Improved modular multipart DNA assembly, development of a DNA part toolkit for E. coli, and applications in traditional biology and bioelectronic systems

DNA assembly and rational design are cornerstones of synthetic biology. While many DNA assembly standards have been published in recent years, only the Modular Cloning standard, or MoClo, has the advantage of publicly available part libraries for use in plant, yeast, and mammalian systems. No multipart modular library has previously been developed for use in prokaryotes. Building upon the existing MoClo assembly framework, we developed a collection of DNA parts and optimized MoClo protocols for use in E. coli. We present this assembly standard and library along with part characterization, design strategies, potential applications, and troubleshooting. Developed as part of the Cross-disciplinary Integration of Design Automation Research (CIDAR) lab collection of tools, the CIDAR MoClo Library is publicly available and contains promoters, ribosomal binding sites, coding sequences, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols. The CIDAR MoClo Library is the first bacterial DNA part library compatible with a multipart assembly standard. To demonstrate the utility of the CIDAR MoClo system in a traditional biology context, we used the library and previous expression data to create a series of dual expression plasmids. In this manner, we produced a dual expression plasmid capable of expressing equimolar amounts of two variants of rabbit aldolase, a His-tagged wildtype protein and a single-amino-acid substitution mutant deficient in binding actin. This expression plasmid will enable the production of dimer-of-dimer heterotetramers needed for structural determination of the actin-aldolase interaction by electron microscopy. To employ CIDAR MoClo in a synthetic biology context, we produced a bioelectronic pH-mediated genetic logic gate with DNA circuits built using MoClo and integrated with Raspberry Pi computers, Twitter, and 3D printed components. Logic gates are an increasingly common biological tool with applications in cellular memory and biological computation. MoClo facilitates rapid iteration of genetic designs, better enabling the development of cellular logic. The CIDAR MoClo Library and assembly standard enable rapid design-build-test cycles in E. coli making this system advantageous for use in many areas of synthetic biology as well as traditional biological research

BBF RFC 94: Type IIS Assembly for Bacterial Transcriptional Units: A Standardized Assembly Method for Building Bacterial Transcriptional Units Using the Type IIS Restriction Enzymes BsaI and BbsI

This RFC94 describes an assembly standard based on the Type IIS restriction enzymes BsaI and BbsI (also called BpiI). This assembly standard is based upon the Modular Cloning (MoClo) assembly strategy, which was introduced in 2011 by Weber et al. [1] and is based upon Golden Gate cloning [2]. In this RFC, we describe our proposed MoClo standard for generating a library of bacterial DNA parts for generating four-part transcriptional units (promoter : 5’UTR : CDS : 3’UTR). In this work, we define 5’UTRs as including ribosomal binding sites (RBS) and bi-cistronic design elements (BCDs) [3], and 3’UTRs as transcriptional terminators. The 2012-2014 BostonU iGEM teams completed this work and a more compact library has also been created based on this work [4]

Contributions of New Hepatocyte Lineages to Liver Growth, Maintenance, and Regeneration in Mice

The contributions that de novo differentiation of new hepatocyte lineages makes to normal liver physiology are unknown. Here a system that uniquely marks cells during a finite period following primary activation of a serum albumin gene promoter/enhancer-driven Cre transgene ( albCre ) was used to investigate birthrates of new hepatocyte lineages from Alb-naïve precursors in mice. Elapsed time was measured using a two-color fluorescent marker-gene that converts from expressing tdTomato (tdT, red-fluorescent) to expressing GFP (green-fluorescent) upon exposure to Cre. Accumulation of GFP and decay of tdT each contributed to a regular fluorescence transition, which was calibrated in vivo . In normal adults, this system revealed that a steady-state level of 0.076% hepatocytes had differentiated within the previous four days from cell lineages that had never previously expressed albCre . As compared to resting adult livers, the relative abundance of these newborn hepatocytes was elevated 3.7-fold in normal growing livers of juveniles and 8.6-fold during liver regeneration following partial hepatectomy in normal adults

CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology

Multipart and modular DNA part libraries and assembly standards have become common tools in synthetic biology since the publication of the Gibson and Golden Gate assembly methods, yet no multipart modular library exists for use in bacterial systems. Building upon the existing MoClo assembly framework, we have developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design, and expression tuning in Escherichia coli. The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library is openly available and contains promoters, ribosomal binding sites, coding sequence, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from that of previously published protocols

Hepatocyte DNA replication in growing liver requires either glutathione or a single allele of txnrd1

Ribonucleotide reductase (RNR) activity requires an electron donor, which in bacteria, yeast, and plants is usually either reduced thioredoxin (Trx) or reduced glutaredoxin. Mice lacking glutathione reductase are viable and, although mice lacking thioredoxin reductase 1 (TrxR1) are embryonic-lethal, several studies have shown that mouse cells lacking the txnrd1 gene, encoding TrxR1, can proliferate normally. To better understand the in vivo electron donor requirements for mammalian RNR, we here investigated whether replication of TrxR1-deficient hepatocytes in mouse livers either employed an alternative source of Trx-reducing activity or, instead, solely relied upon the glutathione (GSH) pathway. Neither normal nor genetically TrxR1-deficient livers expressed substantial levels of mRNA splice forms encoding cytosolic variants of TrxR2, and the TrxR1-deficient livers showed severely diminished total TrxR activity, making it unlikely that any alternative TrxR enzyme activities complemented the genetic TrxR1 deficiency. To test whether the GSH pathway was required for replication, GSH levels were depleted by administration of buthionine sulfoximine (BSO) to juvenile mice. In controls not receiving BSO, replicative indexes were similar in hepatocytes having two, one, or no functional alleles of txnrd1. After BSO treatment, hepatocytes containing either two or one copies of this gene were also normal. However, hepatocytes completely lacking a functional txnrd1 gene exhibited severely reduced replicative indexes after GSH depletion. We conclude that hepatocyte proliferation in vivo requires either GSH or at least one functional allele of txnrd1, demonstrating that either the GSH- or the TrxR1-dependent redox pathway can independently support hepatocyte proliferation during liver growth. ► Mouse hepatocytes genetically lacking TrxR1 exhibit normal proliferation rates. ► Cytosolic variants of TrxR2 do not functionally replace TrxR1 in txnrd1-null livers. ► Wild-type hepatocytes lacking GSH proliferate normally. ► Hepatocytes lacking both TrxR1 and GSH exhibit severely reduced replication. ► S-phase DNA replication in hepatocytes requires either TrxR1 or GSH

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however, their constitutive metabolic state supported more robust GSH biosynthesis, glutathionylation, and glucuronidation systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage. [Display omitted

Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome

Thioredoxin reductases (Txnrd) maintain intracellular redox homeostasis in most organisms. Metazoan Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1 −/− cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated that primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd −/− and txnrd +/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however, mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells

Interactome for Auxiliary Splicing Factor U2AF65 Suggests Diverse Roles

U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is an essential component of the splicing machinery that is composed of two protein subunits, the 35 kD U2AF 35 (U2AF1) and the 65 kD U2AF 65 (U2AF2). U2AF interacts with various splicing factors within this machinery. Here we expand the list of mammalian splicing factors that are known to interact with U2AF 65 as well as the list of nuclear proteins not known to participate in splicing that interact with U2AF 65 . Using a yeast two-hybrid system, we found fourteen U2AF 65 -interacting proteins. The validity of the screen was confirmed by identification of five known U2AF 65 -interacting proteins, including its heterodimeric partner, U2AF 35 . In addition to binding these known partners, we found previously unrecognized U2AF 65 interactions with four splicing related proteins (DDX39, SFRS3, SFRS18, SNRPA), two zinc finger proteins (ZFP809 and ZC3H11A), a U2AF 65 homolog (RBM39), and two other regulatory proteins (DAXX and SERBP1). We report which regions of U2AF 65 each of these proteins interacts with and we and discuss their potential roles in regulation of pre-mRNA splicing, 3’-end mRNA processing, and U2AF 65 sub-nuclear localization. These findings suggest expanded roles for U2AF 65 in both splicing and non-splicing functions