scholarly journals Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Shue Wang ◽  
Sagardip Majumder ◽  
Nicholas J Emery ◽  
Allen P Liu
Keyword(s):  
Synthetic Biology ◽  
Real Time ◽  
Protein Dynamics ◽  
Mammalian Cell ◽  
Locked Nucleic Acid ◽  
Free Expression ◽  
Reporter Protein ◽  
Bottom Up ◽  
Lna Probe

Abstract Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in conjunction with fluorescent proteins in HeLa-based mammalian CFE system provides a versatile in vitro platform for studying mRNA dynamics for bottom-up synthetic biology applications.

scholarly journals Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

2019 ◽  
Vol 2 (2) ◽  
pp. 39 ◽  
Author(s):  
Dohyun Jeong ◽  
Melissa Klocke ◽  
Siddharth Agarwal ◽  
Jeongwon Kim ◽  
Seungdo Choi ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Free Expression ◽  
Dna Nanostructures ◽  
Expression Systems ◽  
Natural Systems ◽  
The Past ◽  
Current Cell ◽  
Recent Developments ◽  
Biological Circuits

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription–translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.

scholarly journals A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G.W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Experimental System ◽  
Free Expression ◽  
Supported Lipid Bilayers ◽  
Linc Complex ◽  
Complex Assembly ◽  
Complex Core

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

scholarly journals An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design

2018 ◽  
Vol 3 (3) ◽  
pp. 186-195 ◽  
Author(s):  
Ting Shi ◽  
Pingping Han ◽  
Chun You ◽  
Yi-Heng P. Job Zhang
Keyword(s):  
Synthetic Biology ◽  
Bottom Up

scholarly journals Direct reconstitution and study of SUN protein interactions in vitro using mammalian cell-free expression

2021 ◽  
Author(s):  
Sagardip Majumder ◽  
Yen-Yu Hsu ◽  
Allen P Liu
Keyword(s):  
Membrane Proteins ◽  
Mammalian Cell ◽  
Protein Function ◽  
Mammalian Cells ◽  
Expression System ◽  
Full Length ◽  
Free Expression ◽  
Lipid Bilayer Membranes ◽  
Linc Complex

SUN proteins are an integral part of LINC (Linker of Nucleoskeleton and Cytoskeleton) complex which spans the nuclear envelope and acts as a physical tether between the cytoskeletal filaments and the nuclear lamina. Several human diseases associated with nuclear deformation are primarily caused by impaired functioning of SUN proteins. Studies in yeast and mammalian cells have illustrated the detrimental effects of different SUN mutants in nuclear positioning and movement. While cell-based studies provide physiological relevance to the functioning of a protein, in vitro reconstitution of isolated proteins is useful in mechanistically dissecting protein function in a biochemically defined environment. In this study, we used a mammalian cell-free expression system to synthesize and reconstitute SUN proteins in artificial lipid bilayer membranes. Building on our previous work demonstrating directional reconstitution of full-length SUN proteins, we deciphered the mechanism of such protein reconstitution and leveraged it to test several theories/models of LINC complex assembly. By using a simple fluorescence-based assay, we revealed the importance of cations such as calcium and the presence of disulfide bonds in the formation of LINC complexes. Through sequential reconstitutions of SUN proteins and soluble luminal domains of SUN proteins, we found that coiled coil domains of SUN proteins are necessary for homomeric and heteromeric interactions of reconstituted SUN proteins. Overall, our results provide mechanistic insights on LINC complex formation and how this might impact cellular mechanotransduction. The facile approach for reconstituting full-length membrane proteins can be extended to study other difficult-to-study membrane proteins in vitro.

scholarly journals Experimental and Biophysical Modeling of Transcription and Translation Dynamics in Bacterial- and Mammalian-based Cell-Free Expression Systems

2021 ◽  
Author(s):  
Yuwen Zhao ◽  
Shue Wang
Keyword(s):  
Fluorescent Protein ◽  
Locked Nucleic Acid ◽  
Biophysical Model ◽  
Free Expression ◽  
Biophysical Modeling ◽  
Gene Circuits ◽  
E Coli ◽  
Quantitative Studies ◽  
The Difference

Cell-free expression (CFE) systems have been used extensively in system and synthetic biology as a promising platform for manufacturing proteins and chemicals. Currently, the most widely used CFE system is in vitro protein transcription and translation platform. As the rapidly increased applications and uses, it is crucial to have a standard biophysical model for quantitative studies of gene circuits, which will provide a fundamental understanding of basic working mechanisms of CFE systems. Current modeling approaches mainly focus on the characterization of E. coli-based CFE systems, a computational model that can be utilized to both bacterial- and mammalian-based CFE has not been investigated. Here, we developed a simple ODE (ordinary differential equation)-based biophysical model to simulate transcription and translation dynamics for both bacterial- and mammalian-based CFE systems. The key parameters were estimated and adjusted based on experimental results. We next tested four gene circuits to characterize kinetic dynamics of transcription and translation in E. coli- and HeLa-based CFE systems. The real-time transcription and translation were monitored using Broccoli aptamer, double stranded locked nucleic acid (dsLNA) probe and fluorescent protein. We demonstrated the difference of kinetic dynamics for transcription and translation in both systems, which will provide valuable information for quantitative genomic and proteomic studies. This simple biophysical model and the experimental data for both E. coli- and HeLa-based CFE will be useful for researchers that are interested in genetic engineering and CFE bio-manufacturing.

scholarly journals The all-E. Coli TXTL Toolbox 3.0: New Capabilities of a Cell-Free Synthetic Biology Platform

2021 ◽  
Author(s):  
David Garenne ◽  
Seth Thompson ◽  
Amaury Brisson ◽  
Aset Khakimzhan ◽  
Vincent Noireaux
Keyword(s):  
Synthetic Biology ◽  
Expression System ◽  
Building Blocks ◽  
Regulatory Elements ◽  
Free Expression ◽  
Batch Mode ◽  
E Coli ◽  
A Genome ◽  
Synthetic Cells

Abstract The new generation of cell-free gene expression systems enables prototyping and engineering biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription-translation (TXTL) platforms remains a major goal to either execute larger DNA programs, or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch mode reactions, synthesis of the fluorescent reporter protein eGFP reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced to concentrations of more than 8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kbp and about 60 genes, is produced at a concentration of 1013 PFU/ml. This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for either testing regulatory elements and circuits, biomanufacturing biologics, or building synthetic cells.

Toward low-cost biomanufacturing through in vitro synthetic biology: bottom-up design

2011 ◽  
Vol 21 (47) ◽  
pp. 18877 ◽  
Author(s):  
Y.-H. Percival Zhang ◽  
Suwan Myung ◽  
Chun You ◽  
Zhiguang Zhu ◽  
Joseph A. Rollin
Keyword(s):  
Synthetic Biology ◽  
Low Cost ◽  
Bottom Up
Sign in / Sign up

Export Citation Format

Share Document