SynBioFromLeukipposInstitute

The methods for establishing synthetic lifeforms with rewritten genetic codes comprising non-canonical amino acids (NCAA) in addition to canonical amino acids (CAA) include proteome-wide replacement of CAA, insertion through suppression of nonsense codon, and insertion via the pyrrolysine and selenocysteine pathways. Proteome-wide reassignments of nonsense codons and sense codons are also under development. These methods enable the application of NCAAs to enrich both fundamental and applied aspects of protein chemistry and biology. Sense codon reassignment to NCAA could incur problems arising from the usage of anticodons as identity elements on tRNA, and possible misreading of NNY codons by UNN anticodons. Evidence suggests that the problem of anticodons as identity elements can be diminished or resolved through removal from the tRNA of all identity elements besides the anticodon, and the problem of misreading of NNY codons by UNN anticodon can be resolved by the retirement of both the UNN anticodon and its complementary NNA codon from the proteome in the event that a restrictive post-transcriptional modification of the UNN anticodon by host enzymes to prevent the misreading cannot be obtained.

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the CRISPR-Cas9 system. First, the functionality of the CRISPR-Cas9 system previously customized for C. beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting on pta and buk, two essential genes for acetate and butyrate production, respectively. The pta, buk single deletion, and the pta and buk double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is < 20%). Therefore, the efficiency was further optimized by evaluating various promoters for the gRNA expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways, however neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield and selectivity were improved in mutants dependent on the fermentation medium. In the pta-buk double deletion mutant, the butanol production reached 19.0 g/l in P2 medium, which is one of the highest among the ever reported from batch fermentations.IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in other close relative microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the metabolic engineering and synthetic biology area. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.

De novo DNA synthesis is one of the key enabling technologies for synthetic biology. Methods for large-scale DNA synthesis, in particular, have transformed many facets of life science research, supporting new discoveries in biology through the design of novel synthetic biological systems. This protocol describes in detail the methods currently being used to synthesize and assemble large pieces of DNA for the synthetic yeast genome project. The protocol includes instructions for building block synthesis as well as chunk assembly, each of which can be used as a stand-alone procedure to generate a synthetic DNA of interest.

Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, L-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology.

Synthetic biology is an emerging science that aims to create new biological functions that do not exist in nature, based on the knowledge acquired in life science over the last century. Since the beginning of this century, several projects in synthetic biology have emerged. The complexity of the developed artificial bio-functions is relatively low so that empirical design methods could be used for the design process. Nevertheless, with the increasing complexity of biological circuits, this is no longer the case and a large number of computer aided design softwares have been developed in the past few years. These tools include languages for the behavioral description and the mathematical modelling of biological systems, simulators at different levels of abstraction, libraries of biological devices and circuit design automation algorithms. All of these tools already exist in other fields of engineering sciences, particularly in microelectronics. This is the approach that is put forward in this paper.

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection.
METHODS:
Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays.
RESULTS:
The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate.
CONCLUSIONS:
Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells. It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system. This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels, including protein engineering, metabolic engineering, and artificial cell engineering. In cell-free protein engineering, the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins, toxic proteins, membrane proteins, and novel proteins with unnatural amino acids. Cell-free systems offer the ability to design metabolic pathways towards the production of desired products. Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.

Genetic circuits have the potential to improve a broad range of metabolic engineering processes and address a variety of medical and environmental challenges. However, in order to engineer genetic circuits that can meet the needs of these real-world applications, genetic sensors that respond to relevant extrinsic and intrinsic signals must be implemented in complex genetic circuits. In this work, we construct the first AND and NAND gates that respond to temperature and pH, two signals that have relevance in a variety of real-world applications. A previously identified pH-responsive promoter and a temperature-responsive promoter were extracted from the E. coli genome, characterized, and modified to suit the needs of the genetic circuits. These promoters were combined with components of the type III secretion system in Salmonella typhimurium and used to construct a set of AND gates with up to 23-fold change. Next, an antisense RNA was integrated into the circuit architecture to invert the logic of the AND gate and generate a set of NAND gates with up to 1,168-fold change. These circuits provide the first demonstration of complex pH- and temperature-responsive genetic circuits, and lay the groundwork for the use of similar circuits in real-world applications.

Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist.

Synthetic biology is revolutionising the biotech industry and is increasingly applied in previously unthought-of markets. Here, we discuss the importance of this industry to the bioeconomy and two of its key factors: the synthetic biology approach to research and development (R&D), and the unique nature of the carefully designed, stakeholder-inclusive, community-directed evolution of the field.

Design automation refers to a category of software tools for designing systems that work together in a workflow for designing, building, testing, and analyzing systems with a target behavior. In synthetic biology, these tools are called bio-design automation (BDA) tools. In this review, we discuss the BDA tools areas—specify, design, build, test, and learn—and introduce the existing software tools designed to solve problems in these areas. We then detail the functionality of some of these tools and show how they can be used together to create the desired behavior of two types of modern synthetic genetic regulatory networks.

The rapid advances in synthetic biology and biotechnology are increasingly demanding high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase. We present a high-throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limitations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets where fluorescence signals are enhanced by 100 times within 24 h of proliferation. Enrichment of functionally-correct genes by employing an error correction method is demonstrated by screening DE droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene. Permeation of isopropyl β-d-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins from at least ∼100 bacteria per droplet generates detectable fluorescence signals to enable fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and labor-intensive cell culture typically required in conventional bulk experiment.