This document summarizes different types of DNA ligase enzymes including their sources, mechanisms, and applications. It discusses bacteriophage T4 DNA ligase, E.coli DNA ligase, Taq DNA ligase, T4 RNA ligase, and mammalian ligases. The mechanism of DNA ligase involves three steps - adenylation of the enzyme using ATP, transfer of AMP to the DNA donor strand, and formation of a phosphodiester bond between the donor and acceptor strands. The types of ligases vary in their substrate specificities and thermal stabilities, making each useful for different molecular biology applications like cloning and DNA amplification.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
transduction is a process which that bacteriophage is transfer the genetic material to one to another bacterial cell .the transduction is have a two types that is generalized and specialized transduction .the two types of phage will be involve in the transduction process that is virulant and temptate pahge
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
transduction is a process which that bacteriophage is transfer the genetic material to one to another bacterial cell .the transduction is have a two types that is generalized and specialized transduction .the two types of phage will be involve in the transduction process that is virulant and temptate pahge
DNA ligase are used known as molecular glues.
There are 3 types of ligases used
1) DNA ligase
2) T4 infected Ecoli DNA ligase
3) terminal deoxynucleotidyl transferase
A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.
Transferring the genetic information to the descendant generation with a high fidelity
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
Key Trends Shaping the Future of Infrastructure.pdfCheryl Hung
Keynote at DIGIT West Expo, Glasgow on 29 May 2024.
Cheryl Hung, ochery.com
Sr Director, Infrastructure Ecosystem, Arm.
The key trends across hardware, cloud and open-source; exploring how these areas are likely to mature and develop over the short and long-term, and then considering how organisations can position themselves to adapt and thrive.
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
Encryption in Microsoft 365 - ExpertsLive Netherlands 2024Albert Hoitingh
In this session I delve into the encryption technology used in Microsoft 365 and Microsoft Purview. Including the concepts of Customer Key and Double Key Encryption.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
LF Energy Webinar: Electrical Grid Modelling and Simulation Through PowSyBl -...DanBrown980551
Do you want to learn how to model and simulate an electrical network from scratch in under an hour?
Then welcome to this PowSyBl workshop, hosted by Rte, the French Transmission System Operator (TSO)!
During the webinar, you will discover the PowSyBl ecosystem as well as handle and study an electrical network through an interactive Python notebook.
PowSyBl is an open source project hosted by LF Energy, which offers a comprehensive set of features for electrical grid modelling and simulation. Among other advanced features, PowSyBl provides:
- A fully editable and extendable library for grid component modelling;
- Visualization tools to display your network;
- Grid simulation tools, such as power flows, security analyses (with or without remedial actions) and sensitivity analyses;
The framework is mostly written in Java, with a Python binding so that Python developers can access PowSyBl functionalities as well.
What you will learn during the webinar:
- For beginners: discover PowSyBl's functionalities through a quick general presentation and the notebook, without needing any expert coding skills;
- For advanced developers: master the skills to efficiently apply PowSyBl functionalities to your real-world scenarios.
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
2. CONTENT
• Introduction
• Mechanism of DNA ligase
• Unit of enzyme activity
• Types of DNA Ligase:
1)Bacteriophage T4 DNA ligase
2)E.coli DNA Ligase
3)Taq DNA Ligase
4)T4 DNA Ligase
• Applications
• Reference
3. Introduction
• DNA ligase catalyses the formation of phosphodiester
bond between the 5’-phosphate of one strand of DNA or
RNA and the 3’-hydroxyl of another.
• This enzyme is used to covalently link or ligate fragments
of DNA together
• The DNA ligase used in molecular cloning differ in their
abilities to ligate noncanonical substrate,such as blunt
ended duplexeDNA:RNA hybrid or ssDNAs.
5. Mechanism of DNA Ligase
• The mechanism of DNA ligase is to form two covalent
phosphodiester bonds between 3' hydroxyl ends of one
nucleotide, ("acceptor") with the 5' phosphate end of
another ("donor").
ATP is required for the ligase reaction, which proceeds in three
steps:
(1) adenylation (addition of AMP) of a residue in the active
center of the enzyme, pyrophosphate is released.
(2) transfer of the AMP to the 5' phosphate so-called
donor, formation of a pyrophosphate bond;
(3) formation of a phosphodiester bond between the 5'
phosphate of the donor and the 3' hydroxyl of the acceptor.
6.
7. • Depending up on the source,the enzyme requires
either ATP or NAD+ as cofactors
8. Unit of Enzyme Activity
• For all ligase, one Weiss unit is the amount of
Enzyme required to convert 1 n mol of radiolabeled
phosphate from pyrophosphate into absorbable
material in 20 min at 370 c under standard assay
condition.
one Weiss unit equals about 67 cohesive end ligation
unit.
9. Bacteriophage T4 DNA Ligase (ATP)
• The most widely used DNA ligase is derived from the
T4 bacteriophage.
• It is a monomeric polypeptide
• MW 68KDa is encoded by bacteriophage gene30.
• It has broder specificity and repairs single strended
Nicks in duplex DNA, RNA or DNA:RNA hybrids.
10.
11. Application:
• 1 ligation of cohesive ends:
2 ligation of blunt ended termini:
this reaction is much slower than ligation of sticky
ends and the ligation is improved by addition of
monovalent cation and low concentration of PEG
.3 Ligation of synthetic linkers or adapter
12.
13. E.Coli DNA ligase
• It is derived from E.coli cell and requires NAD+ as
cofacter.
• It is a monomeric enzyme of MW 74KDa which
catalyzes the formation of the phosphodiester bond
in duplex DNA containing cohesive ends.
• This enzyme has narrower substrate
specificity, making it a useful tool in specific
application.
14. Application:
• 1) ligation of cohesive ends:E.coli DNA ligase is used
to catalyze sticky end ligation.
• 2)cloning of full length cDNA:
E.coli DNA ligase has been employed in a procedure
for high efficiency cloning of full length cDNA
15. Taq DNA ligase [NAD+ ]
The gene encoding thermostable ligases have
been identified from several thermophilic
bacteria.
Several of this ligase have been cloned and
expressed to high levels in E.coli
• It is uses in the detection of mutation as
thermostable DNA ligase retain their activities after
exposure to higher temp for multiple rounds
• it is uses in DNA amplificaton reaction to detect
mutation in mammalian DNA.
16. T4 RNA Ligase
• T4 RNA ligase is the only phage RNA ligase that has
been extensively characterized and used in genetic
engineering.
• This enzyme catalyzed the phosphodiester bond
formation of RNA molecule with hydrolysis of ATP to
PPI
• It is monomeric enzyme with 373 deduced amino
acid residues is a product of the T4 gene 63
17. Application
1) 5’-Tailing of DNA with RNA
2) Radiolabeling of 3’ end of RNA and DNA
3) production of elongated molecules
4) modification of internal Nucleotide
5) stimulation of T4 DNA ligase activity
18. .
Mammalian ligases
.
DNA ligase I: ligates the nascent DNA of the lagging strand after the
DNA polymerase I has removed the RNA primer from the Okazaki
fragments.
DNA ligase II: alternatively spliced form of DNA ligase III found in
non-dividing cells.
DNA ligase III: complexes with DNA repair protein XRCC1 to aid in
sealing base excision mutations and recombinant fragments.
DNA ligase IV:. It catalyzes the final step in the non-homologous end
joining DNA double-strand break repair pathway. It is also required
for V(D)J recombination, the process which generates diversity in
immunoglobulin and T-cell receptor loci during immune system
development.