Mechanistic modeling of a rewritable recombinase addressable data module

Many of the most important applications predicted to arise from Synthetic Biology will require engineered cellular memory with the capability to store data in a rewritable and reversible manner upon induction by transient stimuli. DNA recombination provides an ideal platform for cellular data storage and has allowed the development of a rewritable recombinase addressable data (RAD) module, capable of efficient data storage within a chromosome. Here, we develop the first detailed mechanistic model of DNA recombination, and validate it against a new set of in vitro data on recombination efficiencies across a range of different concentrations of integrase and gp3. Investigation of in vivo recombination dynamics using our model reveals the importance of fully accounting for all mechanistic features of DNA recombination in order to accurately predict the effect of different switching strategies on RAD module performance, and highlights its usefulness as a design tool for building future synthetic circuitry

DNA chisel, a versatile sequence optimizer

Motivation: Accounting for biological and practical requirements in DNA sequence design often results in challenging optimization problems. Current software solutions are problem-specific and hard to combine.Results: DNA Chisel is an easy-to-use, easy-to-extend sequence optimization framework allowing to freely define and combine optimization specifications via Python scripts or Genbank annotations.Availability: The framework is available as a web application (https://cuba.genomefoundry.org/sculpt_a_sequence)or open-source Python library (see at https://github.com/Edinburgh-Genome-Foundry/DNAChisel for code and documentation)

DNA features viewer:A sequence annotation formatting and plotting library for Python

Motivation: While the Python programming language counts many Bioinformatics and Computational Biology libraries, none offers customizable sequence annotation visualizations with layout optimization. Results: DNA Features Viewer is a sequence annotation plotting library which optimizes plot readabilitywhile letting users tailor other visual aspects (colors, labels, highlights, etc.) to their particular use case.Availability: Open-source code and documentation are available on Github under the MIT licence (https://github.com/Edinburgh-Genome-Foundry/DnaFeaturesViewer)

Identifying early predictors of instability in CHO cell lines using the Berkley Lights Beacon® Optofluidic System

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Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor

The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5′ of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting

Control of serine integrase recombination directionality by fusion with the directionality factor

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle

A systematic comparison of triterpenoid biosynthetic enzymes for the production of oleanolic acid in Saccharomyces cerevisiae

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a β-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen β-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of β-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in β-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives

Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7

The CRISPR‐Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram‐positive model organism Bacillus subtilis. A major draw‐back for the commercial use of Cas9 is the IP landscape requiring a licence for its use, as well as reach‐through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7, was released by Inscripta (CO, USA). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93 % and 100 % were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3 % reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR‐based editing mode of action in B. subtilis providing evidence that the nuclease‐mediated DNA double strand break acts as a counterselection mechanism after homologous recombination of the donor DNA