BackgroundPositive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications, for example, in cell-based sensors.ResultsWe developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. We experimentally tested the ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the dynamic range and sensitivity.ConclusionsThe advantage of our design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or tuned by an external inducer. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits.

BackgroundMembrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses.ResultsIn this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration.ConclusionsOur proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

e14529 Background: High tumor mutation burden is a promising biomarker shown in some cancer types to predict positive response to immune checkpoint inhibitors. We show the ability of a targeted cancer research panel to estimate tumor mutation burden per megabase. Methods: We present a single sample analysis workflow for estimating tumor mutation burden from FFPE research samples. Our assay utilizes a PCR-based target enrichment panel that interrogates 409 known key cancer genes covering ~1.7 megabase of genomic space. Our customized workflow requires only 20 ng of input DNA, and enables a 2 day turn-around time from sample to the result. The ease of our workflow enables less than 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Next-generation Sequencing is performed using high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our custom analysis pipeline calls variants with optimized parameters on the tumor sample only, with no matched normal sample required, and applies filters to remove germ-line variants and background noise. Results: Through in silico analysis performed on The Cancer Genome Atlas (TCGA) data we demonstrate that the panel achieves high sensitivity ( > 90%) and specificity ( > 95%) necessary to separate high and low mutation burden samples. Our workflow provides clear separation between allele ratio of somatic and germ-line variants. Our filters consistently eliminate ~98% of germ-line variants from the set of all variants called in single sample analysis workflow. Evidence from tumor-normal analyses on matched tumor and normal samples suggests that our single sample analysis, on the tumor sample only, detects somatic mutations with high sensitivity and specificity with residual of < 3% germ-line variants. Our pipeline identifies mutational signatures consistent with specific mechanisms such as spontaneous deamination of 5-methyl-cytosine, as well as base-damage from FFPE processing. Conclusions: A simple workflow has been developed on the Ion Torrent sequencing platform to estimate per megabase somatic mutational burden from a single tumor FFPE sample. This solution can help advance research in immuno-oncology.

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Title: Analytical performance of a novel next generation sequencing assay for myeloid cancers Nick Khazanov, Wally Zhang, Dinesh Cyanam, Scott P. Myrand, Denis Kaznadzey, Paul Williams, Vinay Mittal, Dan Mazur, Sihong Chen, Jason Wustman, Efren Ballesteros-Villagrana, Goutam Nistala, Jon Sherlock, Michael Hogan, Jim Veitch, John Bishop, Seth Sadis Introduction: Myeloid malignancies contain a diverse and heterogeneous set of genomic alterations that include recurrent somatic mutations in key driver genes as well as frequent and diagnostic chromosomal rearrangements that generate a wide array of gene fusion products. To support clinical and translational research into precision oncology strategies for myeloid cancers, a next-generation sequencing (NGS) assay was generated to detect common and relevant somatic alterations. Methods: To define gene targets that were recurrently altered in myeloid cancers and relevant for clinical and translational research, an extensive survey of investigators at hematology oncology research labs was performed. The gene targets identified by researchers were complemented by a comprehensive survey of literature and genomic databases. Clinical guidelines for myeloid diseases in the US and in Europe were reviewed to ensure representation of relevant alterations. A targeted Ion AmpliSeq panel was generated to support the detection of recurrent single-nucleotide variants, insertions/deletions, and gene fusions from blood or bone marrow samples. The panel was developed for manual or automated library preparation and sequencing on the Ion Torrent PGM or Ion S5 instruments. Results: The Oncomine™ Myeloid Research Assay gene panel included 58 genes and generated an average read depth of &gt;2,000 reads per targeted amplicon with an average uniformity of &gt;95%. Important GC-rich targets such as CEBPA generated sufficient balanced read depth to support variant detection. A cohort of samples positive for FLT3 alterations was analyzed and successful detection of FLT3-internal tandem repeat variants was demonstrated. Several gene fusion transcripts common to myeloid cancers were detected. Comparable results were observed on Ion Torrent PGM and Ion S5 instruments. Conclusions: A novel myeloid specific NGS assay capable of detecting relevant DNA and RNA alterations from the same sample was developed. The assay is useful for characterizing relevant alterations in a range of myeloid diseases including acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelogenous leukemia, chronic myelomonocytic leukemia, and juvenile myelomonocytic leukemia. A review of the analytical studies will be presented. Citation Format: Nickolay Khazanov, Wally Zhang, Dinesh Cyanam, Scott P. Myrand, Denis Kaznadzey, Paul D. Williams, Vinay Mittal, Daniel J. Mazur, Sihong Chen, Jason Wustman, Efren Ballesteros-Villagrana, Goutam Nistala, Santhoshi Bandla, Jim Veitch, Jon Sherlock, John Bishop, Seth Sadis. Analytical performance of a novel next generation sequencing assay for myeloid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3863.