Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain

Synthetic auxotrophs are organisms engineered to require the presence of a particular molecule for viability. They have potential applications in biocontainment and enzyme engineering. We show that these organisms can be generated by engineering ligand-dependence into essential genes. We demonstrate a method for generating a Synthetic auxotroph based on a Ligand-Dependent Essential gene (SLiDE) using 5 essential genes as test cases: <i>pheS</i>, <i>dnaN</i>, <i>tyrS</i>, <i>metG</i>, and <i>adk</i>. We show that a single SLiDE strain can have a 1 × 10⁸-fold increase in viability when chemically complemented with the ligand benzothiazole. The optimized SLiDE strain engineering protocol required less than 1 week and $100 USD. We combined multiple SLiDE strain alleles into the industrial <i>Escherichia coli</i> strain BL21­(DE3), yielding an organism that exceeds the biosafety criteria with an escape frequency below the limit of detection of 3 × 10^–11

Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay

Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies. In this paper, we demonstrate the versatility of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA) and its ability to support high-throughput data acquisition through next-generation sequencing (NGS). Utilizing methyltransferases, we highlight DLEnCA’s ability to rapidly profile an enzyme’s substrate specificity, determine relative enzyme kinetics, detect biosynthetic formation of a target molecule, and its potential to benefit from the scales and standardization afforded by NGS. This improved methodology minimizes the effort in acquiring biosynthetic knowledge by tying biochemical techniques to the rapidly evolving abilities in sequencing and synthesizing DNA

Scalable Plasmid Transfer using Engineered P1-based Phagemids

Dramatic improvements to computational, robotic, and biological tools have enabled genetic engineers to conduct increasingly sophisticated experiments. Further development of biological tools offers a route to bypass complex or expensive mechanical operations, thereby reducing the time and cost of highly parallelized experiments. Here, we engineer a system based on bacteriophage P1 to transfer DNA from one <i>E. coli</i> cell to another, bypassing the need for intermediate DNA isolation (e.g., minipreps). To initiate plasmid transfer, we refactored a native phage element into a DNA module capable of heterologously inducing phage lysis. After incorporating known <i>cis</i>-acting elements, we identified a novel <i>cis</i>-acting element that further improves transduction efficiency, exemplifying the ability of synthetic systems to offer insight into native ones. The system transfers DNAs up to 25 kilobases, the maximum assayed size, and operates well at microliter volumes, enabling manipulation of most routinely used DNAs. The system’s large DNA capacity and physical coupling of phage particles to phagemid DNA suggest applicability to biosynthetic pathway evolution, functional proteomics, and ultimately, diverse molecular biology operations including DNA fabrication

DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity

Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. We demonstrate the DLEnCA methodology using glucosyltransferases as an illustration. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected postreaction using qPCR or a similar means of DNA analysis. OleD and two glucosyltransferases from <i>Arabidopsis</i> were used to verify the assay’s generality toward glucosyltransferases. We further show DLEnCA’s utility by mapping out the substrate specificity for these enzymes

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

<div><p>Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in <i>E</i>. <i>coli</i>. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.</p></div

Expression correction for properly folded soluble proteins.

<p>(A) The synthesized gene library open reading frames (ORFs) are cloned via RE1/RE2 into a vector containing an N-terminal Tat pathway secretion signal (ssTorA) and a C-terminal TEM-1 β-lactamase. (B) Synthesized genes that are expressed, properly folded, and soluble result in the export of mature β-lactamase fusions to the <i>E</i>. <i>coli</i> periplasm, which confers ampicillin resistance. Selection against insoluble protein serves to both remove correct gene sequences which are poorly expressed in <i>E</i>. <i>coli</i> and remove incorrect gene synthesis products as the resulting protein is often insoluble. Expression profiling is characterized with Illumina MiSeq next generation sequencing.</p

Individual Gene Performance in Expression Correction Assay on Ampicillin.

<p>(A) Fold killing on 100 μg/mL of ampicillin (left axis, gray bars) and fraction of soluble protein (right axis, red line) for each individual gene shows an increase in cell survival with increasing soluble protein fraction. Fold killing is calculated based on viable colony counts on solid media with no ampicillin divided by counts at 100 μg/mL ampicillin. Soluble protein fraction was quantified from western blotting of soluble and insoluble protein fractions expressed as C-terminal FLAG tag fusions. (n = 3 for both experiments). Error bars represent standard deviation. (B) Percent representation of each gene per ampicillin selection pool shows enrichment of genes which survive at 100 μg/mL of ampicillin and have higher soluble protein fractions in (A). Percent is calculated as the median number of reads per base pair of each gene compared to the sum of medians for each gene per ampicillin pool.</p

Multiplex Library Synthesis and Expression Correction (MuLSEC) Characterization for a Library of Enzymes.

<p>(A) Survival of clones after multiplex library gene synthesis is presented on the y-axis versus the solid media ampicillin concentration on the x-axis. Fewer clones survive on increasing ampicillin concentrations. (log-log plot). (B) Table of Sanger sequencing results on individual clones. Selection on ampicillin at 5 μg/mL led to a 6x increase in the number of perfect clones. (C) Percent of the 95 genes synthesized detected by next generation sequencing after selection on varying concentrations of ampicillin. Data is presented with a cutoff such that the median number of reads per gene must be ≥ 0.05% of total reads per ampicillin concentration condition (open squares) or with no cutoff (closed circles). Fewer genes are detected with increasing ampicillin concentration (log-log plot). (D) Box-and-whisker plot of pool normalized representation of each gene (reads ≥ 0.05% of group total) for increasing ampicillin concentrations. Black dots represent outliers in the dataset.</p

Enzyme Library Genes Assayed for Individual Performance Analysis.

<p>Five genes from the set of 69 BRENDA enzymes and one control (Efnb2) selected for performance in the expression correction ampicillin-based selection. Efnb2 was previously shown to be insoluble and die as a ssTorA/ β-lactamase fusion on ampicillin[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119927#pone.0119927.ref011" target="_blank">11</a>]. EC refers to enzyme classification number.</p><p>Enzyme Library Genes Assayed for Individual Performance Analysis.</p

Multiplex Library Synthesis of Genes.

<p>(A) Individual genes (top) are assembled from a set of oligonucleotides (arrows) with 15 bp of unique overlapping sequence. Synthons include universal forward (UF) and reverse (UR) primer binding regions common for all genes in the library, and specific forward (SF) and reverse (SR) sites for amplification of individual genes. Synthons include restriction endonuclease sites (RE1 = EcoRI, RE2 = BamHI) for downstream cloning purposes, a random filler sequence (Fi) for length standardization, and are < 950 bp long. (B) Gene synthesis from a pooled library of oligonucleotide starts with high temperature annealing and ligation, then individual genes can either be amplified with SF/SR primers (left) or library of genes is synthesized and amplified (right) with emulsion PCR using UF/UR primers with annealing 5’ and 3’ overhangs to enable suppression PCR, which preferentially amplifies longer synthons. (C) Excitation of 87 GFP variants successfully synthesized, amplified individually, then expressed in <i>E</i>. <i>coli</i> show functional fluorescent protein at various emission wavelengths. Images are combined from ultraviolet (top layer, 50% transparency) and blue light illumination (470nm) with no emission filtering. White X represents the negative control well with media only.</p