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T7Max transcription system

https://doi.org/10.1186/s13036-023-00323-1

Deich, Christopher ; Cash, Brock ; Sato, Wakana ; Sharon, Judee ; Aufdembrink, Lauren ; Gaut, Nathaniel J. ; Heili, Joseph ; Stokes, Kaitlin ; Engelhart, Aaron E. ; Adamala, Katarzyna P. ( December 2023 , Journal of Biological Engineering)

Abstract Background
Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.
Results
Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression withinin vitrosystems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.
Conclusions
The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- basedin vitroprotein expression system.

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Free, publicly-accessible full text available December 1, 2024
New Aequorea Fluorescent Proteins for Cell-Free Bioengineering

https://doi.org/10.1021/acssynbio.3c00057

Deich, Christopher ; Gaut, Nathaniel J. ; Sato, Wakana ; Engelhart, Aaron E. ; Adamala, Katarzyna P. ( April 2023 , ACS Synthetic Biology)

Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms.
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Parasites, Infections, and Inoculation in Synthetic Minimal Cells

https://doi.org/10.1021/acsomega.2c07911

Cash, Brock ; Gaut, Nathaniel J. ; Deich, Christopher ; Johnson, Laura L. ; Engelhart, Aaron E. ; Adamala, Katarzyna P. ( February 2023 , ACS Omega)
Reconstituting Natural Cell Elements in Synthetic Cells

https://doi.org/10.1002/adbi.202000188

Gaut, Nathaniel J. ; Adamala, Katarzyna P. ( February 2021 , Advanced Biology)

Abstract
Building a live cell from non‐living building blocks would be a fundamental breakthrough in biological sciences, and it would enable engineering new lineages of life, not directly descendant of the Last Universal Common Ancestor. Fully engineered synthetic cells will have architectures that can be radically different from the natural cells, yet most life processes reconstituted in synthetic cells so far are built from natural and biosimilar building blocks. Most natural processes have already been reconstituted in synthetic cell chassis. This paper summarizes recent advancements in using non‐living building blocks to reconstitute some of the most crucial features of living systems in a fully engineerable chassis of a synthetic cell.

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