A Novel Framework of a Cell Free Protein System Based on a Psychrotolerant Extremophile
Abstract
Cell-free synthetic biology has emerged as a flexible and powerful tool for bypassing the complexities and difficulties of working with living organisms for biotechnological applications. Composed of a user-made mixture of clarified cell lysates, metabolites, cofactors, and genetic material, cell-free protein synthesis (CFPS) allows for gene expression in an engineerable in vitro reaction environment. The use of CFPS provides unique benefits, as protein synthesis is uncoupled from cell growth and metabolism, allowing for direct control, rapid sampling, and easier manipulation of the system. This represents a significant departure from cell-based processes that rely on microscopic cellular ‘reactors’, allowing us to, in principle, harness all the metabolic resources contained in the cellular cytoplasm. From a prototyping perspective, barrier-free CFPS allows direct access to enzymes and reaction conditions, which dramatically increases the resolution at which we can manipulate and sample the inner cellular environment. Additionally, there is a high degree of flexibility to experimentally isolate biochemical processes from the confounding background of biological networks present in living organisms, as well as the possibility to control the complex set of chemical reactions of native living systems out of the cell. The use of CFPS offers the opportunity to study the details of protein synthesis and its many regulations with high resolution, as it enables a level of control of the variables involved that would be impossible using In vivo systems. This project will study the development of a novel CPFS based on the psychrotolerant (cold loving) microorganism, Serratia sp. I1P molecular chassis with focus on the in vitro protein synthesis at room temperature of Serratia sp. I1P catalase enzyme. To achieve this goal, we will use an interdisciplinary approach which combines microbiology, bioinformatics, molecular biology and biochemical analysis to determine the optimal experimental conditions for the development of this novel CFPS. With the implementation of this CFPS for efficient protein obtention at room temperature of a psychrotolerant enzyme, we will not only lower the number of experimental steps to obtain our target enzyme, but also set a precedent for future developments of this novel technology not previously developed for a low temperature biological system.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 21, 2022
- Source ID
- FA95502210034XX0
Entities
People
- Jenny M Blamey
Organizations
- Air Force Office of Scientific Research
- United States Air Force