SynBioFromLeukipposInstitute

by

Robert Penchovsky

"Here we describe molecular implementations of integrated digital circuits, including a three-input AND logic gate, a two-input multiplexer, and 1-to-2 decoder using allosteric ribozymes. Furthermore, we demonstrate a multiplexer–decoder circuit. The ribozymes are designed to seek-and-destroy specific RNAs with a certain length by a fully computerized procedure. The algorithm can accurately predict one base substitution that alters the ribozyme’s logic function. The ability to sense the length of RNA molecules enables single ribozymes to be used as platforms for multiple interactions. These ribozymes can work as integrated circuits with the functionality of up to five logic gates. The ribozyme design is universal since the allosteric and substrate domains can be altered to sense different RNAs. In addition, the ribozymes can specifically cleave RNA molecules with triplet-repeat expansions observed in genetic disorders such as oculopharyngeal muscular dystrophy. Therefore, the designer ribozymes can be employed for scaling up computing and diagnostic networks in the fields of molecular computing and diagnostics and RNA synthetic biology."
http://bit.ly/Noz6uN

by

Chen Z, Jing C, Gallagher SS, Sheetz MP, Cornish VW.

"Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology."
http://1.usa.gov/OYkJj7

by

Hughes RA, Miklos AE, Ellington AD.

"As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment..."

http://1.usa.gov/MD7tvy

by

Cheemeng Tan , Shih-Jie Lo , Philip LeDuc and Chao-Min Cheng

"The development of optofluidic-based technology has ushered in a new era of lab-on-a-chip functionality, including miniaturization of biomedical devices, enhanced sensitivity for molecular detection, and multiplexing of optical measurements. While having great potential, optofluidic devices have only begun to be exploited in many biotechnological applications. Here, we highlight the potential of integrating optofluidic devices with synthetic biological systems, which is a field focusing on creating novel cellular systems by engineering synthetic gene and protein networks. First, we review the development of synthetic biology at different length scales, ranging from single-molecule, single-cell, to cellular population. We emphasize light-sensitive synthetic biological systems that would be relevant for the integration with optofluidic devices. Next, we propose several areas for potential applications of optofluidics in synthetic biology. Finally, we discuss potential applications of optofluidic devices in the diagnosis of infectious diseases."

http://bit.ly/MxAOYu

by

Todd Peterson

"With the completion of a myriad genome sequencing projects, the emergence of metagenomics and significant progress in understanding and building living systems, synthetic biology engineering has expanded into many applications including the rational optimization of complex pathways, construction and characterization of genetic circuits of increasing complexity and the functional re-design of existing biological systems. Design, construction and validation of genetic circuit, pathway and genome engineering workflows are powered by a comprehensive toolbox that includes intelligent metabolic and genetic design software, sequence verified, scaled, cost-effective gene synthesis and precise, genetic assembly and editing technologies able to address genetic constructs of various size ranges.
We present a growing set of integrated technologies that provide state-of-the-art rational genetic design and simulation, gene synthesis and seamless, simultaneous, flexible, and highly efficient assembly, editing and transfer of genetic elements designed for a wide size range (10s to 100,000s base pairs). The paradigm of next generation deep sequencing to build a foundational understanding at the genomic and transcriptome levels enabling initial metabolic models to facilitate directed engineering will be presented. Scaled, cost-effective gene synthesis methods, formats and process considerations will be discussed. Tools for vector construction, higher order HTP assembly and precision editing of genetic elements in vitro and in vivo will be described. Desk-top and web-based assembly/design software tool and resources to design and manipulate large DNA constructs in silico will be presented in the context of research and applied technologies to enhance the development of synthetic biology engineering capabilities."
http://bit.ly/P964ku

by

Miklos AE, Hughes RA, Ellington AD.

"The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing "in-house" gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides"
http://1.usa.gov/QiZ5Cv

 #igemUKmeetup released! Download PDF here: http://goo.gl/BRXEA @iGEM #SyntheticBiology

http://bit.ly/NowgEs