Biology subjects

Mukhopadhyay, A.

Publications and source records attributed to Mukhopadhyay, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane), and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

synthetic biology↗

bioRxiv2023

Automated Protein Affinity Optimization using a 1D-CNN Deep Learning Model

Functional biologics design is a multi-objective optimization problem often with competing design objectives. We report on a novel deep learning based protein sequence prediction framework, ZymeSwapNet, that can be customized to handle a wide range of quantifiable design objectives, a current limitation of traditional protein design methods. We train a simple convolutional neural network (1D-CNN) on nonredundant curated protein crystal structures, using a set of geometric and topological features that describes a local protein environment, to predict the likelihood of each amino acid type for residue sites in the design region. While the model can be directly used to rank templates derived from mutagenesis campaigns, we extend the scope by developing a sequence/mutation generator that optimizes the desired multivariate distribution using a Monte-Carlo sampling. Using a case study - the design of a stable heterodimeric Fc (HetFc) antibody domain - we show that we can further include a Metropolis criterion to bias the sampling to enhance features such as the heterodimeric binding specificity, in addition to original sampling objective of enhancing stability. We demonstrate that ZymeSwapNet can generate stable HetFc designs, within minutes that had taken several rounds of rational structure and physical force-field based modeling attempts.

bioengineering↗

bioRxiv2023

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

While many heterologous molecules can be produced via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth coupling para-coumarate to indigoidine production and prototype two cut sets in Pseudomonas putida KT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth on p-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY in para-coumarate minimal medium. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/532821v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f0754aorg.highwire.dtl.DTLVardef@a45d24org.highwire.dtl.DTLVardef@df0596org.highwire.dtl.DTLVardef@1e7e140_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2023

A 30-Color Full-Spectrum Flow Cytometry Panel to Characterize the Immune Cell Landscape in Spleen and Tumors within a Syngeneic MC-38 Murine Colon Carcinoma Model

Purpose and Appropriate Sample TypesThis panel was designed to characterize the immune cell landscape in the mouse tumor micro-environment as well as mouse lymphoid tissues (e.g., spleen). Previous Optimized Multicolor Immunofluorescence Panels (OMIP) with conventional cytometry were examples of high-quality fluorescent-based flow cytometry panels to characterize either the T-cell compartment or the myeloid compartment [1, 2]. The advent of spectral cytometry has enabled sufficient markers to be included in a panel to comprehensively characterize the immune cell landscape including both the T-cell and the myeloid cell compartments. In this body of work, we demonstrated that we could measure the frequency and characterize the functional status of CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells, macrophages, granulocytes, monocytes, & dendritic cells. This panel is especially useful for understanding the immune landscape in "cold" preclinical tumor models with very low immune cell infiltration, and for investigating how therapeutic treatments may modulate the immune landscape.

cancer biology↗

bioRxiv2022

A chromosome-scale assembly of Brassica carinata (BBCC) accession HC20 containing resistance to multiple pathogens and an early generation assessment of introgressions into B. juncea (AABB)

Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the Northeastern regions of Africa. Although it is grown in a limited manner, B. carinata is of value as it is resistant/highly tolerant to most of the pathogens affecting cultivated Brassica species of the Us triangle that are grown worldwide as oilseed and vegetable crops. We report a chromosome-scale genome assembly of B. carinata accession HC20 using long-read Oxford Nanopore and Illumina sequencing and BioNano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the available long-read genome assemblies of the six species of the Us triangle and found a highly conserved gene number and collinearity suggesting that B. juncea (AABB), B. napus (AACC), and B. carinata are strict allopolyploids. We cataloged the nucleotide-binding and leucine-rich repeat immune receptor (NLR) repertoire of B. carinata resulting in the identification of 465 NLRs. We investigated the extent and nature of early generation genomic interactions between the subgenomes of B. carinata and B. juncea in interspecific crosses between the two species. We found that C chromosome additions are well tolerated, with homoeologous exchanges occurring between the A and C genomes. Based on the genomic interactions, we propose strategies to utilize the interspecific crosses for transferring disease resistance from B. carinata to B. juncea and other Brassica species.

plant biology↗

bioRxiv2022

Revealing Pentose Catabolism in Pseudomonas putida

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (strain M2), while the third isolate represents a new species in the group (strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPRi system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion. Originality-Significance StatementMembers of the Pseudomonas putida group are intensively studied for their role in plant growth promotion and biomass conversion. Despite this interest, the scope of pentose oxidation, key sugars in plant biomass, in this group is not known. Here, we report targeted isolation of members of the P. putida group that grow by xylose and arabinose oxidation. Using a combined genomic and proteomic approach, we identify gene products involved in pentose oxidation and identify conditionally essential genes for xylose oxidation using a CRISPRi gene repression approach. This work describes a targeted isolation and analysis strategy that may applied for many microbial groups of industrial and agricultural interest.

microbiology↗

bioRxiv2022

Genetic affinities and sub-structuring in Coorg population of Southern India

The Coorgs, also known as Kodavas, are one of the smallest religious and socio-culturally homogenous communities in the world, currently residing in the state of Karnataka, India. Due to a stark contrast with the surrounding subpopulations, their genetic architecture and population & demographic history have been a matter of debate for long. To better understand the population structure and demographic history of this caste group, we analysed the population, using high-resolution autosomal (n=70) as well uniparentally inherited markers (Y-chromosomal and mitochondrial DNA) (n=144). Our first ever findings elucidate that origin of Coorgs traces back to early or middle Bronze Age. We further found population substructure among Coorgs, which manifested as three distinct clusters in a Principal component analysis. One of these subgroups has undergone a rare and immense amount of population-specific drift but all three eventually admixed, both genetically and socio-culturally. The mtDNA analysis showed 40% South Asian-specific mitochondrial lineages among Coorgs; while the Y-chromosomal analysis revealed predominant presence of Eurasian, Middle-Eastern and Indian-specific haplogroups suggesting male-centric migration and eventual assimilation with local females. Our results for the first time identify these ancient and distinct genealogies that make up the contemporary Coorgs and may explain the socio-cultural differences with their immediate and distant neighbours in the country and the prevalent narrative history. In a wider context, the study also reveals an ancient, yet unknown, Middle Eastern source population that might have contributed to an early west to east migration into India.

genetics↗

bioRxiv2022

Modeling Hippocampal Spatial Cells in Rodents navigating in 3D environments

Studies on the neural correlates of navigation in 3D environments are plagued by several unresolved issues. For example, experimental studies show markedly different place cell responses in rats and bats, both navigating in 3D environments. In an effort to understand this divergence, we propose a deep autoencoder network to model the place cells and grid cells in a simulated agent navigating in a 3D environment. We also explore the possibility of a vital role that Head Direction (HD) tuning plays in determining the isotropic or anisotropic nature of the observed place fields in different species. The input layer to the autoencoder network model is the HD layer which encodes the agents HD in terms of azimuth ({theta}) and pitch angles ({phi}). The output of this layer is given as input to the Path Integration (PI) layer, which integrates velocity information into the phase of oscillating neural activity. The output of the PI layer is modulated and passed through a low pass filter to make it purely a function of space before passing it to an autoencoder. The bottleneck layer of the autoencoder model encodes the spatial cell like responses. Both grid cell and place cell like responses are observed. The proposed model is verified using two experimental studies with two 3D environments in each. This model paves the way for a holistic approach of using deep networks to model spatial cells in 3D navigation.

neuroscience↗

bioRxiv2022

Whole exome-sequencing of vitiligo lesions indicate lower burden of somatic variations: implications in risk for non-melanoma skin cancers

Mapping of somatic variations has enabled understanding the progression of clonal variations from healthy skin to cutaneous malignancies. Highlighting, the adaptive nature of pigmentation, germline mutations in albinism amplify skin cancer susceptibility. However, lower incidence of non-melanoma skin cancer among subjects with acquired depigmenting skin disorder vitiligo is enigmatic and a matter of longstanding debate. To address this, we performed high-coverage exome sequencing of matched non-lesional and lesional vitiligo skin along with whole blood to account for germline variations. Our analysis suggests lower burden of somatic cancer-associated variations in exposed depigmented lesional skin compared to the non-lesional skin. A detailed investigation of vitiligo skin transcriptome reveals elevation of DNA repair and cell-proliferation pathways. Validation by comet-assay for DNA damage and cell cycle analysis of epidermal cells suggest undamaged DNA in vitiligo lesions that could be attributed to higher proliferation-coupled repair. Endorsing this, UV-signature variations are not prominent, instead SBS5 associated with endogenous mutational processes is conspicuous in both the vitiligo tissues. Our systematic pilot study indicates lower somatic mutation burden in vitiligo skin and supports the earlier demographic observation on lower risk of non-melanoma skin cancer in vitiligo subjects, providing an opportunity to learn strategies for cancer prevention from vitiligo. Brief SummaryVitiligo skin harbors decreased somatic variation burden in cancer-associated genes and a concomitant augmentation in DNA repair response, explaining the lower incidence of cutaneous malignancies.

genomics↗

bioRxiv2022

A cell non-autonomous FOXO/DAF-16-mediated germline quality assurance program that responds to somatic DNA damage

Germline integrity is critical for progeny fitness. Organisms deploy the DNA damage response (DDR) signalling to protect germline from genotoxic stress, facilitating cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germline quality is well-studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin-IGF-1 signalling (IIS) is low, arrests germline development and induces sterility in a FOXO/DAF-16 transcription factor (TF)-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape arrest to produce poor quality oocytes, showing that the TF imposes strict quality control during low IIS. In response to low IIS in neurons, FOXO/DAF-16 works cell autonomously as well as non-autonomously to facilitate the arrest. Activated FOXO/DAF-16 promotes transcription of checkpoint and DDR genes, protecting germline integrity. However, on reducing DDR during low IIS, the TF decreases ERK/MPK-1 signaling below a threshold, and transcriptionally downregulates genes involved in spermatogenesis-to-oogenesis switch as well as cdk-1/Cyclin B to promote germline arrest. Altogether, our study reveals how cell non-autonomous function of FOXO/DAF-16 promotes germline quality and progeny fitness in response to somatic DNA damage. Significance StatementReproductive decisions are supervised processes that take into account various inputs like cellular energy availability and status of damage repair in order to ensure healthy progeny. In this study, we show that the absence of optimal DNA damage repair in the somatic uterine tissues prevents oocyte development by the cell-autonomous as well non-autonomous function of activated FOXO transcription factor DAF-16. Thus, this study elucidates a new surveillance role of FOXO/DAF-16 in somatic tissues that ensures progeny fitness.

developmental biology↗

bioRxiv2022

Differences in durability of PARP inhibition by clinically approved PARP inhibitors: implications for combinations and scheduling

Five PARP inhibitors (PARPi) are approved for cancer treatment, they exploit cancer-specific defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous PARP inhibition is required for single-agent anticancer activity. PARPi are also being investigated with ATR inhibitors clinically. We previously showed rucaparib caused prolonged PARP inhibition. Here we aimed to determine if this property was unique to rucaparib or common to other PARPis and the implications for scheduling with an ATR inhibitor (VE-821). Durability of PARP inhibition was determined at 0, 1, 24, 48 and 72 h after a 1 h pulse of 1M of rucaparib, olaparib, niraparib, talazoparib or pamiparib in IGROV-1 (human ovarian cancer) cells. Inhibition of PARP was sustained to a variable degree with all inhibitors, but reduced with time. Rucaparib caused the most persistent inhibition of PARP activity, which was maintained at [≥]75% for 72 h after drug withdrawal. In contrast, only 12% inhibition remained at this time with talazoparib and pamiparib and no detectable inhibition with olaparib and niraparib. Rucaparib enhanced VE-821 cytotoxicity to a similar extent in a sequential schedule as in co-exposure studies (PF50: 2.6 vs. 2.7) and there was even an approx. 2-fold enhancement after a 24 h delay between rucaparib and VE-821. Olaparib and niraparib produced similar enhancement of VE-821 cytotoxicity if co-exposed but were ineffective in sequential exposures. These data have clinical implications for both schedules of current PARPi monotherapy and the scheduling of PARPi in combination with ATRi and other cytotoxic drugs. Novelty and ImpactPARPi are a new class of anticancer agent. We demonstrate for the first time that 5 PARPi continue to suppress cellular PARP activity after drug removal to a variable extent. Rucaparib caused the most durable PARP inhibition, olaparib and niraparib the least. Rucaparib enhanced ATR inhibitor cytotoxicity in sequential and co-exposures, olaparib and niraparib were only active in co-exposure settings. These data have implications for the clinical use of PARPi, particularly in combination with other drugs.

pharmacology and toxicology↗

bioRxiv2022

mTORC2 Drives ZFX-mediated Ganglioside Biosynthesis to Promote Breast Cancer Progression.

Sphingolipid and ganglioside metabolic pathways are crucial components of cell signalling, having established roles in tumor cell proliferation, invasion, and migration. However, regulatory mechanisms controlling sphingolipid and ganglioside biosynthesis in mammalian cells is less known. Here, we show that RICTOR, the regulatory subunit of mTORC2, regulates the synthesis of sphingolipids and gangliosides in luminal breast cancer-specific MCF-7 and BT-474 cells through transcriptional and epigenetic mechanisms. RICTOR regulates glucosylceramide levels by modulating the expression of UDP-Glucose Ceramide Glucosyl transferase (UGCG). We identify Zinc Finger protein X-linked (ZFX) as a RICTOR-responsive transcription factor whose recruitment to the UGCG promoter is regulated by DNA methyltransferases and histone demethylase (KDM5A) that are known AKT substrates. We further demonstrate that RICTOR regulates the synthesis of GD3 gangliosides through ZFX and UGCG, and triggers the activation of EGFR signalling pathway, thereby promoting tumor growth. In line with our findings in cell culture and mice models, we observe an elevated expression of RICTOR, ZFX, and UGCG in Indian luminal breast cancer tissues, and in TCGA and METABRIC datasets. Together, we establish a key regulatory circuit, RICTOR-AKT-ZFX-UGCG-Ganglioside-EGFR-AKT, and elucidate its contribution to breast cancer progression.

cancer biology↗

bioRxiv2021

Optimizing a High Performing Multiplex-CRISPRi P. putida strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon ({Delta}phaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the {Delta}phaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.

synthetic biology↗

bioRxiv2021

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3){Delta} pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

biochemistry↗

bioRxiv2021

Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCEUnderstanding the genetic basis of P. putidas diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.

microbiology↗

bioRxiv2021

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation under Ionic Liquid Stress

Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs. absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.

systems biology↗

bioRxiv2021

Using Deep Learning to Count Monarch Butterflies in Dense Clusters

Monarch butterflies display one of the most fascinating migration patterns of all species, traveling over 3000 miles from their North American breeding grounds to reach overwintering sites in Central Mexico. Recent studies have suggested that monarchs have experienced an alarming decline in population size due to a combination of deforestation, loss of native milkweed and nectaring plants, and climate change. An issue that conservation efforts face is the lack of principled mechanisms to accurately estimate and count the population size of monarchs. This difficulty occurs due to their small size and existence in dense overwintering clusters in forests. We create an open-source tool to aid conservationists estimate the count of monarch butterflies from images automatically. To the best of our knowledge, our approach, based on deep convolutional neural networks, is the first automated application that can count small insects like monarch butterflies in dense clusters. We demonstrate that our approach achieves high accuracy in counting the number of butterflies even in the presence of occlusion. We also release an open-source dataset containing high resolution images of monarch butterflies along with human annotations for each butterflys position. Our open-source implementation can be readily used by scientists to estimate monarch numbers in overwintering clusters and could also be adapted for use in other clustering species.

bioengineering↗

bioRxiv2021

Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations-electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the systems capabilities through CRISPR-based engineering of two test cases-1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

synthetic biology↗