Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.     

Computational discovery of soybean promoter cis‐regulatory elements for the construction of soybean cyst nematode‐inducible synthetic promoters

Computational methods offer great hope but limited accuracy in the prediction of functional cis‐regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)‐inducible motif discovery among promoters of 18 co‐expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean–SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co‐localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN‐inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN‐inducible, leading to the discovery of 23 core motifs of 5‐ to 7‐bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W‐AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high‐throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.     

New variants of inducible Cre recombinase: a novel mutant of Cre–PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility

We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val→Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3′-end of Cre. The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.     

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02–1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5′-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.     

Forward engineering of synthetic bio-logical AND gates

The field of synthetic biology has produced genetic circuits capable of emulating functional paradigms seen in digital electronic circuits. Examples are bistable switches, oscillators, and logic gates. The present work combines detailed mechanistic-kinetic models and stochastic simulation techniques as well as the techniques of in vivo molecular biology to study the potential of a synthetic, single promoter AND gate. This device is composed of elements of the tet, lac, and λ-phage promoters and is responsive to the commonly used inducers IPTG and aTc, producing GFP as an output signal. The quantitative behavior of the AND gate phenotype is studied both in numero and in vivo as a function of promoter topology. The model is constructed from kinetic data obtained from the literature and yields clearly defined ON/OFF logical behavior at realistic inducer concentrations. These behaviors are matched with observed in vivo data obtained through fluorescence-activated cell sorting. The effect of incomplete repression by weaker LacI repressor is also investigated and quantified. The simulation results, coupled with in vivo data, not only identify important design degrees of freedom, but also provide parameters that can be used to guide future synthetic designs using these common regulatory elements.     

Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences

During the origin of life, the biological information of nucleic acid polymers must have increased to encode functional molecules (the RNA world). Ribozymes tend to be compositionally unbiased, as is the vast majority of possible sequence space. However, ribonucleotides vary greatly in synthetic yield, reactivity and degradation rate, and their non-enzymatic polymerization results in compositionally biased sequences. While natural selection could lead to complex sequences, molecules with some activity are required to begin this process. Was the emergence of compositionally diverse sequences a matter of chance, or could prebiotically plausible reactions counter chemical biases to increase the probability of finding a ribozyme? Our in silico simulations using a two-letter alphabet show that template-directed ligation and high concatenation rates counter compositional bias and shift the pool toward longer sequences, permitting greater exploration of sequence space and stable folding. We verified experimentally that unbiased DNA sequences are more efficient templates for ligation, thus increasing the compositional diversity of the pool. Our work suggests that prebiotically plausible chemical mechanisms of nucleic acid polymerization and ligation could predispose toward a diverse pool of longer, potentially structured molecules. Such mechanisms could have set the stage for the appearance of functional activity very early in the emergence of life.     

Identification of Novel Inhibitors of 1-Aminocyclopropane-1-carboxylic Acid Synthase by Chemical Screening in Arabidopsis thaliana

Ethylene is a gaseous hormone important for adaptation and survival in plants. To further understand the signaling and regulatory network of ethylene, we used a phenotype-based screening strategy to identify chemical compounds interfering with the ethylene response in Arabidopsis thaliana. By screening a collection of 10,000 structurally diverse small molecules, we identified compounds suppressing the constitutive triple response phenotype in the ethylene overproducer mutant eto1-4. The compounds reduced the expression of a reporter gene responsive to ethylene and the otherwise elevated level of ethylene in eto1-4. Structure and function analysis revealed that the compounds contained a quinazolinone backbone. Further studies with genetic mutants and transgenic plants involved in the ethylene pathway showed that the compounds inhibited ethylene biosynthesis at the step of converting S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Biochemical studies with in vitro activity assay and enzyme kinetics analysis indicated that a representative compound was an uncompetitive inhibitor of ACC synthase. Finally, global gene expression profiling uncovered a significant number of genes that were co-regulated by the compounds and aminoethoxyvinylglycine, a potent inhibitor of ACC synthase. The use of chemical screening is feasible in identifying small molecules modulating the ethylene response in Arabidopsis seedlings. The discovery of such chemical compounds will be useful in ethylene research and can offer potentially useful agrochemicals for quality improvement in post-harvest agriculture.