One approach to understanding components in living organisms is to attempt to create them artificially, using principles of chemistry, engineering and genetics. A suite of powerful techniques—collectively referred to as synthetic biology—have been used to produce self-replicating molecules, artificial pathways in living systems and organisms bearing synthetic genomes.
In a new twist, John Chaput, a researcher at Arizona State University’s Biodesign Institute and colleagues at the Department of Pharmacology, Midwestern University, Glendale, Ariz. have fabricated an artificial protein in the laboratory and examined the surprising ways living cells respond to it.
The phosphodiester bonds of ATP contain the energy necessary to drive reactions in living systems, giving up their stored energy when these bonds are chemically cleaved. The depletion of available intracellular ATP by DX binding disrupts normal metabolic activity in the cells, preventing them from dividing, (though they continue to grow).
After exposure to DX, the normally spherical E. coli bacteria develop into elongated filaments. Within the filamentous bacteria, dense intracellular lipid structures act to partition the cell at regular intervals along its length (see figure 1). These unusual structures, which the authors call endoliposomes, are an unprecedented phenomenon in such cells.
Producing a synthetic protein like DX, which can mimic the elaborate folding characteristics of naturally occurring proteins and bind with a key metabolite like ATP is no easy task. As Chaput explains, a clever strategy known as mRNA display was used to produce, fine-tune and amplify synthetic proteins capable of binding ATP with high affinity and specificity, much as a naturally occurring ATP-binding protein would.
First, large libraries of random sequence peptides are formed from the four nucleic acids making up DNA, with each strand measuring around 80 nucleotides in length. These sequences are then transcribed into RNA with the help of an enzyme—RNA polymerase. If a natural ribosome is then introduced, it attaches to the strand and reads the random sequence RNA as though it was a naturally-occurring RNA, generating a synthetic protein as it migrates along the strand. In this way, synthetic proteins based on random RNA sequences can be generated.
The main trick involves an earlier step in the process. A molecular linker is chemically attached to the RNA templates, such that each RNA strand forms a bond with its newly translated protein. The mRNA-protein hybrids are exposed to selection targets (like ATP) over consecutive rounds of increasing stringency. After each round of selection, those library members that remain bound to the target are reverse-transcribed into cDNA (using their conveniently attached RNA messages), and then PCR amplified.
The research indicates that there is still a great deal to learn about bacterial behavior and the repertoire of responses available when such cells encounter novel situations, such as an unfamiliar, synthetic protein.
This story is reprinted from material from Arizona State University, with editorial changes made by Materials Today. The views expressed in this article do not necessarily represent those of Elsevier. Link to original source.