Importance of the 5′ regulatory region to bacterial synthetic biology applications

Tietze, Lisa; Lale, Rahmi

doi:10.1111/1751-7915.13868

Cited by 11 publications

(12 citation statements)

References 225 publications

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“… 52 In general, the expression of functional components is affected by short adjacent upstream/downstream sequences. 41 , 42 , 53 Calculator tools such as the RBS calculator ( ) 37 , 54 now require 35 bp upstream and 60 bp downstream to estimate the translation rate of an RBS.…”

Section: Resultsmentioning

confidence: 99%

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PASIV: A Pooled Approach-Based Workflow to Overcome Toxicity-Induced Design of Experiments Failures and Inefficiencies

et al. 2022

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We present here a newly developed workflow—which we have called PASIV—designed to provide a solution to a practical problem with design of experiments (DoE) methodology: i.e., what can be done if the scoping phase of the DoE cycle is severely hampered by burden and toxicity issues (caused by either the metabolite or an intermediary), making it unreliable or impossible to proceed to the screening phase? PASIV—standing for pooled approach, screening, identification, and visualization—was designed so the (viable) region of interest can be made to appear through an interplay between biology and software. This was achieved by combining multiplex construction in a pooled approach (one-pot reaction) with a viability assay and with a range of bioinformatics tools (including a novel construct matching tool). PASIV was tested on the exemplar of the lycopene pathway—under stressful constitutive expression—yielding a region of interest with comparatively stronger producers.

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Section: Resultsmentioning

confidence: 99%

“…h i b i t e d . downstream sequences 41,42,53. Calculator tools such as the RBS calculator (https://www.denovodna.com/)37,54 now require 35 bp upstream and 60 bp downstream to estimate the translation rate of an RBS.…”

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confidence: 99%

PASIV: A Pooled Approach-Based Workflow to Overcome Toxicity-Induced Design of Experiments Failures and Inefficiencies

et al. 2022

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“…Of all composition factors, the interfacing of adjacent regions is the most important for QC. Expression of functional components is indeed affected by short adjacent sequences ( Espah Borujeni et al, 2014 ; Taylor et al, 2019 ; Tietze and Lale, 2021 ). A calculator such as the RBS calculator ( Salis et al, 2009 ) ( https://www.denovodna.com/ ) now requires 35 bp upstream and 60 downstream to estimate the translation rate of an RBS.…”

Section: Introductionmentioning

confidence: 99%

Removing the Bottleneck: Introducing cMatch - A Lightweight Tool for Construct-Matching in Synthetic Biology

Casas

Bultelle

Motraghi

et al. 2022

Front. Bioeng. Biotechnol.

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We present a software tool, called cMatch, to reconstruct and identify synthetic genetic constructs from their sequences, or a set of sub-sequences—based on two practical pieces of information: their modular structure, and libraries of components. Although developed for combinatorial pathway engineering problems and addressing their quality control (QC) bottleneck, cMatch is not restricted to these applications. QC takes place post assembly, transformation and growth. It has a simple goal, to verify that the genetic material contained in a cell matches what was intended to be built - and when it is not the case, to locate the discrepancies and estimate their severity. In terms of reproducibility/reliability, the QC step is crucial. Failure at this step requires repetition of the construction and/or sequencing steps. When performed manually or semi-manually QC is an extremely time-consuming, error prone process, which scales very poorly with the number of constructs and their complexity. To make QC frictionless and more reliable, cMatch performs an operation we have called “construct-matching” and automates it. Construct-matching is more thorough than simple sequence-matching, as it matches at the functional level-and quantifies the matching at the individual component level and across the whole construct. Two algorithms (called CM_1 and CM_2) are presented. They differ according to the nature of their inputs. CM_1 is the core algorithm for construct-matching and is to be used when input sequences are long enough to cover constructs in their entirety (e.g., obtained with methods such as next generation sequencing). CM_2 is an extension designed to deal with shorter data (e.g., obtained with Sanger sequencing), and that need recombining. Both algorithms are shown to yield accurate construct-matching in a few minutes (even on hardware with limited processing power), together with a set of metrics that can be used to improve the robustness of the decision-making process. To ensure reliability and reproducibility, cMatch builds on the highly validated pairwise-matching Smith-Waterman algorithm. All the tests presented have been conducted on synthetic data for challenging, yet realistic constructs - and on real data gathered during studies on a metabolic engineering example (lycopene production).

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“…To this end, genetic parts that influence translation are modified to alter absolute and relative expression levels to engineer biosystems through control of individual genes, pathways and even entire metabolic networks (1)(2)(3). In prokaryotes, initiation of translation is the rate-limiting step in the translational process, during which ribosomes assemble on the mRNA to start the templated elongation of the nascent polypeptide (4)(5)(6)(7). At the onset of this step, the 30S ribosomal subunit attaches to the ribosome binding site (RBS) in the 5'-untranslated region (5'-UTR) upstream of the coding sequence (CDS).…”

Section: Introductionmentioning

confidence: 99%

Ultradeep characterisation of translational sequence determinants refutes rare-codon hypothesis and unveils quadruplet base pairing of initiator tRNA and transcript

Höllerer

Jeschek

2022

Preprint

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Translation is a key determinant of gene expression and an important biotechnological engineering target. In bacteria, 5’-untranslated region (5’-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. However, the complex interaction of 5’-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. Herein, we systematically assess the dynamic translation from over 1.2 million 5’-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. This allows us to disentangle and precisely quantify effects of known and hypothetical sequence determinants of translation. We find that 5’-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. Moreover, the obtained large-scale data clearly point to a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation.

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Importance of the 5′ regulatory region to bacterial synthetic biology applications

Cited by 11 publications

References 225 publications

PASIV: A Pooled Approach-Based Workflow to Overcome Toxicity-Induced Design of Experiments Failures and Inefficiencies

PASIV: A Pooled Approach-Based Workflow to Overcome Toxicity-Induced Design of Experiments Failures and Inefficiencies

Removing the Bottleneck: Introducing cMatch - A Lightweight Tool for Construct-Matching in Synthetic Biology

Ultradeep characterisation of translational sequence determinants refutes rare-codon hypothesis and unveils quadruplet base pairing of initiator tRNA and transcript

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