Biology subjects

Voigt, C.

Publications and source records attributed to Voigt, C..

8 recordsLinked to original sources

bioRxiv2024

A chloroplast cell-free system for measuring ribosome binding site strengths

Plastid engineering offers the potential to carry multi-gene traits in plants, however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth make it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 {+/-} 1 g/mL luciferase in batch reactions. We apply our system to test a library of 104 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold range of luciferase expression normalized by mRNA expression, as assessed by the malachite green aptamer (relative luminescence units per relative fluorescence units). We also find a positive correlation between the observed expression in chloroplast extracts and the predictions made by the RBS calculator. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic programs in plant chloroplasts for diverse applications.

synthetic biology↗

bioRxiv2023

Disentangling gene expression burden identifies generalizable phenotypes induced by synthetic gene networks

Large-scale genetic circuits are rapidly becoming critical components for the next generation of biotechnologies and living therapeutics. However, the relationship between synthetic and host gene expression is poorly understood. To reveal the impact of genetic circuits on their host, we measure the transcriptional response of wild-type and engineered E. coli MG1655 subject to seven genomically integrated circuits and two plasmid-based circuits across 4 growth time points and 4 circuit input states resulting in 1007 transcriptional profiles. We train a classifier to distinguish profiles from wild-type or engineered strains and use the classifier to identify synthetic construct burdened genes, i.e., genes whose dysregulation is dependent on the presence of a genetic circuit and not what is encoded on the circuit. We develop a deep learning architecture, capable of disentangling influence of combinations of perturbations, to model the impact that synthetic genes have on their host. We use the model to hypothesize a generalizable, synthetic cell state phenotype and validate the phenotype through antibiotic challenge experiments. The synthetic cell state results in increased resistance to {beta}-lactam antibiotics in gram-negative bacteria. This work enhances our understanding of circuit impact by quantifying the disruption of host biological processes and can guide the design of robust genetic circuits with minimal burden or uncover novel biological circuits and phenotypes.

systems biology↗

bioRxiv2022

A living conductive marine biofilm engineered to sense and respond to small molecules

Engineered electroactive bacteria have potential applications ranging from sensing to biosynthesis. In order to advance the use of engineered electroactive bacteria, it is important to demonstrate functional expression of electron transfer modules in chassis adapted to operationally relevant conditions, such as non-freshwater environments. Here, we use the Shewanella oneidensis electron transfer pathway to induce current production in a marine bacterium, Marinobacter atlanticus, during biofilm growth in artificial seawater. Genetically encoded sensors optimized for use in E. coli were used to control protein expression in planktonic and biofilm attached cells. Significant current production required addition of menaquinone, which M. atlanticus does not produce, for electron transfer from the inner membrane to the expressed electron transfer pathway. Current through the S. oneidensis pathway in M. atlanticus was observed when inducing molecules were present during biofilm formation. Electron transfer was also reversible, indicating electron transfer into M. atlanticus could be controlled. These results show that an operationally relevant marine bacterium can be genetically engineered for environmental sensing and response using an electrical signal.

microbiology↗

bioRxiv2021

A synthetic distributed genetic multi-bit counter

A design for genetically-encoded counters is proposed via repressor-based circuits. An N -bit counter reads sequences of input pulses and displays the total number of pulses, modulo 2N . The design is based on distributed computation, with specialized cell types allocated to specific tasks. This allows scalability and bypasses constraints on the maximal number of circuit genes per cell due to toxicity or failures due to resource limitations. The design starts with a single-bit counter. The N -bit counter is then obtained by interconnecting (using diffusible chemicals) a set of N single-bit counters and connector modules. An optimization framework is used to determine appropriate gate parameters and to compute bounds on admissible pulse widths and relaxation (inter-pulse) times, as well as to guide the construction of novel gates. This work can be viewed as a step toward obtaining circuits that are capable of finite-automaton computation, in analogy to digital central processing units. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/468063v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@19a5330org.highwire.dtl.DTLVardef@cbb5c2org.highwire.dtl.DTLVardef@a0b5borg.highwire.dtl.DTLVardef@16f9d07_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Prediction of Whole-Cell Transcriptional Response with Machine Learning

Applications in synthetic and systems biology can benefit from measuring whole-cell response to biochemical perturbations. Execution of experiments to cover all possible combinations of perturbations is infeasible. In this paper, we present the host response model (HRM), a machine learning approach that takes the cell response to single perturbations as the input and predicts the whole cell transcriptional response to the combination of inducers. We find that the HRM is able to qualitatively predict the directionality of dysregulation to a combination of inducers with an accuracy of >90% using data from single inducers. We further find that the use of known prior, known cell regulatory networks doubles the predictive performance of the HRM (an R2 from 0.3 to 0.65). This tool will significantly reduce the number of high-throughput sequencing experiments that need to be run to characterize the transcriptional impact of the combination of perturbations on the host.

bioinformatics↗

bioRxiv2020

Distributed implementation of Boolean functions by transcriptional synthetic circuits

Starting in the early 2000s, a sophisticated technology has been developed for the rational construction of synthetic genetic networks that implement specified logical functionalities. Despite impressive progress, however, the scaling necessary in order to achieve greater computational power has been hampered by many constraints, including repressor toxicity and the lack of large sets of mutually-orthogonal repressors. As a consequence, a typical circuit contains no more than roughly seven repressor-based gates per cell. A possible way around this scalability problem is to distribute the computation among multiple cell types, which communicate among themselves using diffusible small molecules (DSMs) and each of which implements a small sub-circuit. Examples of DSMs are those employed by quorum sensing systems in bacteria. This paper focuses on systematic ways to implement this distributed approach, in the context of the evaluation of arbitrary Boolean functions. The unique characteristics of genetic circuits and the properties of DSMs require the development of new Boolean synthesis methods, distinct from those classically used in electronic circuit design. In this work, we propose a fast algorithm to synthesize distributed realizations for any Boolean function, under constraints on the number of gates per cell and the number of orthogonal DSMs. The method is based on an exact synthesis algorithm to find the minimal circuit per cell, which in turn allows us to build an extensive database of Boolean functions up to a given number of inputs. For concreteness, we will specifically focus on circuits of up to 4 inputs, which might represent, for example, two chemical inducers and two light inputs at different frequencies. Our method shows that, with a constraint of no more than seven gates per cell, the use of a single DSM increases the total number of realizable circuits by at least 7.58-fold compared to centralized computation. Moreover, when allowing two DSMs, one can realize 99.995% of all possible 4-input Boolean functions, still with at most 7 gates per cell. The methodology introduced here can be readily adapted to complement recent genetic circuit design automation software.

synthetic biology↗

bioRxiv2019

Genetic encoding of targeted MRI contrast agents for in vivo tumor imaging

Tumor-selective contrast agents have the potential to aid in the diagnosis and treatment of cancer using noninvasive imaging modalities such as magnetic resonance imaging (MRI). Such contrast agents can consist of magnetic nanoparticles incorporating functionalities that respond to cues specific to tumor environments. Genetically engineering magnetotactic bacteria to display peptides has been investigated as a means to produce contrast agents that combine the robust image contrast effects of magnetosomes with transgenic targeting peptides displayed on their surface. This work reports the first use of magnetic nanoparticles that display genetically-encoded pH low insertion peptide (pHLIP), a long peptide intended to enhance MRI contrast by targeting the extracellular acidity associated with the tumors. To demonstrate the modularity of this versatile platform to incorporate diverse targeting ligands by genetic engineering, we also incorporated the cyclic v integrin-binding peptide iRGD into separate magnetosomes. Specifically, we investigate their potential for enhanced binding and tumor imaging both in vitro and in vivo. Our experiments indicate that these tailored magnetosomes retain their magnetic properties, making them well-suited as T2 contrast agents, while exhibiting increased binding compared to wild-type magnetosomes.

synthetic biology↗

bioRxiv2017

Surface-driven RNA-refolding by the OB-fold proteins of the Trypanosoma brucei editosome

RNA editing in African trypanosomes represents an RNA-processing reaction that generates functional mitochondrial transcripts from sequence-deficient pre-mRNAs. The reaction is catalyzed by a macromolecular protein complex known as the editosome. Editosomes have been demonstrated to execute RNA-chaperone activity to overcome the highly folded nature of pre-edited substrate mRNAs. The molecular basis of this activity is unknown. Here we test five OB-fold proteins of the editosome as potential candidates. We show that the different proteins interact by hetero-oligomerization and we demonstrate that all proteins execute RNA-chaperone activity. Activity differences correlate with the surface areas of the proteins and map predominantly to the intrinsically disordered subdomains of the polypeptides. To provide a structural context for our findings we present a coarse-grained model of the editosome. The model suggests that an inner core of catalytically active editosome components is separated from an outer shell of IDP-domains that act as RNA-remodeling sites.

molecular biology↗