The document discusses synthetic biology and some of its tools and applications. It describes techniques like zinc fingers, siRNA, and recombinases that can be used to engineer biological systems. Examples of engineered circuits include toggle switches, counters, and systems using Cre/FLPe recombinases. Potential applications mentioned include whole-cell biosensors, phage-based microbial engineering, biological containment, and iPSC control, though issues around modularity and biological knowledge need further consideration.
Flash introduction to Qiime2 -- 16S Amplicon analysisAndrea Telatin
Review of basic concepts in the 16S Amplicon analysis workflow for microbial community characterization, and brief introdution to Qiime and Qiime 2 concepts.
BiteSized seminar at Quadram Institute, UK
Presentation in the "Whole genome sequencing for clinical microbiology:Translation into routine applications" Symposium , Basel , Switzerland, 2 Sep 2017
Saha UC Davis Plant Pathology seminar Infrastructure for battling the Citrus ...Surya Saha
Rapidly spreading invasive diseases in systems with little or no prior experimental data or resources pose a unique set of challenges for growers, scientists as well as regulators. As a part of a USDA NIFA CAPS project focused on the psyllid, Diaphorina citri, we have released improved genomics resources including high quality genome assemblies and annotation. We have also created an open access web portal for analyses around the Citrus Greening/Huanglongbing disease complex. Citrusgreening.org includes pathosystem-wide resources and bioinformatics tools for multiple Citrus spp. hosts, the Asian citrus psyllid vector (ACP, Diaphorina citri), and multiple pathogens including Candidatus Liberibacter asiaticus (CLas). To the best of our knowledge, this is the first example of a database to use the pathosystem as a holistic framework to understand an insect transmitted plant disease. Users can submit relevant data sets to enable sharing and allow the community to leverage their data within an integrated system. The system includes the metabolic pathway databases CitrusCyc and DiaphorinaCyc with organism specific pathways that can be used to mine metabolomics, transcriptomics and proteomics results to identify pathways and regulatory mechanisms involved in disease response. The Psyllid Expression Network (PEN) contains expression profiles of ACP genes from multiple life stages, tissues, conditions and hosts. The Citrus Expression Network (CEN) contains public expression data from multiple tissues and conditions for various citrus hosts. All tools connect to a central database. The portal also includes electrical penetration graph (EPG) recordings, information about citrus rootstock trials and metabolomics data in addition to traditional omics data types with a goal of combining and mining all information related to the Huanglongbing pathosystem. User-friendly manual curation tools will allow the continuous improvement of knowledge base as more experimental research is published. The portal can be accessed at https://citrusgreening.org/.
Flash introduction to Qiime2 -- 16S Amplicon analysisAndrea Telatin
Review of basic concepts in the 16S Amplicon analysis workflow for microbial community characterization, and brief introdution to Qiime and Qiime 2 concepts.
BiteSized seminar at Quadram Institute, UK
Presentation in the "Whole genome sequencing for clinical microbiology:Translation into routine applications" Symposium , Basel , Switzerland, 2 Sep 2017
Saha UC Davis Plant Pathology seminar Infrastructure for battling the Citrus ...Surya Saha
Rapidly spreading invasive diseases in systems with little or no prior experimental data or resources pose a unique set of challenges for growers, scientists as well as regulators. As a part of a USDA NIFA CAPS project focused on the psyllid, Diaphorina citri, we have released improved genomics resources including high quality genome assemblies and annotation. We have also created an open access web portal for analyses around the Citrus Greening/Huanglongbing disease complex. Citrusgreening.org includes pathosystem-wide resources and bioinformatics tools for multiple Citrus spp. hosts, the Asian citrus psyllid vector (ACP, Diaphorina citri), and multiple pathogens including Candidatus Liberibacter asiaticus (CLas). To the best of our knowledge, this is the first example of a database to use the pathosystem as a holistic framework to understand an insect transmitted plant disease. Users can submit relevant data sets to enable sharing and allow the community to leverage their data within an integrated system. The system includes the metabolic pathway databases CitrusCyc and DiaphorinaCyc with organism specific pathways that can be used to mine metabolomics, transcriptomics and proteomics results to identify pathways and regulatory mechanisms involved in disease response. The Psyllid Expression Network (PEN) contains expression profiles of ACP genes from multiple life stages, tissues, conditions and hosts. The Citrus Expression Network (CEN) contains public expression data from multiple tissues and conditions for various citrus hosts. All tools connect to a central database. The portal also includes electrical penetration graph (EPG) recordings, information about citrus rootstock trials and metabolomics data in addition to traditional omics data types with a goal of combining and mining all information related to the Huanglongbing pathosystem. User-friendly manual curation tools will allow the continuous improvement of knowledge base as more experimental research is published. The portal can be accessed at https://citrusgreening.org/.
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In this lesson, students will mine data from Araport.org to design and propose a reverse genetics experiment using a known Arabidopsis mutant. They will select a treatment to reveal phenotypic dfifferences between wild type and mutant Arabidopsis. Student handout and teacher resources are available at www.Araport.org, teacher resources. Suitable for grades 9-12 or first year undergraduate students.
This presentation is a thorough guide to the use of Web Apollo, with details on User Navigation, Functionality, and the thought process behind manual annotation.
During this workshop, participants:
- Learn to identify homologs of known genes of interest in a newly sequenced genome.
- Become familiar with the environment and functionality of the Web Apollo genome annotation editing tool.
- Learn how to corroborate or modify automatically annotated gene models using available evidence in Web Apollo.
- Understand the process of curation in the context of genome annotation.
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Mining Phenotypes: How to set up a reverse genetics experiment with an Arabid...adcobb
In this lesson, students will mine data from Araport.org to design and propose a reverse genetics experiment using a known Arabidopsis mutant. They will select a treatment to reveal phenotypic dfifferences between wild type and mutant Arabidopsis. Student handout and teacher resources are available at www.Araport.org, teacher resources. Suitable for grades 9-12 or first year undergraduate students.
This presentation is a thorough guide to the use of Web Apollo, with details on User Navigation, Functionality, and the thought process behind manual annotation.
During this workshop, participants:
- Learn to identify homologs of known genes of interest in a newly sequenced genome.
- Become familiar with the environment and functionality of the Web Apollo genome annotation editing tool.
- Learn how to corroborate or modify automatically annotated gene models using available evidence in Web Apollo.
- Understand the process of curation in the context of genome annotation.
Next Generation Sequencing Informatics - Challenges and OpportunitiesChung-Tsai Su
Genetic data is the foundation of precision medicine. Next Generation Sequencing(NGS) enable us to get our whole genome data in affordable cost. How to process huge amount of NGS data effectively ?
ICAR 2015
Workshop 10 (TUESDAY, JULY 7, 2015, 4:30-6:00 PM)
The Arabidopsis information portal for users and developers
Nick Provart (University of Toronto)
A Community Collaborator Perspective: Case study 1 - BioAnalytic Resource
Development of FDA MicroDB: A Regulatory-Grade Microbial Reference DatabaseNathan Olson
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Utilizing system biology resources to decipher a tritrophic disease complex presented at 2017 Annual meeting of Entomological Society of America at Denver, Colorado
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"Development of FDA MicroDB: A Regulatory-Grade
Microbial Reference Database" presentation at the Standards for Pathogen Identification via NGS (SPIN) workshop hosted by National Institute for Standards and Technology October 2014 by Heike Sichtig, PhD from the FDA and Luke Tallon from IGS UMSOM.
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A Global Commons for Scientific Data: Molecules and Wikidatapetermurrayrust
Methods for extracting facts from the scientific literature, and linking them to Wikidata IDs. Wikidata is introduced by an architectural example and bioscience. Then we explore how data can be extracted from text and from images
6. Toolbox
• Zinc-finger
• siRNA
• Recombinase
http://www.scholarpedia.org/article/Gene_assembly_in_Ciliates Alonso J C et al. J. Biol. Chem. 1995;270:2938-2945
28. Oh, the possibilities!
• Whole-cell biosensors
• Microbial engineering using circuit carrying
bacteriophages
• Biological containment
• iPSC control
29. Issues of consideration
• Modularity
vs.
• Biological knowledge
• Probes to assay and characterize in vivo
operation of synthetic gene circuits
http://estherspace.wordpress.com/2008/01/21/ http://naturalorder.info/
30. References
• Friedland et al. Synthetic Gene Netowrks
that Count. Science (2009)
• Lu, Khalil, and Collins. Next-generation
synthetic gene networks. Nature
Biotechnology (2009)
• Gardner, Canter, and Collins. Construction
of a genetic toggle switch in Escherichia
coli. Nature (2000)
Editor's Notes
Instead of having circuits with switches and oscillators, you are building a circuit made of biological counter-parts. Goal: Whole-cell biosensors: made with e.coli. Focus of talk: variety of different networks and switches that are used and how they are constructed
Control at: Signal Repressor Translation Structure of Operon Activation of proteins
Zinc finger domain: bind DNA Proteins are modular
Zinc finger domain: bind DNA Proteins are modular
Memory elements and genetic counters Go to board to explain recombinatiom ~100 natural recombinases are known Recombinases recognize a short DNA sequence motif, and they do so by contact at a few a.a. residues Easily genetically engineered for greater diversity and sequence specificity
Ribosome-binding Site A sequence that is complementary to the ribosome binding site (cis-repressor sequence) is placed upstream of it, so that transcription of this RNA leads to hairpin This secondary structure blocks 30S ribosomal subunit binding to RBS->inhibit translation However, if you express a short trans-activating non-coding RNA, called a taRNA, that contains a sequence that is complementary to the cis-regulatory sequence Then this sequence will bind to the cis-repressor sequence, disrupting the hairpin structure and relieving the ribosome binding site cis-acting generally means "acting from the same molecule " ( i.e. , intramolecular). It may be considered the opposite of trans-acting which generally means "acting from a different molecule" ( i.e. , intermolecular).
This is the motivating factor for the toggle switch The toggle switch is composed of two repressors and two promoters. Each promoter is inhibited by the repressor that is transcribed by the opposing promoter If both promoters have then same strength, then this results in a network with bistable behaviour
The toggle switch is composed of two repressors and two promoters. Each promoter is inhibited by the repressor that is transcribed by the opposing promoter If both promoters have then same strength, then this results in a network with bistable behaviour
Adaptive learning networks Natural networks in bacteria can exhibit anticipatory behaviour for related environmental simuli If we design this into our circuit, we could code the network to “learn” Synaptic interconnections between neurons Activator A and B are expressed in response to a different stimulus Suppose both transcriptional activators drive expression of effector proteins (Effector A and Effector B) which contorl distinct genetic pathways … explain circuit This creates associative memory
In another example of a learning network We could design a “winner take all” behaviour in detecting stimuli In the future, will respond with an output only in the presence of the single type of stimuli This system could be adapted to create chenmotactic bacteria that remember a particular location or landmark and only respond to gradient of one chemoattractant
Protein based circuits Advantage: react with shorter time scales (transcription takes time, so does translation and folding; activation is just conformational change) Advantage: target synthetic activities to subcellular locations Here we have a protein circuit that exhibits Amyloid-based memory (CONST is a constitutive promoter, meaning that it’s always on. ON and OFF are inducible) Prionogenic domain (PD) fused to an effector gene, such as a transcriptional activator Prions propagate by transmitting a mis-folded protein state induces pre-existing normal forms of the protein to convert into the rogue form. this triggers a chain reaction that produces large amounts of the prion form Prions are the cause of a number of diseases in a variety of mammals, including bovine spongiform encephalopathy(BSE, also known as "mad cow disease") Chaperones disaggregate amyloids
Light responsive elements Bacteria engineered to seek out hazardous chemicals or heavy metals in the environment, perform cleanup and return to origin to report hazardous sites encountered via analysis by microfluidic devices Production of chemoattractant receptors aka chemotaxis network
One example of this is used to Engineered circuits for biological containment For ensuring genetically modified orgnaisms do not spread throughout natural environment Call active containment: Cells engineered to express toxic compounds when located out of their target environments, for programmed cell death
Here we have a two-counter: P-BAD = arabinose promoter Both genes T7RNAP and T3RNAP are regulated by riboregulators. Recall that these contain a cis-repressor sequence (cr) upstrem of the ribosome binding site Cr is placed between transcription start site and ribosome binding site. The first arabinose pulse translates taRNA, which relieves RBS and allows translation of T7 RNAP Remove arabinose from cell environment What happens if you don’t remove the arabinose? ALSO: Notice basal signal –> Why do you think this is? (arabinose)
Three-counter Notice that the peak of GFP expression is at 150 minutes. Why do you think this takes so long? (Counter limited by rate of transcription and protein translation)
Mathematical model predictions. Used model to analyze the effects of pulse frequency and pulse length on performance of the RTC 2-counter and 3-counter Color scale = expression level/fluorescence Maximum expression occurs with pulse lengths of 20-30min and intervals of 10-40min
DNA Invertase Cascade (DIC) counter Cre FLPe ssrA tag causes rapid protein degradation Term – transcription terminus
DNA Invertase Cascade (DIC) counter Cre FLPe
DNA Invertase Cascade (DIC) counter Cre FLPe
Multiple-inducible DIC three-counter
Notice basal level again: why do you think this is? (In this case, not clearing of chemical inducers. All inducible promoters are a little leaky aka constitutive transcription)
Analog-to-digital converters could translate external analog inputs, such as inducer concentrations or exposure times, into internal digital representations for biological processing Depending on the level of analog inputs, different genetic pathways could be activated Cells with these type of converters could be used as biosensors in medical and environmental settings. For example, whole-cell biosensors in the cut could be engineered to generate different reporter molecules based on the level of gastrointestinal bleeding, and this could be measured in stool Not binary
Alternatively, we could have digital to analog to translate digital representations into analog outputs Eg. Instead of fine-tuning transcriptional activity with the perfect amount of chemical inducers, cell uptake, etc, we could have a digital to analog converter Which in this case takes in 3 different signals So you would have three genetic switches, each sensitive to a different inducer Allow you to generate a gradient of responses Here, we have a bank of recombinase-based switches, known as single invertase memory modules (SIMMs) Recall: recombinase is an enzyme that will recombine homologous regions, recognize specific motif Explain recombinase circuit If each promoter is of a different strength, then digital combinations of inducers can be used to program defined levels of transcriptional activities Use for reliable expression of different pathways