λ-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5′-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required.

ncRNA plays an important role in regulating gene expression by various mechanisms.Many approaches,such as bioinformatics,genomic SELEX technology,and microarray analysis,were applied to study ncRNA,resulting in the discovery of a large number of new ncRNAs in recent five years.Here we briefly summarize these methods for identifying and validating ncRNA.

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3′-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)6-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

With the growing concern regarding worsening environment, microbial cell factories have been built for the biorefinery of many products including chemicals, biofuel, nutraceuticals, and pharmaceuticals from renewable biomass resources. In this chapter, we introduced the mainly strategies for the design, construction, and optimization of metabolic pathway. In addition, the applications of modular metabolic engineering and dynamic regulation for the further optimization and regulation of metabolic network were discussed. Furthermore, we also highlighted the role of system biology such as omics technology, genomics-based metabolic model, and genome-scale engineering tools. Finally, future perspectives on the design and construction of microbial cell factories were provided.

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria.

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates.

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.

We have developed an improved and rapid genomic engineering procedure for the construction of custom-designed microorganisms. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via markerless deletion of selected genomic regions. The deletion process was mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (i) an arabinose-inducible promoter that drives expression of λ-Red recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (ii) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which stimulates deletion of the introduced marker by double-strand breakage-mediated intramolecular recombination. This genomic deletion was performed successively with only one plasmid, pREDI, simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette were nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.

We have developed an easy, reliable two-step method for the insertion of large DNA fragments into any desired location in the E. coli chromosome. The method is based on the recombineering of a small (~1.3 kbp) “Landing Pad” into the chromosome at the insertion site, to which the large construct is subsequently delivered via I-SceI endonuclease excision from a donor plasmid. To demonstrate the power of this method, we here show the insertion of a fragment containing the entire lac operon (~9 kbp) into four predefined novel locations in the E. coli chromosome, a feat not possible with existing technologies. In addition, the chromosomal breaks induced by landing pad excision provide sufficient selective pressure that positive selection by antibiotics is unnecessary, making precise, exact insertion without extraneous sequence possible.

We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.

We have constructed a set of plasmids that can be used to express recombineering functions in some gram-negative bacteria, thereby facilitating in vivo genetic manipulations. These plasmids include an origin of replication and a segment of the bacteriophage lambda genome comprising the red genes (exo, bet and gam) under their native control. These constructs do not require the anti-termination event normally required for Red expression, making their application more likely in divergent species. Some of the plasmids have temperature-sensitive replicons to simplify curing. In creating these vectors we developed two useful recombineering applications. Any gene linked to a drug marker can be retrieved by gap-repair using only a plasmid origin and target homologies. A plasmid origin of replication can be changed to a different origin by targeted replacement, to potentially alter its copy number and host range. Both these techniques will prove useful for manipulation of plasmids in vivo. Most of the Red plasmid constructs catalyzed efficient recombination in E. coli with a low level of uninduced background recombination. These Red plasmids have been successfully tested in Salmonella, and we anticipate that that they will provide efficient recombination in other related gram-negative bacteria.

We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of beta-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (10(10)) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range.

We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

We demonstrate that the bacteriophage λ Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage λ to create specific changes in the viral genome. Point mutations, deletions, and gene replacements have been created. While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change. DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis.

Voluntary control of movement relies on activity in a subclass of layer 5B pyramidal neurons projecting to the spinal cord—corticospinal neurons (also called Betz cells or upper motor neurons) (Figure 1). In motor cortex, corticospinal neurons are one of the many classes of pyramidal neurons distributed over multiple layers and sublayers with diverse local and long-range projections. Studies in primates, the motor systems of which so closely resemble our own, have provided a wealth of information about the spiking activities of motor cortex neurons during action planning and execution. However, in vivo recording methods usually preclude identification of the precise laminar locations of neurons and their synaptic connectivity, and in vitro analysis is impractical in primates. Consequently, much remains to be learned about how local circuits are organized according to projection target and sublayer (Brown and Hestrin, 2009; Anderson et al, 2010), and how this organization influences spiking activity in different subclasses of projection neurons in motor cortex. Figure 1 Corticospinal neurons in layer 5B of mouse motor cortex. Neurons were retrogradely labeled in vivo by injecting red fluorescent microspheres into the cervical spinal cord. Brain slices containing motor cortex were prepared, and a single corticospinal ... Recently, several groups have established the feasibility of motor behavior experiments with rodents, using not only electrophysiology, but with calcium imaging, also large-scale optical recordings (Dombeck et al, 2009). It is remarkable that this has now been combined with sophisticated within-session learning paradigms (Komiyama et al, 2010). There are, of course, limitations. One of the limitations is depth: only neurons in the upper layers of the cortex are easily imaged. However, this is hardly uninteresting, as microcircuit mapping studies have shown that layer 2/3 is the main source of synaptic output within motor cortex circuits, and is the major source of input to corticospinal neurons (Anderson et al, 2010). Another intriguing issue is whether circuits in the remaining frontal agranular cortex—presumably involved in more ‘cognitive' aspects of motor control—are organized in the form of primary motor cortex. If “thinking is the evolutionary internalization of movement” (Llinas, 2002), then one might expect so. In this view the cortical extent of the motor system extends beyond primary motor areas. Behavior, after all, is essentially synonymous with the activity of the motor system. Indeed, a compelling case can be made for the viewpoint that ‘layer 5 is motor everywhere' (Diamond, 1979); in other words, layer 5B is ‘motor cortex'. Should we regard layer 2/3 as ‘premotor cortex'? We expect that as research on motor/frontal cortex goes forward, a general framework for understanding behavioral ‘control' mechanisms at the level of cortical circuits will emerge, which will link multiple levels of neural organization and have useful roles both in assimilating the rapidly accruing new information and in inspiring testable hypotheses. New opportunities for the neuropsychopharmacology of movement disorders are also likely to arise. For example, many cognitive disorders have a motor component, and vice versa; apraxias are classic examples. Also, the expression patterns of ion channel and intracellular signaling molecules is highly diverse in layer 5B (Allen Brain Atlas). This diversity presents a fertile substrate for exploring pharmacological strategies to selectively target pathological mechanisms in specific subclasses of cortical projection neurons involved in different aspects of voluntary movement.

Understanding how diversity emerges in a single niche is not fully understood. Rugged fitness landscapes and epistasis between beneficial mutations could explain coexistence among emerging lineages. To provide an experimental test of this notion, we investigated epistasis among four pleiotropic mutations in rpoS, mglD, malT, and hfq present in two coexisting lineages that repeatedly fixed in experimental populations of Escherichia coli. The mutations were transferred into the ancestral background individually or in combination of double or triple alleles. The combined competitive fitness of two or three beneficial mutations from the same lineage was consistently lower than the sum of the competitive fitness of single mutants—a clear indication of negative epistasis within lineages. We also found sign epistasis (i.e., the combined fitness of two beneficial mutations lower than the ancestor), not only from two different lineages (i.e., hfq and rpoS) but also from the same lineage (i.e., mglD and malT). The sign epistasis between loci of different lineages indeed indicated a rugged fitness landscape, providing an epistatic explanation for the coexistence of distinct rpoS and hfq lineages in evolving populations. The negative and sign epistasis between beneficial mutations within the same lineage can further explain the order of mutation acquisition.

Transcriptional biosensors emerged as powerful tools for protein and strain engineering as they link inconspicuous production phenotypes to easily measurable output signals such as fluorescence. When combined with fluorescence-activated cell sorting, transcriptional biosensors enable high throughput screening of vast mutant libraries. Interestingly, even though many published manuscripts describe the construction and characterization of transcriptional biosensors, only very few studies report the successful application of transcriptional biosensors in such high-throughput screening campaigns. Here, we describe construction and characterization of the trans-cinnamic acid responsive transcriptional biosensor pSenCA for Escherichia coli and its application in a FACS based screen. In this context, we focus on essential methodological challenges during the development of such biosensor-guided high-throughput screens such as biosensor cross-talk between producing and nonproducing cells, which could be minimized by optimization of expression and cultivation conditions. The optimized conditions were applied in a five-step FACS campaign and proved suitable to isolate phenylalanine ammonia lyase variants with improved activity in E. coli and in vitro. Findings from this study will help researchers who want to profit from the unmatched throughput of fluorescence-activated cell sorting by using transcriptional biosensors for their enzyme and strain engineering campaigns.

Together with metabolites, proteins and RNAs form complex biological systems through highly intricate networks of physical and functional interactions. Large-scale studies aimed at a molecular understanding of the structure, function, and dynamics of proteins and RNAs in the context of cellular networks require novel approaches and technologies. This Special Issue of Genome Research features strategies for the high-throughput construction and manipulation of complete sets of protein-encoding open reading frames (ORFeome), gene promoters (promoterome), and noncoding RNAs, as predicted from genome and transcriptome sequences. Here we discuss the use of a recombinational cloning system that allows efficiency, adaptability, and compatibility in the generation of ORFeome, promoterome, and other resources.

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5′ of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5′ of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS‐induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read‐through transcription into a cryptic prophage encoding kilR. The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS‐mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.