scholarly journals Cas9-Assisted Biological Containment of a Genetically Engineered Human Commensal Bacterium and Genetic Elements

2021 ◽  
Author(s):  
Naoki Hayashi ◽  
Yong Lai ◽  
Mark Mimee ◽  
Timothy K Lu
Keyword(s):  
Gene Expression ◽  
Gene Cassette ◽  
Genetically Engineered ◽  
Commensal Bacterium ◽  
Human Gut ◽  
Genetic Elements ◽  
Thymidine Auxotrophy ◽  
Engineered Bacteria

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination for at least 10 days. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

HMGN proteins play roles in DNA repair and gene expression in mammalian cells

2004 ◽  
Vol 32 (6) ◽  
pp. 918-919 ◽  
Author(s):  
K.L. West
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Knockout Mice ◽  
Mammalian Cells ◽  
High Mobility ◽  
Genetically Engineered ◽  
Expression In Mammalian Cells ◽  
Transcription And Replication

HMGN (high-mobility-group N) family members are vertebrate proteins that unfold chromatin and promote transcription and replication of chromatin templates in vitro. However, their precise roles in vivo have been elusive until recently. This paper summarizes recent advances from studies of Hmgn1 knockout mice and genetically engineered cell lines that are beginning to reveal the diverse roles that HMGN proteins play in DNA repair and transcription within mammalian cells.

scholarly journals Metatranscriptome profiling of the dynamic transcription of mRNA and sRNA of a probioticLactobacillusstrain in human gut

2018 ◽  
Author(s):  
Jicheng Wang ◽  
Zhihong Sun ◽  
Jianmin Qiao ◽  
Dong Chen ◽  
Chao Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactobacillus Casei ◽  
Lactic Acid Production ◽  
Expression Patterns ◽  
Probiotic Bacteria ◽  
Gi Tract ◽  
Human Gut ◽  
Bacterial Gene

AbstractMetatranscriptomic sequencing has recently been applied to study how pathogens and probiotics affect human gastrointestinal (GI) tract microbiota, which provides new insights into their mechanisms of action. In this study, metatranscriptomic sequencing was applied to deduce thein vivoexpression patterns of an ingestedLactobacillus caseistrain, which was compared with itsin vitrogrowth transcriptomes. Extraction of the strain-specific reads revealed that transcripts from the ingestedL. caseiwere increased, while those from the residentL. paracaseistrains remained unchanged. Mapping of all metatranscriptomic reads and transcriptomic reads toL. caseigenome showed that gene expressionin vitroandin vivodiffered dramatically. About 39% (1163) mRNAs and 45% (93) sRNAs ofL. caseiwell-expressed were repressed after ingested into human gut. Expression of ABC transporter genes and amino acid metabolism genes was induced at day-14 of ingestion; and genes for sugar and SCFA metabolisms were activated at day-28 of ingestion. Moreover, expression of sRNAs specific to thein vitrolog phase was more likely to be activated in human gut. Expression of rli28c sRNA with peaked expression during thein vitrostationary phase was also activated in human gut; this sRNA repressedL. caseigrowth and lactic acid productionin vitro. These findings implicate that the ingestedL. caseimight have to successfully change its transcription patterns to survive in human gut, and the time-dependent activation patterns indicate a highly dynamic cross-talk between the probiotic and human gut including its microbe community.ImportanceProbiotic bacteria are important in food industry and as model microorganisms in understanding bacterial gene regulation. Although probiotic functions and mechanisms in human gastrointestinal tract are linked to the unique probiotic gene expression, it remains elusive how transcription of probiotic bacteria is dynamically regulated after being ingested. Previous study of probiotic gene expression in human fecal samples has been restricted due to its low abundance and the presence of of closely related species. In this study, we took the advantage of the good depth of metatranscriptomic sequencing reads and developed a strain-specific read analysis method to discriminate the transcription of the probioticLactobacillus caseiand those of its resident relatives. This approach and additional bioinformatics analysis allowed the first study of the dynamic transcriptome profiles of probioticL casei in vivo. The novel findings indicate a highly regulated repression and dynamic activation of probiotic genome in human GI tract.

scholarly journals Detection and analysis of gene expression during infection by in vivo expression technology

2000 ◽  
Vol 355 (1397) ◽  
pp. 587-599 ◽  
Author(s):  
D. Scott Merrell ◽  
Andrew Camilli
Keyword(s):  
Gene Expression ◽  
Gene Fusion ◽  
Ex Vivo ◽  
Gene Cassette ◽  
Chromosome Loss ◽  
In Vivo Expression ◽  
Reporter Systems ◽  
A Site

Many limitations associated with the use of in vitro models for study of bacterial pathogenesis can be overcome by the use of technologies that detect pathogen gene expression during the course of infection within an intact animal. in vivo expression technology (IVET) accomplishes this with versatility: it has been developed with a variety of reporter systems which allow for either in vivo selection or ex vivo screening. Selectable gene fusion systems generally allow for the complementation of a bacterial metabolic defect that is lethal in vivo, or for antibiotic resistance during the course of in vivo antibiotic challenge. In contrast, the screenable gene fusion system uses a site–specific DNA recombinase that, when expressed in vivo, excises a selectable gene cassette from the bacterial chromosome. Loss of this cassette can then be either screened or selected for ex vivo . The recombinase–based IVET can be used to detect genes that are transcriptionally induced during infection, including those expressed transiently or at low levels and, in addition, can be used to monitor the spatial and temporal expression of specific genes during the course of infection.

Effect of X-irradiation on gene expression of rat liver chemokines: in vivo and in vitro studies

2008 ◽  
Vol 46 (01) ◽  
Author(s):  
F Moriconi ◽  
H Christiansen ◽  
H Christiansen ◽  
N Sheikh ◽  
J Dudas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
In Vitro Studies ◽  
X Irradiation

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle

2017 ◽  
Vol 95 (3) ◽  
pp. 1313 ◽  
Author(s):  
L. Zhang ◽  
L. F. Schütz ◽  
C. L. Robinson ◽  
M. L. Totty ◽  
L. J. Spicer
Keyword(s):  
Gene Expression ◽  
Tight Junction ◽  
Tight Junction Proteins ◽  
Junction Proteins

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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