More Like this

1. Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget

   https://doi.org/10.1093/nar/gkaa734  

   Davey, James A ; Wilson, Corey J ( September 2020 , Nucleic Acids Research)

   Abstract Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden – as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength. 

   more » « less
2. Maize ANT1 modulates vascular development, chloroplast development, photosynthesis, and plant growth

   https://doi.org/10.1073/pnas.2012245117  

   Liu, Wen-Yu ; Lin, Hsin-Hung ; Yu, Chun-Ping ; Chang, Chao-Kang ; Chen, Hsiang-June ; Lin, Jinn-Jy ; Lu, Mei-Yeh Jade ; Tu, Shih-Long ; Shiu, Shin-Han ; Wu, Shu-Hsing ; et al ( September 2020 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1 , GNC , and AN3 , which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis. 

   more » « less
3. Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites

   https://doi.org/10.1002/cpz1.705  

   Murray, Andrew ; Vollmers, Christopher ; Schmitz, Robert J. ( March 2023 , Current Protocols)

   Promoters and the noncoding sequences that drive their function are fundamental aspects of genes that are critical to their regulation. The transcription preinitiation complex binds and assembles on promoters where it facilitates transcription. The transcription start site (TSS) is located downstream of the promoter sequence and is defined as the location in the genome where polymerase begins transcribing DNA into RNA. Knowing the location of TSSs is useful for annotation of genes, identification of non‐coding sequences important to gene regulation, detection of alternative TSSs, and understanding of 5′ UTR content. Several existing techniques make it possible to accurately identify TSSs, but are often difficult to perform experimentally, require large amounts of input RNA, or are unable to identify a large number of TSSs from a single sample. Many of these protocols take advantage of template switching reverse transcriptases (TSRTs), which reliably place an adaptor at the 5′ end of a first strand synthesis of cDNA. Here, we introduce a protocol that exploits TSRT activity combined with rolling circle amplification to identify TSSs with several unique advantages over existing methods. Sequence adaptors are placed on the 5′ and 3′ end of the full‐length cDNA copy of a transcript. A splint compatible with those adaptors is then used to circularize the full‐length cDNA. Linear DNA containing concatemers of the cDNA are generated using rolling circle amplification, and a sequencing library is formed by fragmenting the concatemers. This protocol is straightforward to execute, requiring limited bench time with relatively stable reagents. Using extremely low amounts of RNA input, this protocol produces large numbers of accurate, deduplicated TSSs genome wide. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

   Basic Protocol 1: Splint generation

   Basic Protocol 2: RNA extraction

   Basic Protocol 3: cDNA synthesis

   Basic Protocol 4: cDNA circularization and amplification

   Basic Protocol 5: Library generation

    

   more » « less
4. Lithium niobate photonic-crystal electro-optic modulator

   https://doi.org/10.1038/s41467-020-17950-7  

   Li, Mingxiao ; Ling, Jingwei ; He, Yang ; Javid, Usman A. ; Xue, Shixin ; Lin, Qiang ( August 2020 , Nature Communications)

   Modern advanced photonic integrated circuits require dense integration of high-speed electro-optic functional elements on a compact chip that consumes only moderate power. Energy efficiency, operation speed, and device dimension are thus crucial metrics underlying almost all current developments of photonic signal processing units. Recently, thin-film lithium niobate (LN) emerges as a promising platform for photonic integrated circuits. Here, we make an important step towards miniaturizing functional components on this platform, reporting high-speed LN electro-optic modulators, based upon photonic crystal nanobeam resonators. The devices exhibit a significant tuning efficiency up to 1.98 GHz V⁻¹, a broad modulation bandwidth of 17.5 GHz, while with a tiny electro-optic modal volume of only 0.58μm³. The modulators enable efficient electro-optic driving of high-Q photonic cavity modes in both adiabatic and non-adiabatic regimes, and allow us to achieve electro-optic switching at 11 Gb s⁻¹with a bit-switching energy as low as 22 fJ. The demonstration of energy efficient and high-speed electro-optic modulation at the wavelength scale paves a crucial foundation for realizing large-scale LN photonic integrated circuits that are of immense importance for broad applications in data communication, microwave photonics, and quantum photonics.

    

   more » « less
5. Genome expansion by a CRISPR trimmer-integrase

   https://doi.org/10.1038/s41586-023-06178-2  

   Wang, Joy Y. ; Tuck, Owen T. ; Skopintsev, Petr ; Soczek, Katarzyna M. ; Li, Gary ; Al-Shayeb, Basem ; Zhou, Julia ; Doudna, Jennifer A. ( June 2023 , Nature)

   CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity¹. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient^2–5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation^6,7, but many CRISPR–Cas systems lack Cas4⁸. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited^9,10exonucleases for faithful acquisition of new CRISPR immune sequences.

    

   more » « less