SynBioFromLeukipposInstitute

11/01/2015, 
Big Sky,  
Montana

by
Zhanar Abil , Xiong Xiong , and Huimin Zhao

"Synthetic biology is a relatively new field with the key aim of designing and constructing biological systems with novel functionalities. Today, synthetic biology devices are making their first steps in contributing new solutions to a number of biomedical challenges, such as emerging bacterial antibiotic resistance and cancer therapy. This review discusses some synthetic biology approaches and applications that were recently used in disease mechanism investigation and disease modeling, drug discovery and production, as well as vaccine development, treatment of infectious diseases, cancer, and metabolic disorders."


http://bit.ly/1ya322V

by
Lu Y, Liang FX, Wang X

"Signaling in the ancestral branch of the unfolded protein response (UPR) is initiated by unconventional splicing of HAC1/XBP1 mRNA during endoplasmic reticulum (ER) stress. In mammals, IRE1α has been known to cleave the XBP1 intron. However, the enzyme responsible for ligation of two XBP1 exons remains unknown. Using an XBP1 splicing-based synthetic circuit, we identify RtcB as the primary UPR RNA ligase. In RtcB knockout cells, XBP1 mRNA splicing is defective during ER stress. Genetic rescue and in vitro splicing show that the RNA ligase activity of RtcB is directly required for the splicing of XBP1 mRNA. Taken together, these data demonstrate that RtcB is the long-sought RNA ligase that catalyzes unconventional RNA splicing during the mammalian UPR."

 http://bit.ly/XExPd0

by
Thomas H Segall‐Shapiro, Adam J Meyer, Andrew D Ellington, Eduardo D Sontag, Christopher A Voigt

"Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co‐expressed to function. The DNA‐binding loop is encoded in a C‐terminal 285‐aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601‐aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67‐aa N‐terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids."

 http://bit.ly/1scT9la

by
Yi Liu , Brian R. Fritz , Mark J. Anderson , Jennifer A. Schoborg , and Michael C. Jewett

"Complete cell-free synthesis of ribosomes could make possible minimal cell projects and the construction of variant ribosomes with new functions. Recently, we reported the development of an integrated synthesis, assembly, and translation (iSAT) method for in vitro construction of Escherichia coli ribosomes. iSAT allows simultaneous ribosomal RNA synthesis, ribosome assembly, and reporter protein expression as a measure of ribosome activity. Here we explore causes of iSAT reaction termination to improve efficiency and yields. We discovered that phosphoenolpyruvate (PEP), the secondary energy substrate, and nucleoside triphosphates (NTPs) were rapidly degraded during iSAT reactions. In turn, we observed a significant drop in the adenylate energy charge and termination of protein synthesis. Further, we identified that the accumulation of inorganic phosphate is inhibitory to iSAT. Fed-batch replenishment of PEP and magnesium glutamate (to offset the inhibitory effects of accumulating phosphate by repeated additions of PEP) prior to energy depletion prolonged the reaction duration 2-fold and increased yields by 75%. By adopting a semi-continuous method, where passive diffusion enables substrate replenishment and byproduct removal, we prolonged iSAT reaction duration 5-fold and increased superfolder green fluorescent protein yield 7-fold to 7.5 ± 0.7 μmol L-1. This protein yield is the highest ever reported for iSAT reactions. Our results underscore the critical role energy substrates play in iSAT and highlight the importance of understanding metabolic processes that influence substrate depletion for cell-free synthetic biology."

 http://bit.ly/1qQh9fF

by
Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepez J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, Golden JW

"Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains."


 http://bit.ly/1u4px8Q

by
Joseph P Torella, Florian Lienert, Christian R Boehm, Jan-Hung Chen,  Jeffrey C Way & Pamela A Silver

"Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies—for example, repeated terminator and insulator sequences—that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)–guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2–3 d. If the DNA parts must be generated from scratch, an additional 2–5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques."

 http://bit.ly/1u6RzUh

http://bit.ly/1sBBp35

 http://bit.ly/1sB6Iu4

by
Savas Konur , Marian Gheorghe , Ciprian Dragomir , Laurentiu Mierla , Florentin Ipate , and Natalio Krasnogor

"Computational models are perceived as an attractive alternative to mathematical models, e.g. ordinary differential equations. These models incorporate a set of methods for specifying, modelling, testing and simulating biological systems. In addition, they can be analysed using algorithmic techniques, e.g. formal verification. This paper shows how formal verification is utilised in systems and synthetic biology through qualitative vs quantitative analysis. Here, we choose two well known case studies: quorum sensing in P. aeruginosas and pulse generator. The paper reports verification analysis of two systems carried out using some model checking tools, integrated to the Infobiotics Workbench platform, where system models are based on stochastic P systems."

 http://bit.ly/1lAQETO

by
David L. Shis, Faiza Hussain, Sarah Meinhardt, Liskin Swint-Kruse, and Matthew R. Bennett 

"In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits."

 http://bit.ly/XygrXx

by
David L Shis, Matthew R Bennett

"Split T7 RNA polymerase provides new avenues for creating synthetic gene circuits that are decoupled from host regulatory processes—but how many times can this enzyme be split, yet retain function? New research by Voigt and colleagues (Segall‐Shapiro et al, 2014) indicates that it may be more than you think."

http://bit.ly/XpHowm

by
Andrew Adamatzky and Theresa Schubert

"We demonstrate how logical operations can be implemented in ensembles of protoplasmic tubes of acellular slime mold Physarum polycephalum. The tactile response of the protoplasmic tubes is used to actuate analogs of two- and four-input logical gates and memory devices. The slime mold tube logical gates display results of logical operations by blocking flow in mechanically stimulated tube fragments and redirecting the flow to output tube fragments. We demonstrate how XOR and NOR gates are constructed. We also exemplify circuits of hybrid gates and a memory device. The slime mold based gates are non-electronic, simple and inexpensive, and several gates can be realized simultaneously at sites where protoplasmic tubes merge."


 http://bit.ly/Pfzxxt