Versioning Biological Cells for Trustworthy Cell Engineering

Abstract“Full-stack”biotechnology platforms for cell line (re)programming are on the horizon, due mostly to (a) advances in gene synthesis and editing techniques as well as (b) the growing integration with informatics, the internet of things and automation. These emerging platforms will accelerate the production and consumption of biological products. Hence, transparency, traceability and -ultimately-trustworthiness is required -from cradle to grave- for engineered cell lines and their engineering processes. We report here the first version control system for cell engineering that integrates a new cloud-based version control software for cell lines’ digital footprint with molecular barcoding of living samples. We argue that version control for cell engineering marks a significant step towards more open, reproducible, easier to trace and share, and more trustworthy engineering biology.One Sentence SummaryWe demonstrate a transparent and open way of engineering and sharing cell lines.

DNA Supercoiling Drives a Transition between Collective Modes of Gene Synthesis

Recent experiments showed that multiple copies of the molecular machine RNA polymerase (RNAP) can efficiently synthesize mRNA collectively in the active state of the promoter. However, environmentally-induced promoter repression results in long-distance antagonistic interactions that drastically reduce the speed of RNAPs and cause a quick arrest of mRNA synthesis. The mechanism underlying this transition between cooperative and antagonistic dynamics remains poorly understood. In this Letter, we introduce a continuum deterministic model for the translocation of RNAPs, where the speed of an RNAP is coupled to the local DNA supercoiling as well as the density of RNAPs on the gene. We assume that torsional stress experienced by individual RNAPs is exacerbated by high RNAP density on the gene and that transcription factors act as physical barriers to the diffusion of DNA supercoils. We show that this minimal model exhibits two transcription modes mediated by the torsional stress: a fluid mode when the promoter is active and a torsionally stressed mode when the promoter is repressed, in quantitative agreement with experimentally observed dynamics of co-transcribing RNAPs. Our work provides an important step towards understanding the collective dynamics of molecular machines involved in gene expression.

Simple and efficient custom transcription activator-like effector gene synthesis via twin primer assembly

Transcription activator-like effector (TALE) nucleases (TALENs) efficiently recognize and cleave DNA in a sequence-dependent manner. However, current TALE custom synthesis methods are either complicated or expensive. Here we report a simple and low-cost method for TALE construct assembly. This method utilizes the denaturation/reannealing nature of double-stranded DNA to create a unique single-stranded DNA overhang for proper ordering of TALE monomers in an engineered multimer. We successfully synthesized two TALEN pairs targeting the endogenous TET1 locus in human embryonic kidney cells and demonstrated their editing efficiency. Our method provides an alternative simple, low-cost method for effective TALEN assembly, which may improve the application of TALE-based technology.

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.

De-extinction and Gene Drives: The Engineering of Anthropocene Organisms

Advances in gene reading, gene synthesis, and genome editing are making possible a number of radical new practices for transforming animal futures in the Anthropocene. De-extinction may make it possible to bring back lost species. Gene drives may enable the sending of desirable traits through wild populations of organisms. The hype accompanying these promises can make each of them look ethically irresistible. This chapter investigates the ‘speculative ethics’ that has arisen around these technologies, asking questions about both their viability and the approach to animals they contain. Reductive and non-relational thinking is identified as one potential problem with the thinking behind these techniques. The neglect of non-human agency is identified as another. After indicating some of the problems these two ways of conceptualizing an animal and its genome can create, a brief suggestion is made about how to better conceptualize animal futures in the Anthropocene.

A GENE SYNTHESIS REGIME FOR MALAYSIA TO EMULATE IN SECURING FUTURE BIOPRINTED VACCINES

The merger between synthetic biology and bioprinting will someday enable vaccines to be bioprinted utilising genetic material. Unregulated gene synthesis companies may unwittingly supply genetic material to a terrorist if there is no verification of purchasers’ personal identity and affiliation with a legitimate research organisation. This study has the objective of focusing on whether Malaysia regulates and conducts Deoxyribonucleic Acid (DNA) sequence screening among gene synthesis companies which are meant for bioprinting vaccines that can be misused for bioterrorism. This study is qualitative. Gene synthesis guidelines from the United States (US), the International Gene Synthesis Consortium (IGSC), the Nuclear Threat Initiative-World Economic Forum (NTI-WEF) Biosecurity Innovation and Risk Reduction report were referred as examples for changes in Malaysia’s draft National Code of Conduct for Biosecurity [thereafter known as Code]. These soft law documents constitute the regime for gene synthesis and a form of transnational new governance. The findings indicate that in the absence of a specific binding regulation, Malaysia’s draft Code must be amended to incorporate the need to screen customers, genetic sequences and address the cyberbiosecurity of biological life in digitalised form besides the physical biosecurity of laboratories which houses seedstocks from being stolen for malicious intent.

An improved gene synthesis method with asymmetric directions of oligonucleotides designed using a simulation program

Artificial gene synthesis based on oligonucleotide augmentation is known as overlap extension PCR which generates a variety of intermediate synthetic products. The orientation and concentration of oligomers can be adjusted to reduce the synthesis of intermediates and optimize the full-length process of DNA synthesis, using a simulation program for serial oligomer extension. The efficiency of the serial oligomer extension process is predicted to be greatest when oligomers are in a ‘forward-reverse-reverse-reverse’ direction. Oligomers with such designed directions demonstrated generation of the desired product in the shortest time (number of cycles) by repeated annealing and elongation. This method, named Asymmetric Extension supported by a Simulator for Oligonucleotide Extension (AESOE), has shown efficiency and effectiveness with potentials for future improvements and optimal usage in DNA synthesis.

DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here, we improve DropSynth, a low-cost, multiplexed method that builds gene libraries by compartmentalizing and assembling microarray-derived oligonucleotides in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity.