Expanding the Genetic Code of a Photoautotrophic Organism

Biotech & Bioengineering

Kushmaro

et al. 2019

Self Cite

Photosynthesis is one of the most fundamental and complex mechanisms in nature. It is a well‐studied process, however, some photosynthetic mechanisms are yet to be deciphered. One of the many proteins that take part in photosynthesis, cytochrome bd, is a terminal oxidase protein that plays a role both in photosynthesis and in respiration in various organisms, specifically, in cyanobacteria. To clarify the role of cytochrome bd in cyanobacteria, a system for the incorporation of an unnatural amino acid into a genomic membrane protein cytochrome bd was constructed in Synechococcus sp. PCC7942. N‐propargyl‐ l‐lysine (PrK) was incorporated into mutants of cytochrome bd. Incorporation was verified and the functionality of the mutant cytochrome bd was tested, revealing that both electrochemical and biochemical activities were relatively similar to those of the wild‐type protein. The incorporation of PrK was followed by a highly specific labeling and localization of the protein. PrK that was incorporated into the protein enabled a “click” reaction in a bio‐orthogonal manner through its alkyne group in a highly specific manner. Cytochrome bd was found to be localized mostly in thylakoid membranes, as was confirmed by an enzyme‐linked immunosorbent assay, indicating that our developed localization method is reliable and can be further used to label endogenous proteins in cyanobacteria.

Section: Introductionmentioning

confidence: 99%

Cellular localization of cytochrome bd in cyanobacteria using genetic code expansion

Cohen

Biotech & Bioengineering

Kushmaro

et al. 2019

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“…Therefore, it is necessary to To date, live-cell imaging can be obtained through different approaches, however genetic code expansion, the reassignment of codons and incorporation of an unnatural amino acid (Uaa) into proteins 17 , displays advantages over other methodologies, and is gaining increasing exposure and momentum [18][19][20][21] . Genetic code expansion systems are being constantly improved, expanded and adapted to a growing number of organisms [22][23][24][25] . This technique aids in improving imaging.…”

Section: Mainmentioning

confidence: 99%

“…All plasmids for initial method establishment were constructed by a standard yeast assembly protocol 22 . The upstream and downstream regions from P. aeruginosa's leucyl translational system were chosen for OTS expression.…”

Section: Plasmids Constructionmentioning

confidence: 99%

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

Yaniv

Chetrit

et al. 2020

Preprint

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The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for studying biofilm formation. In order to elucidate the role of the bacteria flagella in biofilm formation, we developed a new tool for flagella bio-tracking. We have site-specifically labeled the bacterial flagella by incorporating an unnatural amino acid into the flagella monomer via genetic code expansion. This enabled us to label and track the bacterial flagella during biofilm maturation. Direct, live imaging revealed for the first-time presence and synthesis of flagella throughout the biofilm lifecycle. To ascertain the possible role of the flagella in the strength of a biofilm we produced a “flagella knockout” strain and compared its biofilm to that of the wild type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison to the flagella knockout strain. This suggests a newly discovered structural role for bacterial flagella in biofilm structure, possibly acting as a scaffold. Based on our findings we suggest a new model for biofilm maturation dynamic and underscore the importance of direct evidence from within the biofilm.

“…Thereby enabling the rational enhancement of the chemical and physical properties of proteins. This methodology was established in the early years of the current century in E. coli [1] and gradually was adapted to many other organisms in both eukarya [2][3][4][5][6][7] and prokarya [8,9].…”

Section: Introductionmentioning

confidence: 99%

Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

Chemla

Alfonta

et al. 2019

Preprint

Self Cite

Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thioboc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.