currently deciding on his next steps and finishing up a few projects in the lab. Congrats and best wishes, Wil!

 

Fisk Lab Publication!

"Hyperthermostable Binding Molecules on Phage: Assay Components for Point-of-Care Diagnostics for Active Tuberculosis Infection"

In our FEBS J manuscript, we described the utilization of the hyperthermostable protein Sso7d as a scaffold for identification of tight specific binding molecules for a variety of protein targets by phage display. Here, we report identification of an Sso7d variant that binds a biomarker for active tuberculosis infection which is secreted in the urine of patients with an active Tb infection. We are working to eliminate antibodies from molecular diagnostics for tuberculosis infection, as the antibody components are often costly and unstable. Binding molecules based on hyperthermostable protein scaffolds, such as Sso7d, could lower the cost of production and improve the physical characteristics of diagnostic devices for use in resource-limited environments.

Congratulations to Ning and Meg for this exciting publication!

Analytical Biochemistry, In press. Accepted December 23, 2016

 

Fisk Lab Publication Selected as Highlighted Article!  January 2017 Virology

"Simulation of the M13 life cycle I: Assembly of a genetically-structures deterministic chemical kinetic simulation"

The editors of Virology selected the first paper on the computational model as a Highlighted Article for the January 2017 issue! This is terrific news and speaks volumes about the detailed difficult work done by Steve, Ronnie and Meg in assembling the model and writing the manuscript. Congratulations to all the authors!!!!!

We'll also be writing an entry for the Virology Highlights blog, which was started to feature Virology's best articles and provide authors an opportunity to elaborate on the back story of their work.

Edit, December 27, 2016: You can read more about our work on the Virology Highlights Blog.  Our entry was posted on December 22, 2016.

 

Fisk Lab Publication!

"Modification of Orthogonal tRNAs: Unexpected Consequences for Sense Codon Reassignment "

In our Biochemistry manuscript describing our simple screen for sense codon reassignment, we reported nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon. We suspected a modification of the tRNA anticodon was responsible for the anomalous lack of codon discrimination. In this manuscript, we demonstrate that adenosine 34 of the orthogonal tRNA with an AUG anticodon is converted to inosine. We identified anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues infiltrate the genetic codes of diverse organisms.

Congratulations to Wil and Meg for this exciting publication!

Nucleic Acids Research, In press. Accepted October 7, 2016DOI: 10.1093/nar/gkw948

 

Fisk Lab Publications!

"Simulation of the M13 life cycle I: Assembly of a genetically-structures deterministic chemical kinetic simulation" and "Simulation of the M13 life cycle II: Investigation of the control mechanisms of M13 infection and establishment of the carrier state"

A pair of papers on the development and implementation of an in silico simulation of the M13 phage life cycle from infection to release of progeny phage were accepted for back-to-back publication in Virology! These manuscripts represent a great deal of hard work and combing 50 years of research on filamentous phages to assemble as complete a model as is possible with the quantitative data available today.

Congratulations to Steve, Ronnie and Meg for all your hard work on the simulations and preparation of these complex, thorough manuscripts.

DOI: 10.1016/j.virol.2016.08.015 and 10.1016/j.virol.2016.08.017

 

Genetic Code Engineering 2016

Dr. Fisk and graduate students Wil Biddle and David Schwark attended the first GCE conference in August 2016 at Oregon State University.

All three attendees presented recent Fisk lab research during the poster session.

Wil, Dave and Nick attended a very special tour of the Linus Pauling archives at Oregon State.  The tour was a rare experience, as they got to view Pauling's notebooks, original handwritten manuscripts, and correspondence (from back when correspondence *didn't* all happen via email!).  They also got to hold both of his original Nobel prizes for Chemistry and Peace!  A truly amazing experience at an equally productive conference!

 

Synthetic Biology: Engineering, Evolution and Design 2016

Dr. Fisk, research scientist Dr. Meg Schmitt, and graduate student Wil Biddle attended SEED 2016 in July 2016 in Chicago, IL.

Dr. Schmitt and Wil Biddle presented recent Fisk lab research during two poster sessions.

 

Congratulations Dave and Trish!

Graduate student David Schwark married occupational therapist Trish in July 2016. The entire Fisk lab was in attendance. The reception took place in a natural stone amphitheater near Denver. A great time was had by all!

 

Congratulations Dr. Ning Zhao!

Ning Zhao successfully defended her Ph.D. thesis in June 2016! Ning worked on developing a phage-based biosensor for active tuberculosis infection, which began with an evaluation of a series of alternative scaffold proteins for phage library generation. Ning's work provided a lot of valuable DNA and phage-related infrastructure for the Fisk lab for future work on both improved tuberculosis sensing and other biomarkers of interest.  Ning is currently deciding on her next steps and is choosing between several postdoctoral research opportunities. Congrats and best wishes, Ning!

 

Fisk Lab Publication

"Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library."

Congratulations Ning and Meg!

FEBS J. 2016 Apr;283(7):1351-67. doi: 10.1111/febs.13674. Epub 2016 Mar 6.

 

American Chemical Society Meeting, Spring 2016

Dr. Fisk was invited to give a talk at the Spring 2016 ACS National Meeting in San Diego, CA. His talk was presented in the Biotechnological Chemistry Division, Biomolecular and Biophysical Processes: Protein Structure and Function section.

 

Fisk Lab Publication

"Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen"

This manuscript was selected by the Editors of Biochemistry for free open access as an Editors' Choice manuscript. One article per issue is selected for its potential for wide audience interest and scientific impact.

Congratulations Wil and Meg!

Biochemistry, 2015, 54 (50), pp 7355–7364

DOI: 10.1021/acs.biochem.5b00870

 

Funding! NSF Grant Awarded, May 2015!

The National Science Foundation has awarded John Fisk funding to evaluate and improve sense codon reassignment! This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences and the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

Under this award, we will study the expansion of the genetic code through sense codon reassignment. The research project will employ a fluorescence-based screen to measure the extent to which sense codons that naturally read through wobble interactions in E. coli can be reassigned using the orthogonal tRNA/aaRS pairs most commonly employed for nonsense suppression. The same fluorescence screen will be further employed to evolve the tRNA/aaRS systems to better decode reassigned sense codons, to evaluate the plasticity of aminoacyl tRNA synthetase-tRNA recognition domains, and explore the extent to which the machinery of translation can be molded to accommodate expanded genetic codes. The systems identified and optimized to reassign sense codons will be combined to generate genetic codes containing 22 and 23 amino acids. The combined experimental aims of the project provide a host of new measurements to better map the plasticity of the translational system in E. coli and evaluate the degree to which representative tRNA/aaRS pairs can be modified to reassign the meaning of sense codons.

 

Congratulations Steven Smeal, Masters of Science in Engineering awarded March 2014!

Steve successfully defended his masters' thesis in March 2014. Steve was responsible for developing the computational model of the M13 life cycle for the Fisk lab. His excellent work has enabled many many evaluations of factors that influence the M13 life cycle in a way never possible until 50 years of biochemical data were combined into a single set of differential equations. Thanks for your amazing work, Steve! Congrats and best wishes in your future endeavors!

 

Funding! Colorado Bioscience Association, Discovery Evaluation grant awarded, November 2012!

The Fisk lab was awarded a Bioscience Discovery Evaluation grant through the Colorado Bioscience Association.

 

Funding! Dr. Fisk named a Boettcher Foundation, Webb-Waring Biomedical Investigator, July 2011!

Dr. Fisk was selected from young faculty with biomedical research interests across the State of Colorado as 1 of 5 Boettcher Investigators for 2011. The grant from the Boettcher Foundation will allow Fisk to continue the development of bacterial virus particles with a technological utility – specifically improving and expanding on the sensor development project initially funded by the Gates Foundation.

 

Funding! Infectious Disease Supercluster at Colorado State grant awarded, July 2011!

The Fisk lab was awarded a grant from the IDSC at Colorado State University.

 

Funding! Bill and Melinda Gates Foundation, Grand Challenges Explorations grant awarded, May 2010!

This award supported the initiation of the Fisk lab's work towards the development of a phage-based sensor for active tuberculosis infection. The sensors involve engineered phage particles that display combinations of binding molecules and sensing molecules in which binding of a protein analyte triggers signal generation.