Synthetic Biology in the Real World

Microbial biosensors detect water contaminants in 3 minutes.

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Bacteria are powerful, little machines. They’ve been engineered to detect environmental contaminants, spot inflammatory signals inside the body, and ‘sniff’ out certain cancers in mice. I’ve written about each of these applications at some point, and the programmability of life surprises me every time.

In using bacteria as living biosensors, though, we are fundamentally limited. Biology is slow. It takes at least 30 minutes for a protein to detect a signal, relay that message into a cell, produce mRNAs encoding a fluorescent protein, and coerce ribosomes to make a protein that a scientist can detect under a microscope.

The dream is to make bacteria that can detect any metabolite in minutes, with no fancy equipment required.

For a new study in Nature, researchers from Rice University and the Lawrence Berkeley National Laboratory engineered E. coli bacteria to sense water contaminants, such as endocrine disruptors, and emit an electrical signal in less than three minutes. This is a remarkable feat of biological engineering; probably the best paper I’ve seen in six months.

Each bacterial cell was first modified to express a synthetic electron transport chain, made up of more than a dozen finely-tuned genes. The transport chain is made up of three modules: Input, Coupling, and Output.

To detect a water contaminant, such as thiosulfate, the input module reduces the metabolite, the coupling module converts byproducts to a quinol, and the output module strips quinol of its electrons and passes them onto an electrode. The full genetic circuit and schematic are shown below; marvel at its engineering.

Schematic of the electron transport chain (top) and genetic constructs (B). From Atkinson JT et al. in Nature (2022).

Bacterial cells with this synthetic electron transport chain could sensitively detect thiosulfate — a toxic water contaminant — and other chemicals. In the laboratory, engineered cells detected as little as 0.1 mM of thiosulfate, and exhibited a linear increase in current with increasing concentrations, “within 2–10 min of exposure,” according to the paper.

These bacteria are also adaptable. By swapping out the input module, they were programmed to detect other molecules, including an endocrine disruptor called 4-HT, or 4-hydroxytamoxifen, four-fold faster than prior versions of microbial biosensors.

The beauty of this paper, though, lies not in its engineering or adaptability, but in its real-world adaptation. The researchers took these biosensing cells out of the lab, drove to three different beaches around Houston, Texas, and tested them in water with differing pH and pollution levels. And in all three cases, spiking the water with a bit of thiosulfate or 4-HT “resulted in a significant increase in signal intensity” in 6.5 minutes for the former, and 3 minutes for the latter.

This paper is a pivotal part of a storied history of bioelectronic devices. Earlier this year, a group at MIT built a similar device — a tiny capsule — that houses both bacterial biosensors and intricate electronic circuits. The microbes detect nitric oxide gas in the gut, relay those signals to an electronic circuit, and a wireless antenna transmits real-time data on gut inflammation to a smartphone.

In merging bacteria and electronics, we get the best of both worlds: A powerful chemical interface and reliable circuits.

And by bringing these creations into the world, we can demonstrate — as scientists — that engineered life forms can tangibly improve the lives of people. Too often, we run experiments in the lab, show proofs-of-concept, and then abandon our ideas, or roll them into a company that avoids talking to consumers altogether — therapeutics and diagnostics.

Synthetic biology is the invisible wheel that powers our foods and medicines, and yet people still fear it. We need to showcase consumer-facing projects out in the real-world. This paper hits the mark.

Thanks for reading.

— Niko McCarty

A variant of the Cas13a protein, an RNA-guided RNA cutter first found in a rod-shaped bacterium called Leptotrichia buccalis, can be used to edit phage genomes with startling efficiency. This protein does not have any PAM sequence requirements — meaning it can theoretically edit any target sequence — and can also tolerate mismatches between its guide RNA and the target.

For a new study from Jennifer Doudna’s group, this Cas13a variant was used to edit the genomes of three different phages. It successfully removed genes and swapped out a single codon, all with 100% efficiency. This Cas13a variant could drastically speed up phage engineering and help create new therapies. Nature Microbiology (Link)

Phage genome edits made using the Cas13a protein, including deletions, large deletions, and many different single-nucleotide edits. Each was done with 100% efficiency.

Look at two cells, side by side. Each is glowing a soft, translucent green. One might say, “Cell A is 1.5 times brighter than Cell B,” an arbitrary comparison, and record those values in a research paper. To make synthetic biology a true engineering discipline, though, we must instead move toward absolute units; “140 proteins per cell” is a lot better than “1.3-fold brighter” because it helps others check and replicate your work more consistently. Now, a tool called FPCount (and a software companion, called FPCountR), makes absolute protein quantification simple, and it requires only a plate reader — no fancy microscopes or RNA-sequencing! A full protocol is available online, and the software package is open source and written in the R programming language. Nature Communications (Link)


There have been probably a dozen papers about Cas 7-11 in the last few weeks. I wrote about one of the early structures of these RNA-guided protein-cleaving enzymes, but the papers keep coming. Last week, back-to-back studies in Science reported that a protein called Csx29 cozies up with Cas7-11 and, together, they cleave target proteins. This protein complex can be “activated” using a strand of RNA. Both of these new studies include cryo-electron microscopy structures of the protein complexes, and both indicate that these proteins can be used for programmable RNA sensing applications in cell culture and mammalian cells. This is truly a blistering pace of progress; the first structures came out just a few weeks ago! Science (Link and Link)


The Index 🔻

A collection of research papers published in the last week.

(↑ = recommended article, * = open access, † = review, comment, etc. )

Basic Research

↑*Genome-wide base editor screen identifies regulators of protein abundance in yeast. Schubert OT…Kruglyak L. eLife. Link

↑*Translation and natural selection of micropeptides from long non-canonical RNAs. Patraquim P…Couso JP. Nature Communications. Link

  • About 30% of long, noncoding RNAs in fruit flies are translated by ribosomes into short micropeptides, ranging from 100 to 300 amino acids in length.

*Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences. Miura F…Ito T. BMC Biotechnology. Link

Manipulating the nature of embryonic mitotic waves. Hayden L…Di Talia S. Current Biology. Link

*Spatiotemporal and genetic regulation of A-to-I editing throughout human brain development. Cuddleston WH…Breen MS. Cell Reports. Link

Biomanufacturing, Metabolic Engineering & Biomaterials

↑*Directed assembly of genetically engineered eukaryotic cells into living functional materials via ultrahigh-affinity protein interactions. Yi Q…Sun F. Science Advances. Link

*Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales. Puzet S…Hersen P. bioRxiv (preprint). Link

Post-translational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units. Kuroda T…Suga H. Nature Chemistry. Link

Engineering a Prokaryotic Non-P450 Hydroxylase for 3′-Hydroxylation of Flavonoids. Wang H…Yuan Q. ACS Synthetic Biology. Link

*An arrayed CRISPR screen reveals Myc depletion to increase productivity of difficult-to-express complex antibodies in CHO cells. Bauer N…Ausländer S. Synthetic Biology. Link

*†Recent progress in adaptive laboratory evolution of industrial microorganisms. Wang G…Yang X. Journal of Industrial Microbiology and Biotechnology. Link

Microbial production of the plant-derived fungicide physcion. Qi F…Lu X. Metabolic Engineering. Link

Systems engineering of Escherichia coli for high-level shikimate production. Li Z…Liu L. Metabolic Engineering. Link

Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli. Wang Z…Xia X. Metabolic Engineering. Link

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation. Turlin J…Nikel PI. Metabolic Engineering. Link

*Exploration of a novel and efficient source for production of bacterial nanocellulose, bioprocess optimization and characterization. El-Naggar N.E-A…Mohammed ABA. Scientific Reports. Link

*Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol. Loll-Krippleber R…Brown GW. bioRxiv (preprint). Link

Unusual Class I Lanthipeptides from the Marine Bacteria Thalassomonas viridans. Vermeulen R…Trindade M. ACS Synthetic Biology. Link

Biosensors

↑Real-time bioelectronic sensing of environmental contaminants. Atkinson JT…Ajo-Franklin CM. Nature. Link

*Probing coenzyme A homeostasis with semisynthetic biosensors. Xue L…Johnsson K. Nature Chemical Biology. Link

*A genetically encoded fluorescent biosensor for detecting itaconate with subcellular resolution in living macrophages. Sun P…Li X. Nature Communications. Link

†*Olfactory Receptors as an Emerging Chemical Sensing Scaffold. Patel A & Peralta-Yahya P. Biochemistry. Link

Build-A-Cell

†*Bottom-up assembly of viral replication cycles. Staufer O…Spatz JP. Nature Communications. Link

Computational Tools & Models

↑*A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies. Hoberecht L…Fortin J-P. Nature Communications. Link

  • A suite of R packages to design guide RNAs for gene activation, repression, or editing. Includes tools for Cas9, Cas12, and Cas13. Available on GitHub.

↑*REBASE: a database for DNA restriction and modification: enzymes, genes and genomes. Roberts RJ…Macelis D. Nucleic Acids Research. Link

*GenomicKB: a knowledge graph for the human genome. Feng F…Liu J. Nucleic Acids Research. Link

*TmAlphaFold database: membrane localization and evaluation of AlphaFold2 predicted alpha-helical transmembrane protein structures. Dobson L…Tusnády GE. Nucleic Acids Research. Link

*Independent mutation effects enable design of combinatorial protein binding mutants. Ding D. bioRxiv (preprint). Link

*Tuned Fitness Landscapes for Benchmarking Model-Guided Protein Design. Thomas N…Colwell LJ. bioRxiv (preprint). Link

*De novo analysis of bulk RNA-seq data at spatially resolved single-cell resolution. Liao J…Fan X. Nature Communications. Link

Agent-Based Modeling of Microbial Communities. Nagarajan K, Ni C & Lu T. ACS Synthetic Biology. Link

*Controlling microbial co-culture based on substrate pulsing can lead to stability through differential fitness advantages. Martinez JA…Delvigne F. PLOS Computational Biology. Link

*Logistic Regression-Guided Identification of Cofactor Specificity-Contributing Residues in Enzyme with Sequence Datasets Partitioned by Catalytic Properties. Sugiki S…Shimizu H. ACS Synthetic Biology. Link

CRISPR, Gene Editing & Control

↑RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease. Kato K…Nishimasu H. Science. Link

↑RNA-activated protein cleavage with a CRISPR-associated endopeptidase. Strecker J…Zhang F. Science. Link

↑*Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering. Esquerra-Ruvira B…Mojica FJM. bioRxiv (preprint). Link

↑Bacteriophage genome engineering with CRISPR–Cas13a. Guan J…Bondy-Denomy J. Nature Microbiology. Link

↑*Structural basis for Cas9 off-target activity. Pacesa M…Jinek M. Cell. Link

↑*Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing. Adler BA…Doudna JA. Nature Microbiology. Link

A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences. Xing D…Dunham R. ACS Synthetic Biology. Link

*Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data. Ciciani M…Segata N. Nature Communications. Link

*A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration. Wu Y…Xie G. Nucleic Acids Research. Link

*Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors. Chen F…Lai L. Communications Biology. Link

*Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system. Zhang B…Ouyang S. Nucleic Acids Research. Link

DNA Sequencing, Synthesis & Assembly

†*A User’s Guide to Golden Gate Cloning Methods and Standards. Bird JE, Marles-Wright J & Giachino A. ACS Synthetic Biology. Link

Genetic Circuits

†Synthetic biology by controller design. Barajas C & Del Vecchio D. Current Opinion in Biotechnology. Link

Medicine & Diagnostics

↑*Targeting AAV vectors to the CNS via de novo engineered capsid-receptor interactions. Huang Q…Deverman BE. bioRxiv (preprint). Link

↑Safety and Efficacy of a Monoclonal Antibody against Malaria in Mali. Kayentao K…Mali Malaria mAb Trial Team. The New England Journal of Medicine. Link

On-demand cell-autonomous gene therapy for brain circuit disorders. Qiu Y…Lignani G. Science. Link

*A split, conditionally active mimetic of IL-2 reduces the toxicity of systemic cytokine therapy. Quijano-Rubio A…Baker D. Nature Biotechnology. Link

*Ligand-switchable nanoparticles resembling viral surface for sequential drug delivery and improved oral insulin therapy. Yang T…Gan Y. Nature Communications. Link

*Selective decrease of donor-reactive Tregs after liver transplantation limits Treg therapy for promoting allograft tolerance in humans. Tang Q…Feng S. Science Translational Medicine. Link

*Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification. Xu Z…Liu M. Nature Communications. Link

*Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression. Antoniou P…Miccio A. Nature Communications. Link

*Reprogramming of pancreatic adenocarcinoma immunosurveillance by a microbial probiotic siderophore. Chaib M…Chauhan SC. Communications Biology. Link

*speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing. Castellanos-Rueda R…Reddy ST. Nature Communications. Link

Nanocellulose coated paper diagnostic to measure glucose concentration in human blood. Hossain L…Garnier G. Frontiers in Bioengineering and Biotechnology. Link

Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation. Zhu X…Fan G. Cell Chemical Biology. Link

†*Unlocking the promise of mRNA therapeutics. Rohner E…Chien KR. Nature Biotechnology. Link

*A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice. Williams AJ…DeLisa MP. bioRxiv (preprint). Link

*Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A. Panova EA…Gintsburg AL. bioRxiv (preprint). Link

Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure. Metzger D…Saldanha R. ACS Synthetic Biology. Link

Plants

*High-efficiency retron-mediated single-stranded DNA production in plants. Jiang W…Mahfouz MM. Synthetic Biology. Link

*Simultaneous editing of three homoeologs of TaCIPK14 confers broad-spectrum resistance to stripe rust in wheat. He F…Guo J. Plant Biotechnology Journal. Link

*Optimization of biomass and target protein yield for Phase III clinical trial to evaluate Angiotensin Converting Enzyme 2 expressed in lettuce chloroplasts to reduce SARS-CoV-2 infection and transmission. Ganesan PK…Daniell H. Plant Biotechnology Journal. Link

*Overexpression of ZmKL9 increases maize hybrid hundred kernel weight. Gong D…Qiu F. Plant Biotechnology Journal. Link

Protein Engineering

*Engineering transmembrane signal transduction in synthetic membranes using two-component systems. Peruzzi JA, Galvez NR & Kamat NP. bioRxiv (preprint). Link

*Dual-function artificial molecular motors performing rotation and photoluminescence. Pfeifer L…Feringa BL. Science Advances. Link

*Extension of the short wavelength side of fluorescent proteins using hydrated chromophores, and its application. Sugiura K & Nagai T. Communications Biology. Link

Tools, Toolkits & Technology

↑*Absolute protein quantification using fluorescence measurements with FPCountR. Csibra E & Stan G-B. Nature Communications. Link

↑*Inducible plasmid copy number control for synthetic biology in commonly used E. coli strains. Joshi S.H-N, Yong C & Gyorgy A. Nature Communications. Link

*Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag. Meers MP…Henikoff S. Nature Biotechnology. Link

Engineering an SspB-mediated degron for novel controllable protein degradation. Lei Y…Tang S-Y. Metabolic Engineering. Link

*Demonstration of intracellular real-time molecular quantification via FRET-enhanced optical microcavity. Wang Y…Wang P. Nature Communications. Link

Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus. Yoon J…Nagasawa K. PNAS. Link

*A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids. Sockell A…Fordyce PM. bioRxiv (preprint). Link

Synthetic Platforms for Characterizing and Targeting of SARS-CoV-2 Genome Capping Enzymes. Ornelas MY…Mehta AP. ACS Synthetic Biology. Link

Other Interesting Stuff

*Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy. Liu Z…Ouyang H. Gene. Link

Single-Dose Psilocybin for a Treatment-Resistant Episode of Major Depression. Goodwin GM…Malievskaia E. The New England Journal of Medicine. Link

*Electronic photoreceptors enable prosthetic visual acuity matching the natural resolution in rats. Wang B-Y…Palanker D. Nature Communications. Link

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem. van Spanning RJM…Bitter W. Nature Microbiology. Link

*Effect of resistance to third-generation cephalosporins on morbidity and mortality from bloodstream infections in Blantyre, Malawi: a prospective cohort study. Lester R…Mangochi H. The Lancet Microbe. Link

*Wireless neuromodulation in vitro and in vivo by intrinsic TRPC-mediated magnetomechanical stimulation. Su C-L…Chiang P-H. Communications Biology. Link