Genome-scale metabolic modeling reveals key features of a minimal gene set.

Mesoplasma florum, a fast-growing near-minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing ˜ 30% of its protein-coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species-specific constraints to produce the functional iJL208 genome-scale model (GEM) of metabolism. Genome-wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine iJL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. iJL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0 revealed key features of a minimal gene set. iJL208 is a stepping-stone toward model-driven whole-genome engineering.

Integrative characterization of the near-minimal bacterium Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.

Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5'-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

Development of oriC-Based Plasmids for Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10-6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florumoriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10-6 and 8.44 × 10-7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

Complete Genome Sequence of the Mesoplasma florum W37 Strain.

Mesoplasma florum is a small-genome fast-growing mollicute that is an attractive model for systems and synthetic genomics studies. We report the complete 825,824-bp genome sequence of a second representative of this species, M. florum strain W37, which contains 733 predicted open reading frames and 35 stable RNAs.

Fast and easy protocol for the purification of recombinant S-layer protein for synthetic biology applications.

A goal of synthetic biology is to make biological systems easier to engineer. One of the aims is to design, with nanometer-scale precision, biomaterials with well-defined properties. The surface-layer protein SbpA forms 2D arrays naturally on the cell surface of Lysinibacillus sphaericus, but also as the purified protein in solution upon the addition of divalent cations. The high propensity of SbpA to form crystalline arrays, which can be simply controlled by divalent cations, and the possibility to genetically alter the protein, make SbpA an attractive molecule for synthetic biology. To be a useful tool, however, it is important that a simple protocol can be used to produce recombinant wild-type and modified SbpA in large quantities and in a biologically active form. The present study addresses this requirement by introducing a mild and non-denaturing purification protocol to produce milligram quantities of recombinant, active SbpA.

Assembly of BioBrick standard biological parts using three antibiotic assembly.

An underlying goal of synthetic biology is to make the process of engineering biological systems easier and more reliable. In support of this goal, we developed BioBrick assembly standard 10 to enable the construction of systems from standardized genetic parts. The BioBrick standard underpins the distributed efforts by the synthetic biology research community to develop a collection of more than 6000 standard genetic parts available from the Registry of Standard Biological Parts. Here, we describe the three antibiotic assembly method for physical composition of BioBrick parts and provide step-by-step protocols. The method relies on a combination of positive and negative selection to eliminate time- and labor-intensive steps such as column cleanup and agarose gel purification of DNA during part assembly.

Introduction of customized inserts for s-treamlined assembly and optimization of BioBrick synthetic genetic circuits.

BACKGROUND: BioBrick standard biological parts are designed to make biological systems easier to engineer (e.g. assemble, manipulate, and modify). There are over 5,000 parts available in the Registry of Standard Biological Parts that can be easily assembled into genetic circuits using a standard assembly technique. The standardization of the assembly technique has allowed for wide distribution to a large number of users -- the parts are reusable and interchangeable during the assembly process. The standard assembly process, however, has some limitations. In particular it does not allow for modification of already assembled biological circuits, addition of protein tags to pre-existing BioBrick parts, or addition of non-BioBrick parts to assemblies. RESULTS: In this paper we describe a simple technique for rapid generation of synthetic biological circuits using introduction of customized inserts. We demonstrate its use in Escherichia coli (E. coli) to express green fluorescent protein (GFP) at pre-calculated relative levels and to add an N-terminal tag to GFP. The technique uses a new BioBrick part (called a BioScaffold) that can be inserted into cloning vectors and excised from them to leave a gap into which other DNA elements can be placed. The removal of the BioScaffold is performed by a Type IIB restriction enzyme (REase) that recognizes the BioScaffold but cuts into the surrounding sequences; therefore, the placement and removal of the BioScaffold allows the creation of seamless connections between arbitrary DNA sequences in cloning vectors. The BioScaffold contains a built-in red fluorescent protein (RFP) reporter; successful insertion of the BioScaffold is, thus, accompanied by gain of red fluorescence and its removal is manifested by disappearance of the red fluorescence. CONCLUSIONS: The ability to perform targeted modifications of existing BioBrick circuits with BioScaffolds (1) simplifies and speeds up the iterative design-build-test process through direct reuse of existing circuits, (2) allows incorporation of sequences incompatible with BioBrick assembly into BioBrick circuits (3) removes scar sequences between standard biological parts, and (4) provides a route to adapt synthetic biology innovations to BioBrick assembly through the creation of new parts rather than new assembly standards or parts collections.

Engineering a family of synthetic splicing ribozymes.

Controlling RNA splicing opens up possibilities for the synthetic biologist. The Tetrahymena ribozyme is a model group I self-splicing ribozyme that has been shown to be useful in synthetic circuits. To create additional splicing ribozymes that can function in synthetic circuits, we generated synthetic ribozyme variants by rationally mutating the Tetrahymena ribozyme. We present an alignment visualization for the ribozyme termed as structure information diagram that is similar to a sequence logo but with alignment data mapped on to secondary structure information. Using the alignment data and known biochemical information about the Tetrahymena ribozyme, we designed synthetic ribozymes with different primary sequences without altering the secondary structure. One synthetic ribozyme with 110 nt mutated retained 12% splicing efficiency in vivo. The results indicate that our biochemical understanding of the ribozyme is accurate enough to engineer a family of active splicing ribozymes with similar secondary structure but different primary sequences.

Engineering BioBrick vectors from BioBrick parts.

BACKGROUND: The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. RESULTS: Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. CONCLUSION: We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals.

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.

Reclassification of Mesoplasma pleciae as Acholeplasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data.

Genomic DNA sequence data for the 16S rRNA gene and the gyrB gene of Mesoplasma pleciae PS-1(T) (=ATCC 49582(T)=NBRC 100476(T)) demonstrate a much closer relationship to Acholeplasma laidlawii and Acholeplasma oculi than to other species in the order Entomoplasmatales. In addition, the preferred use of UGG rather than UGA as the codon for tryptophan in the gyrB sequence probably places the organism outside the order Entomoplasmatales. It is proposed that M. pleciae be reclassified in the genus Acholeplasma, as Acholeplasma pleciae comb. nov.