A5023345723- CRISPR and Genetic Engineering
- Advanced biosensing and bioanalysis techniques
- Bacteriophages and microbial interactions
- Monoclonal and Polyclonal Antibodies Research
- Transgenic Plants and Applications
- DNA and Nucleic Acid Chemistry
- RNA and protein synthesis mechanisms
- Viral Infectious Diseases and Gene Expression in Insects
- Plant tissue culture and regeneration
- Biochemical and Molecular Research
- RNA modifications and cancer
- Animal Genetics and Reproduction
- Molecular Biology Techniques and Applications
- CAR-T cell therapy research
- Enzyme Structure and Function
- Bacterial Genetics and Biotechnology
- Advanced Biosensing Techniques and Applications
- Glycosylation and Glycoproteins Research
- RNA Research and Splicing
- RNA Interference and Gene Delivery
- Protein Structure and Dynamics
University of Colorado Boulder
2020-2024
A general method to generate biosensors for user-defined molecules could provide detection tools a wide range of biological applications. Here, we describe an approach the rapid engineering using PYR1 (Pyrabactin Resistance 1), plant abscisic acid (ABA) receptor with malleable ligand-binding pocket and requirement ligand-induced heterodimerization, which facilitates construction sense-response functions. We applied this platform evolve 21 sensors nanomolar micromolar sensitivities small...
Abstract Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly synthetic DNA cassettes employ disparate, system‐specific methodology. Here we present a standardized method building user‐defined libraries. We demonstrate that 25 μL reaction, using 40 fmol input DNA, can generate library on order 1 × 10 6 members and reaction volume or concentration be scaled up with no losses in transformation efficiency. Such constructed from dsDNA...
Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the
Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment sequence-binding datasets may not contain mutagenic, antigenic, or sequence diversity necessary for deep learning approaches to capture recognition. In part, this is because limited experimental platforms exist assessing quantitative and simultaneous sequence-function...
For many enzymes, activity is regulated post-translationally by endogenous metabolites. Designing liganded control of essential activities like transcription would advance predictive biological processes, a fundamental goal synthetic biology. Here we demonstrate that full-length, single subunit T7-derived RNA polymerases (T7 RNAP) can be controlled physiologically relevant concentrations indoles. We used rational design and directed evolution to identify T7 RNAP variants with minimal...
Generating combinatorial libraries of specific sets mutations are essential for addressing protein engineering questions involving contingency in molecular evolution, epistatic relationships between mutations, as well functional antibody and enzyme engineering. Here we present optimization a mutagenesis method template-based nicking mutagenesis, which allows the generation with >99% coverage tens thousands user-defined variants. The non-optimized resulted low library coverage, could be...
Construction of user-defined long circular single stranded DNA (cssDNA) and linear (lssDNA) is important for various biotechnological applications. Many current methods synthesis these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology generating cssDNA employing Golden Gate assembly, nickase, exonuclease degradation. Our technique demonstrated three plasmids with insert sizes ranging from 2.1 3.4 kb, requires no specialized equipment, can be...
Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking (NM) allows the user to rapidly construct libraries of all possible single mutations in target sequence from plasmid DNA one-pot procedure. Briefly, one strand degraded using nicking restriction endonuclease exonuclease treatment. Mutagenic primers encoding desired are annealed resulting circular single-stranded DNA, extended with high-fidelity polymerase, ligated into covalently...
Abstract Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly synthetic DNA cassettes employ disparate, system specific methodology. Here we benchmark a broadly applicable method building user-defined libraries. We demonstrate that 25 μl reaction, using 40 fmol input DNA, can generate library on order 1×10 6 members and reaction volume or concentration be scaled up with no losses in transformation efficiency. Such constructed from...
ABSTRACT Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought set of generalized mutations would provide stability affecting functionality for variants with ligand-binding capabilities. To do this we used global multi-mutant analysis (GMMA) approach, which can identify substitutions have stabilizing effects not lower function....
Precise, stringent, post-translational activation of enzymes is essential for many synthetic biology applications. For example, even a few intracellular molecules unregulated T7 RNA polymerase can result in growth cessation bacterium. We sought to mimic the properties natural enzymes, where activity regulated ubiquitously by endogenous metabolites. Here we demonstrate that full-length, single subunit T7-derived polymerases (T7 RNAP) be activated physiologically relevant concentrations...
Abstract T7 RNA polymerase (T7 RNAP) biosensors, in which RNAP transcribes some reporter gene or signal response to external stimuli, have wide applications synthetic biology and metabolic engineering. We adapted a biochemical reaction network model used an vitro transcription assay determine parameters for different constructs. Under conditions where template DNA is limiting, the EC 50 values of native engineered RNAPs ranged from 33 nM (29 -37 95% c.i.) 570 (258 -714 (wild-type RNAP). The...