Biology subjects

Gallegos, J. E.

Publications and source records attributed to Gallegos, J. E..

5 recordsLinked to original sources

bioRxiv2020

Rapid, robust plasmid verification by de novo assembly of short sequencing reads

Plasmids are a foundational tool for basic and applied research across all subfields of biology. Increasingly, researchers in synthetic biology are relying on and developing massive libraries of plasmids as vectors for directed evolution, combinatorial gene circuit tests, and for CRISPR multiplexing. Verification of plasmid sequences following synthesis is a crucial quality control step that creates a bottleneck in plasmid fabrication workflows. Crucially, researchers often elect to forego the cumbersome verification step, potentially leading to reproducibility and— depending on the application—security issues. In order to facilitate plasmid verification to improve the quality and reproducibility of life science research, we developed a fast, simple, and open source pipeline for assembly and verification of plasmid sequences from Illumina reads. We demonstrate that our pipeline, which relies on de novo assembly, can also be used to detect contaminating sequences in plasmid samples. In addition to presenting our pipeline, we discuss the role for verification and quality control in the increasingly complex life science workflows ushered in by synthetic biology.Competing Interest StatementJP holds an equity stake in GenoFAB Inc., a company that may benefit or may be perceived to benefit from the publication of this article. MR and CC are GenoFAB employees.View Full Text

bioinformatics↗

bioRxiv2019

Genetic interactions derived from high-throughput phenotyping of 7,350 yeast cell cycle mutants

Over the last 30 years, computational biologists have developed increasingly realistic mathematical models of the regulatory networks controlling the division of eukaryotic cells. These models capture data resulting from two complementary experimental approaches: low-throughput experiments aimed at extensively characterizing the functions of small numbers of genes, and large-scale genetic interaction screens that provide a systems-level perspective on the cell division process. The former is insufficient to capture the interconnectivity of the genetic control network, while the latter is fraught with irreproducibility issues. Here, we describe a hybrid approach in which the genetic interactions between 36 cell-cycle genes are quantitatively estimated by high-throughput phenotyping with an unprecedented number of biological replicates. Using this approach, we identify a subset of high-confidence genetic interactions, which we use to refine a previously published mathematical model of the cell cycle. We also present a quantitative dataset of the growth rate of these mutants under six different media conditions in order to inform future cell cycle models.\n\nAuthor SummaryThe process of cell division, also called the cell cycle, is controlled by a highly complex network of interconnected genes. If this process goes awry, diseases such as cancer can result. In order to unravel the complex interactions within the cell cycle control network, computational biologists have developed mathematical models that describe how different cell cycle genes are related. These models are built using large datasets describing the effect of mutating one or more genes within the network. In this manuscript, we present a novel method for producing such datasets. Using our method, we generate 7,350 yeast mutants to explore the interactions between key cell cycle genes. We measure the effect of the mutations by monitoring the growth rate of the yeast mutants under different environmental conditions. We use our mutants to revise an existing model of the yeast cell cycle and present a dataset of [~]44,000 gene by environment combinations as a resource to the yeast genetics and modeling communities.

systems biology↗

bioRxiv2019

Securing the exchange of synthetic genetic constructs using digital signatures

Synthetic biology relies on an ever-growing supply chain of synthetic genetic material. Technologies to secure the exchange of this material are still in their infancy. Solutions proposed thus far have focused on watermarks, a dated security approach that can be used to claim authorship, but is subject to counterfeit, and does not provide any information about the integrity of the genetic material itself. We describe how data encryption and digital signature algorithms can be used to ensure the integrity and authenticity of synthetic genetic constructs. Using a pilot software that generates digital signatures and other encrypted data for plasmids, we demonstrate that we can predictably extract information about the author, the identity, and the integrity of plasmid sequences from sequencing data alone without a reference sequence, all without compromising the function of the plasmids. We discuss how this technology can be improved, applied, and expanded to support the new bioeconomy.

synthetic biology↗

bioRxiv2018

Intron-mediated enhancement is not limited to introns

Certain introns strongly increase mRNA accumulation by a poorly understood mechanism known as Intron-Mediated Enhancement (IME). Introns that boost expression by IME have no effect when located upstream of or more than ~1 Kb downstream from the start of transcription. The sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, can convert a non-stimulating intron into one that strongly increases mRNA accumulation. We tested the ability of an intron containing this motif to stimulate expression from different locations and found that it had the same positional requirements as naturally occurring IME introns. The motif also stimulated gene expression from within the 5-UTR and coding sequences of an intronless construct. Furthermore, the 5-UTR of another gene increased expression when inserted into an otherwise non-stimulating intron in coding sequences. These results demonstrate that splicing is not required for intron-mediated enhancement, and suggest that other sequences downstream of the transcription start site in addition to introns may stimulate expression by a similar mechanism.

molecular biology↗

bioRxiv2016

Intron-driven gene expression in the absence of a core promoter in Arabidopsis thaliana

In diverse eukaryotes, certain introns increase mRNA accumulation through the poorly understood mechanism of intron-mediated enhancement (IME). A distinguishing feature of IME is that these introns have no effect from upstream or more than 1 Kb downstream of the transcription start site (TSS). To more precisely define the intron position requirements for IME in Arabidopsis, we tested the effect of the UBQ10 intron on gene expression from 6 different positions surrounding the TSS of a TRP1:GUS fusion. The intron strongly increased expression from all transcribed positions, but had no effect when 204 nt or more upstream of the 5-most TSS. When the intron was located in the 5 UTR, the TSS unexpectedly changed, resulting in longer transcripts. Remarkably, deleting 303 nt of the core promoter, including all known TSSs and all but 18 nt of the 5 UTR, had virtually no effect on the level of gene expression as long as a stimulating intron was included in the gene. When the core promoter was deleted, transcription initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. Together, these results suggest that certain introns play unexpectedly large roles in directing transcription initiation and represent a previously unrecognized type of downstream regulatory elements for genes transcribed by RNA polymerase II. This study also demonstrates considerable flexibility in the sequences surrounding the TSS, indicating that the TSS is not determined by promoter sequences alone. These findings are relevant in practical applications where introns are used to increase gene expression and contribute to our general understanding of gene structure and regulation in eukaryotes.

molecular biology↗