The document discusses various types of bacteriophage vectors that can be used for cloning genomic DNA, including their structure and applications. Phage derivatives like lambda phages and M13 phages have been developed as cloning vectors since they allow large DNA fragments to be cloned and can package millions of recombinant phage particles. The document describes different types of phage vectors like insertion vectors, replacement vectors, and hybrid plasmid-phage vectors. It also discusses various bacteriophages like lambda phage and M13 phage used to create these vectors, along with their genome structure and life cycle. Cosmids are also introduced as hybrid vectors containing phage and plasmid elements.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
Transportable elements are DNA Sequences that move from one location in a chromosome to another within the same chromosome or into another chromosome.
These are DNA Sequences that move from one location in a chromosome to another within the same chromosome or into another chromosome.
These are DNA Sequences that move from one location in a chromosome to another within the same chromosome or into another chromosome.
These are also known as “Jumping genes”.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
Transportable elements are DNA Sequences that move from one location in a chromosome to another within the same chromosome or into another chromosome.
These are DNA Sequences that move from one location in a chromosome to another within the same chromosome or into another chromosome.
These are DNA Sequences that move from one location in a chromosome to another within the same chromosome or into another chromosome.
These are also known as “Jumping genes”.
in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.
A bacteriophage (informally, phage) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.
Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface,] and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection
in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.
A bacteriophage (informally, phage) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.
Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface,] and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection
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
this is done by me and my team mates of Wayamba University Sri Lanka for our project.From now we decided to allow download this file.I would be greatful if you could send your comments..
And I'm willing to help you in similar works.I'm in final year of my degree(.BSc Biotechnology)..
pubudu_gokarella@yahoo.com
It is the basics of vector cloning which necessary for every and each student who is intrested in biotechnology. It is only starting, if you want to more than this then please comment on it.
Gene Cloning Vectors - Plasmids, Bacteriophages and Phagemids.Ambika Prajapati
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.
They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
Types -
1.Plasmid vectors.
2.Bacteriophage vectors .
3.Phagemids.
description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
Food hygiene is more than cleanliness ......
Protecting food from risk of contamination, including harmful bacteria, poison and other foreign bodies.
Preventing any bacteria present multiplying to an extent which would result in the illness of consumers or the early spoilage of the food.
Destroying any harmful bacteria in the food by thorough cooking
or processing.
Discarding unfit or contaminated food.
T-Cell Activation
• Concept of immune response
• T cell-mediated immune response
• B cell-mediated immune response
I. Concept of immune response
• A collective and coordinated response to the introduction of foreign substances in an individual mediated by the cells and molecules in the immune system.
II. T cell-mediated immune response
• Cell-mediated immunity is the arm of the adaptive immune response whose role is to combat infection of intracellular pathogens, such as intracellular bacteria (mycobacteria, listeria monocytogens), viruses, protozoa, etc.
Major Histocompatibility Complex
MHC:
• Major Histocompatibility Complex
– Cluster of genes found in all mammals
– Its products play role in discriminating self/non-self
– Participant in both humoral and cell-mediated immunity
• MHC Act As Antigen Presenting Structures
• In Human MHC Is Found On Chromosome 6
– Referred to as HLA complex
• In Mice MHC Is Found On Chromosome 17
– Referred to as H-2 complex
• Genes Of MHC Organized In 3 Classes
– Class I MHC genes
• Glycoproteins expressed on all nucleated cells
• Major function to present processed Ags to TC
– Class II MHC genes
• Glycoproteins expressed on macrophages, B-cells, DCs
• Major function to present processed Ags to TH
– Class III MHC genes
• Products that include secreted proteins that have immune functions. Ex. Complement system, inflammatory molecules
Antigen Processing and Presentation MID
Antigens and “foreignness”
• Antigens (or, more properly, immunogens) have a series of features which confer immunogenicity.
• One of these features is “foreignness.”
• So, we can infer that – most often – antigens – ultimately – originate externally.
• (There are exceptions, of course. Some cells become transformed by disease [e. g., cancer] or by aging. In such instances, the antigens have an internal origin.)
Extinction of a particular animal or plant species occurs when there are no more individuals of that species alive anywhere in the world - the species has died out. This is a natural part of evolution. But sometimes extinctions happen at a much faster rate than usual. Natural Causes of Extinction.
Difference between In-Situ and Ex-Situ conservation
Conservation of biodiversity and genetic resources helps protect, maintain and recover endangered animal and plant species. There are mainly two strategies for the conservation of wildlife: In-situ conservation and Ex-situ conservation. Although, both the strategies aim to maintain and recover endangered species, they are different from each other. Let us see how they differ from each other!
Evolution Of Bacteria
Bacteria have existed from very early in the history of life on Earth. Bacteria fossils discovered in rocks date from at least the Devonian Period (419.2 million to 358.9 million years ago), and there are convincing arguments that bacteria have been present since early Precambrian time, about 3.5 billion years ago. Bacteria were widespread on Earth at least since the latter part of the Paleoproterozoic, roughly 1.8 billion years ago, when oxygen appeared in the atmosphere as a result of the action of the cyanobacteria. Bacteria have thus had plenty of time to adapt to their environments and to have given rise to numerous descendant forms.
Impact of Environment on Loss of Genetic Diversity and Speciation
Genetic variation describes naturally occurring genetic differences among individuals of the same species. This variation permits flexibility and survival of a population in the face of changing environmental circumstances. Consequently, genetic variation is often considered an advantage, as it is a form of preparation for the unexpected. But how does genetic variation increase or decrease? And what effect do fluctuations in genetic variation have on populations over time?
GENE ENVIRONMENT INTERACTION
Subtle differences in one person’s genes can cause them to respond differently to the same environmental exposure as another person. As a result, some people may develop a disease after being exposed to something in the environment while others may not.
As scientists learn more about the connection between genes and the environment, they pursue new approaches for preventing and treating disease that consider individual genetic codes.
How to store food in hot
The Good News
To maximize benefit of preservation, keep your food as fresh as possible for as long as possible. You can do this, even in the heat, by creating a “cooler” made from two basic terra cotta pots, one larger than the other. Put the smaller pot in the larger one, fill the gap with sand, and saturate the sand with water. Then cover it with a cloth. To add additional insulation from the heat, bury the pot up to its rim. The evaporation of moisture from the wet sand will cool the air around the food and help keep it fresh.
What is IUPAC naming?
In order to give compounds a name, certain rules must be followed. When naming organic compounds, the IUPAC (International Union of Pure and Applied Chemistry) nomenclature (naming scheme) is used. This is to give consistency to the names. It also enables every compound to have a unique name, which is not possible with the common names used (for example in industry). We will first look at some of the steps that need to be followed when naming a compound, and then try to apply these rules to some specific examples.
IUPAC Nomenclature
IUPAC nomenclature uses the longest continuous chain of carbon atoms to determine the basic root name of the compound. The root name is then modified due to the presence of different functional groups which replace hydrogen or carbon atoms in the parent structure.
Hybridization describes the bonding atoms from an atom's point of view. For a tetrahedral coordinated carbon (e.g. methane CH4), the carbon should have 4 orbitals with the correct symmetry to bond to the 4 hydrogen atoms.
INTRODUCTION:
Hybrid Orbitals
Developed by Linus Pauling, the concept of hybrid orbitals was a theory created to explain the structures of molecules in space. The theory consists of combining atomic orbitals (ex: s,p,d,f) into new hybrid orbitals (ex: sp, sp2, sp3).
1. Why Firefly give light during night?
2. Why atomic mass and Atomic numbers are given to elements ?
3. Why elements have been characterized and classified into different groups?
4. What is the transition of elements and what they play their role in elements stability?
Observability Concepts EVERY Developer Should Know -- DeveloperWeek Europe.pdfPaige Cruz
Monitoring and observability aren’t traditionally found in software curriculums and many of us cobble this knowledge together from whatever vendor or ecosystem we were first introduced to and whatever is a part of your current company’s observability stack.
While the dev and ops silo continues to crumble….many organizations still relegate monitoring & observability as the purview of ops, infra and SRE teams. This is a mistake - achieving a highly observable system requires collaboration up and down the stack.
I, a former op, would like to extend an invitation to all application developers to join the observability party will share these foundational concepts to build on:
Alt. GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using ...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.
Le nuove frontiere dell'AI nell'RPA con UiPath Autopilot™UiPathCommunity
In questo evento online gratuito, organizzato dalla Community Italiana di UiPath, potrai esplorare le nuove funzionalità di Autopilot, il tool che integra l'Intelligenza Artificiale nei processi di sviluppo e utilizzo delle Automazioni.
📕 Vedremo insieme alcuni esempi dell'utilizzo di Autopilot in diversi tool della Suite UiPath:
Autopilot per Studio Web
Autopilot per Studio
Autopilot per Apps
Clipboard AI
GenAI applicata alla Document Understanding
👨🏫👨💻 Speakers:
Stefano Negro, UiPath MVPx3, RPA Tech Lead @ BSP Consultant
Flavio Martinelli, UiPath MVP 2023, Technical Account Manager @UiPath
Andrei Tasca, RPA Solutions Team Lead @NTT Data
Essentials of Automations: The Art of Triggers and Actions in FMESafe Software
In this second installment of our Essentials of Automations webinar series, we’ll explore the landscape of triggers and actions, guiding you through the nuances of authoring and adapting workspaces for seamless automations. Gain an understanding of the full spectrum of triggers and actions available in FME, empowering you to enhance your workspaces for efficient automation.
We’ll kick things off by showcasing the most commonly used event-based triggers, introducing you to various automation workflows like manual triggers, schedules, directory watchers, and more. Plus, see how these elements play out in real scenarios.
Whether you’re tweaking your current setup or building from the ground up, this session will arm you with the tools and insights needed to transform your FME usage into a powerhouse of productivity. Join us to discover effective strategies that simplify complex processes, enhancing your productivity and transforming your data management practices with FME. Let’s turn complexity into clarity and make your workspaces work wonders!
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
SAP Sapphire 2024 - ASUG301 building better apps with SAP Fiori.pdfPeter Spielvogel
Building better applications for business users with SAP Fiori.
• What is SAP Fiori and why it matters to you
• How a better user experience drives measurable business benefits
• How to get started with SAP Fiori today
• How SAP Fiori elements accelerates application development
• How SAP Build Code includes SAP Fiori tools and other generative artificial intelligence capabilities
• How SAP Fiori paves the way for using AI in SAP apps
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.
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
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/
Climate Impact of Software Testing at Nordic Testing DaysKari Kakkonen
My slides at Nordic Testing Days 6.6.2024
Climate impact / sustainability of software testing discussed on the talk. ICT and testing must carry their part of global responsibility to help with the climat warming. We can minimize the carbon footprint but we can also have a carbon handprint, a positive impact on the climate. Quality characteristics can be added with sustainability, and then measured continuously. Test environments can be used less, and in smaller scale and on demand. Test techniques can be used in optimizing or minimizing number of tests. Test automation can be used to speed up testing.
Welcome to the first live UiPath Community Day Dubai! Join us for this unique occasion to meet our local and global UiPath Community and leaders. You will get a full view of the MEA region's automation landscape and the AI Powered automation technology capabilities of UiPath. Also, hosted by our local partners Marc Ellis, you will enjoy a half-day packed with industry insights and automation peers networking.
📕 Curious on our agenda? Wait no more!
10:00 Welcome note - UiPath Community in Dubai
Lovely Sinha, UiPath Community Chapter Leader, UiPath MVPx3, Hyper-automation Consultant, First Abu Dhabi Bank
10:20 A UiPath cross-region MEA overview
Ashraf El Zarka, VP and Managing Director MEA, UiPath
10:35: Customer Success Journey
Deepthi Deepak, Head of Intelligent Automation CoE, First Abu Dhabi Bank
11:15 The UiPath approach to GenAI with our three principles: improve accuracy, supercharge productivity, and automate more
Boris Krumrey, Global VP, Automation Innovation, UiPath
12:15 To discover how Marc Ellis leverages tech-driven solutions in recruitment and managed services.
Brendan Lingam, Director of Sales and Business Development, Marc Ellis
Why You Should Replace Windows 11 with Nitrux Linux 3.5.0 for enhanced perfor...SOFTTECHHUB
The choice of an operating system plays a pivotal role in shaping our computing experience. For decades, Microsoft's Windows has dominated the market, offering a familiar and widely adopted platform for personal and professional use. However, as technological advancements continue to push the boundaries of innovation, alternative operating systems have emerged, challenging the status quo and offering users a fresh perspective on computing.
One such alternative that has garnered significant attention and acclaim is Nitrux Linux 3.5.0, a sleek, powerful, and user-friendly Linux distribution that promises to redefine the way we interact with our devices. With its focus on performance, security, and customization, Nitrux Linux presents a compelling case for those seeking to break free from the constraints of proprietary software and embrace the freedom and flexibility of open-source computing.
Pushing the limits of ePRTC: 100ns holdover for 100 daysAdtran
At WSTS 2024, Alon Stern explored the topic of parametric holdover and explained how recent research findings can be implemented in real-world PNT networks to achieve 100 nanoseconds of accuracy for up to 100 days.
3. PHAGESPHAGES
• Derivatives of phage have been developed as cloning vectors since the earlyDerivatives of phage have been developed as cloning vectors since the early
days of gene technology.days of gene technology.
• The phage derivatives are considered to be the most suitable cloning vehiclesThe phage derivatives are considered to be the most suitable cloning vehicles
for cloning genomic eukaryotic DNA because of the following advantages overfor cloning genomic eukaryotic DNA because of the following advantages over
the plasmids.the plasmids.
Thousands of phage plaques can be obtained in a single petri dish.Thousands of phage plaques can be obtained in a single petri dish.
Selection bySelection by DNA-DNA hybridizationDNA-DNA hybridization is possible.is possible.
In vitro packagingIn vitro packaging into empty phage head is possible thus increasing phageinto empty phage head is possible thus increasing phage
infectivityinfectivity
Size selection of the packaged DNA is possible.Size selection of the packaged DNA is possible.
Millions of independently cloned virus particle can be constituted to form aMillions of independently cloned virus particle can be constituted to form a
gene library.gene library.
.
4. BACTERIOPHAGESBACTERIOPHAGES • Bacteriophage is aBacteriophage is a genetically complexgenetically complex butbut
very extensively studied virus ofvery extensively studied virus of E.coliE.coli..
• The DNA of phage, is aThe DNA of phage, is a linear duplex moleculelinear duplex molecule
of 48502 bpof 48502 bp (~49kb) in length.(~49kb) in length.
• The DNA isolated from virus particles is aThe DNA isolated from virus particles is a
double stranded linear molecule with shortdouble stranded linear molecule with short
complementary single stranded projections ofcomplementary single stranded projections of
12 nucleotides at its 5’ ends.12 nucleotides at its 5’ ends.
• These cohesive termini, also referred to asThese cohesive termini, also referred to as coscos
sites,sites, allow the DNA to be circularizedallow the DNA to be circularized afterafter
infection of the host cell and also packaging ofinfection of the host cell and also packaging of
the DNA.the DNA.
.
5. GENERAL STRUCTURE OF BACTERIOPHAGE DNAGENERAL STRUCTURE OF BACTERIOPHAGE DNA
• The genetic map of phage comprises approximately 40 genes which areThe genetic map of phage comprises approximately 40 genes which are
organized in functional clusters.organized in functional clusters.
• Genes coding for head and tail are proteins (genesGenes coding for head and tail are proteins (genes A-JA-J) are on the left of the) are on the left of the
linear map.linear map.
• The central region contains genes, such asThe central region contains genes, such as int, xis, exoint, xis, exo etc., which areetc., which are
responsible forresponsible for lysogenisationlysogenisation i.ei.e the process leading to the integration of viralthe process leading to the integration of viral
DNA and other recombination events.DNA and other recombination events.
• Much of this central region is not essential for lytic growth.Much of this central region is not essential for lytic growth.
• Genes to the right of the central region compriseGenes to the right of the central region comprise six regulatory genessix regulatory genes, two, two
genes (genes (OO andand PP) which are essential) which are essential for DNA replicationfor DNA replication during lytic growth andduring lytic growth and
two more genes (two more genes (SS andand RR) which are required) which are required for the lysis of the cellularfor the lysis of the cellular
membranes.membranes.
.
6. • In the phage DNA, larger central region is not essential for phage growth andIn the phage DNA, larger central region is not essential for phage growth and
replication.replication.
• This region of phage can be deleted or replaced without seriously impairingThis region of phage can be deleted or replaced without seriously impairing
the phage growth cycle.the phage growth cycle.
• Using this non-essential region of phage, several phage vector derivatives haveUsing this non-essential region of phage, several phage vector derivatives have
been constructed for efficient gene cloning.been constructed for efficient gene cloning.
.
7. TYPES OF PHAGE VECTORSTYPES OF PHAGE VECTORS
• Wild type phage DNA itself cannot be used as a vector since it contains tooWild type phage DNA itself cannot be used as a vector since it contains too
many restriction sites.many restriction sites.
• Further, these sites are often located within the essential regions for phage'sFurther, these sites are often located within the essential regions for phage's
growth and development.growth and development.
• From these wild phages, derivatives with single target sites and two targetFrom these wild phages, derivatives with single target sites and two target
sites have been synthesized.sites have been synthesized.
• Phage vectors whichPhage vectors which contain single sitecontain single site for the insertion of foreign DNA havefor the insertion of foreign DNA have
been designated asbeen designated as Insertional vectorsInsertional vectors;;
• vectors withvectors with two cleavage sitestwo cleavage sites, which allow foreign DNA to be substituted for, which allow foreign DNA to be substituted for
the DNA sequences between those sites, are known asthe DNA sequences between those sites, are known as replacement vectorsreplacement vectors..
.
8. INSERTIONAL VECTORSINSERTIONAL VECTORS
• A large segment of the non-essential region hasA large segment of the non-essential region has
been deleted, and the two arms ligated together.been deleted, and the two arms ligated together.
• An insertion vector possesses at least one uniqueAn insertion vector possesses at least one unique
restriction site into which new DNA can be inserted.restriction site into which new DNA can be inserted.
• Two popular insertion vectors are:Two popular insertion vectors are:
• Egt10 :Egt10 : which can carrywhich can carry up to 8 kbup to 8 kb of new DNA,of new DNA,
inserted into ainserted into a uniqueunique EcoEcoRIRI site located in thesite located in the ccII
gene.gene.
• Insertional inactivation of this gene means thatInsertional inactivation of this gene means that
recombinants are distinguished as clear rather thanrecombinants are distinguished as clear rather than
turbid plaques.turbid plaques.
• EZAPIIEZAPII : insertion of up to 10 kb DNA into any of 6: insertion of up to 10 kb DNA into any of 6
restriction sites withinrestriction sites within a polylinkera polylinker inactivates theinactivates the
lacZlacZ gene carried by the vector.′ gene carried by the vector.′
• Recombinants give clear rather than blue plaquesRecombinants give clear rather than blue plaques
on X-gal agar.on X-gal agar.
.
9. E.COLI/E.COLI/λλ REPLACEMENT VECTORSREPLACEMENT VECTORS
ExamplesExamples:: EMBL3 andEMBL3 and λλ DASH.DASH.
A representative scheme for cloning:A representative scheme for cloning:
1. The1. The λλ vector DNAvector DNA is cleaved withis cleaved with
BamH1BamH1 and the long (19 kb) andand the long (19 kb) and
short (9 kb) arms are purified;short (9 kb) arms are purified;
2. The2. The targettarget fragments are preparedfragments are prepared
by digestion, also withby digestion, also with BamBamH1 or aH1 or a
compatible enzyme (compatible enzyme (SauSau3A);3A);
3. The target fragments are treated3. The target fragments are treated
withwith alkaline phosphatasealkaline phosphatase to preventto prevent
them ligating to each other;them ligating to each other;
4. The4. The λλ armsarms and theand the targettarget
fragmentsfragments are ligated together atare ligated together at
relatively high concentration to formrelatively high concentration to form
long linear products.long linear products.
B B
∼ 20kb
B
Can not
Parking
infect
E.coli
Long armLong arm
ShortShort
armarmReplace.
48.5 kb
.
10. PACKAGING AND INFECTIONPACKAGING AND INFECTION
TheThe RecombinantsRecombinants that canthat can notnot be packaged:be packaged:
1. Ligated1. Ligated λλ ends which do not contain an insert;ends which do not contain an insert;
2. The insert is much smaller or larger than the 20 kb;2. The insert is much smaller or larger than the 20 kb;
3. The recombinants with two left or right arms.3. The recombinants with two left or right arms.
in vivo
B
Replication→ concata-mers
cleave individual λ genomes
in vitro
A mixture of phage coat proteins and
the phage DNA-processing enzymes
Packaging:
Packaging
phage
particles
Infection of E. coli
109
recombinants per mg of vector DNA..
11. FORMATION OF PLAQUESFORMATION OF PLAQUES
Plaques are the analogs of single bacterial colonies.Plaques are the analogs of single bacterial colonies.
FormationFormation::
The infectedThe infected E.coliE.coli cells from a packaging reaction are spread oncells from a packaging reaction are spread on
an agar plate,an agar plate,
The plate has been pre-spread with uninfected cells, which willThe plate has been pre-spread with uninfected cells, which will
grow to form a continuous lawn.grow to form a continuous lawn.
After incubation, phage-infected cells result in clear areas, thatAfter incubation, phage-infected cells result in clear areas, that
are plaques, where cycles of lysis and re-infection haveare plaques, where cycles of lysis and re-infection have
prevented the cells from growing.prevented the cells from growing.
Recombinant λ DNA may be purified:
• from phage particles isolated from plaques or
• from the supernatant of a culture infected
with a specific recombinant plaque.
E.coli lawn
Plaques
.
12. RF
BACTERIOPHAGE M13BACTERIOPHAGE M13
Genome features:Genome features: Size is small (6.7 kb); Single-stranded;Size is small (6.7 kb); Single-stranded;
Circular genome; DNA; Positive-sense.Circular genome; DNA; Positive-sense.
g3pg6p
g7p
g8p
g9p
Host
enzymes
end
ini
Infection: M13 particles attach specifically to E.coli sex pili (encoded by a
plasmid called F factor), through a minor coat protein (g3p). Binding of g3p
induces a structural change in the major capsid protein. This causes the whole
particle to shorten, injecting the viral DNA into the host cell.
.
13. E.COLIE.COLI/M13 PHAGE/M13 PHAGE
VECTORSVECTORS
StructureStructure:: ♣♣ TheThe phage particlesphage particles
containcontain a 6.7 kb circulara 6.7 kb circular ssDNAssDNA.. ♣♣
After infection of a sensitiveAfter infection of a sensitive E. coliE. coli
host, the complementary strand ishost, the complementary strand is
synthesized, like asynthesized, like a plasmidplasmid, and the, and the
DNA replicated as aDNA replicated as a dsDNAdsDNA, the, the
replicative form (RF).replicative form (RF).
FeaturesFeatures:: ♣♣ The host cells can continueThe host cells can continue
to grow slowly.to grow slowly.
• ssDNAssDNA: The single-stranded forms are: The single-stranded forms are
continuously packaged and releasedcontinuously packaged and released
from the cells as new phage particles.from the cells as new phage particles.
ssDNA has a number of applications,ssDNA has a number of applications,
includingincluding ♣♣ DNA sequencing andDNA sequencing and ♣♣
site-directed mutagenesis.site-directed mutagenesis.
• dsDNAdsDNA: The RF (dsDNA) can be: The RF (dsDNA) can be
purifiedpurified in vitroin vitro and manipulatedand manipulated
exactly like a plasmid.exactly like a plasmid..
14. CLONING IN M13CLONING IN M13
PurposePurpose: When the: When the single-stranded DNAsingle-stranded DNA of a fragment is required, a M 13of a fragment is required, a M 13
vector can be used as a common cloning tool.vector can be used as a common cloning tool.
PreparationPreparation of ssDNA:of ssDNA:
1.1. CloningCloning: standard plasmid cloning method can be used to incorporate: standard plasmid cloning method can be used to incorporate
recombinant DNA into M13 vectors;recombinant DNA into M13 vectors;
2.2. TransformationTransformation: the M13 then infects sensitive: the M13 then infects sensitive E. coliE. coli cells;cells;
3.3. PlatingPlating: the host cells grow to form the plaques;: the host cells grow to form the plaques;
4.4. IsolationIsolation: the ssDNA may then be isolated from phage particles in the: the ssDNA may then be isolated from phage particles in the
growth medium of the plate.growth medium of the plate.
ScreeningScreening: Blue-white screening using MCSs and lacZ' has been engineered: Blue-white screening using MCSs and lacZ' has been engineered
into M13 vectors.into M13 vectors.
ExamplesExamples: The: The M13mpl8M13mpl8 andand M13mp19M13mp19, which are a pair of vectors in which, which are a pair of vectors in which
the MCS are in opposite orientations relative to the M13 origin ofthe MCS are in opposite orientations relative to the M13 origin of
replication.replication.
.
15. HYBRID PLASMID-M13 VECTORSHYBRID PLASMID-M13 VECTORS
DefinitionDefinition: A number of small plasmid vectors, for example pBlue-script, have: A number of small plasmid vectors, for example pBlue-script, have
been developed to incorporate M13 functionality.been developed to incorporate M13 functionality.
StructureStructure: They contain both plasmid and M13 origins of replication, but do not: They contain both plasmid and M13 origins of replication, but do not
possess the genes required for the full phage life cycle.possess the genes required for the full phage life cycle.
Working waysWorking ways::
1.1. Plasmid wayPlasmid way: they normally propagate as true plasmids, and have the: they normally propagate as true plasmids, and have the
advantages of rapid growth and easy manipulation of plasmid vectors;advantages of rapid growth and easy manipulation of plasmid vectors;
2.2. Phage wayPhage way: they can be induced to produce single-stranded phage particles: they can be induced to produce single-stranded phage particles
by co-infection with a fully functionalby co-infection with a fully functional helper phagehelper phage, which provides the gene, which provides the gene
products required for single-strand production and packaging.products required for single-strand production and packaging.
.
16. COSMIDSCOSMIDS
• Cosmids areCosmids are hybrids betweenhybrids between aa phagephage DNA molecule and aDNA molecule and a
bacterial plasmidbacterial plasmid, and their design centers on the fact that, and their design centers on the fact that
the enzymes that package thethe enzymes that package the λλ DNA molecule into theDNA molecule into the
phage protein coat need only the cos sites in order tophage protein coat need only the cos sites in order to
function.function.
• The in vitro packaging reaction works not only withThe in vitro packaging reaction works not only with λλ
genomes, but also with any molecule thatgenomes, but also with any molecule that carriescarries coscos sitessites
separated by 37–52 kb of DNA.separated by 37–52 kb of DNA.
• A cosmid is basically a plasmid that carries aA cosmid is basically a plasmid that carries a coscos site .site .
• It also needs aIt also needs a selectable markerselectable marker, such as the, such as the ampicillinampicillin
resistance gene, and a plasmidresistance gene, and a plasmid origin of replicationorigin of replication, as, as
cosmids lack all thecosmids lack all the λλ genes and so do not produce plaques.genes and so do not produce plaques.
• Instead colonies are formed on selective media, just asInstead colonies are formed on selective media, just as
with a plasmid vector.with a plasmid vector.
.
17. • The following table provides a list cosmid vectorsThe following table provides a list cosmid vectors
and their structural features.and their structural features.
• Cosmid Size(kb)Cosmid Size(kb) Cleavage sites Size ofCleavage sites Size of
insertion (kb)insertion (kb)
• MUA3MUA3 4.764.76 EcoRI/PstI/PvuII/PvuIEcoRI/PstI/PvuII/PvuI 40 – 4840 – 48
• pJB8pJB8 5.405.40 BamHIBamHI 32 – 4532 – 45
• Homer I 5.40Homer I 5.40 EcoRI/ClaIEcoRI/ClaI 30 – 4730 – 47
• Homer II 6.38Homer II 6.38 SstISstI 32 – 4432 – 44
• pJC79pJC79 6.406.40 EcoRI/ClaI/BamH IEcoRI/ClaI/BamH I 32 – 4432 – 44
.
18. BACTERIAL ARTIFICIALBACTERIAL ARTIFICIAL
CHROMOSOMES (BAC)CHROMOSOMES (BAC)
BACs are based on bacterial mini-FBACs are based on bacterial mini-F
plasmids, which are small pieces ofplasmids, which are small pieces of
episomal bacterial DNA that give theepisomal bacterial DNA that give the
bacteria the ability to initiate conjugationbacteria the ability to initiate conjugation
with adjacent bacteria. They have awith adjacent bacteria. They have a
cloning limit of 75-300 kb.cloning limit of 75-300 kb.
.
19. YEAST ARTIFICIAL CHROMOSOMES (YAC)YEAST ARTIFICIAL CHROMOSOMES (YAC)
YACs are artificial chromosomes that replicate in yeast cells. They consist ofYACs are artificial chromosomes that replicate in yeast cells. They consist of
TelomeresTelomeres, which are ends of chromosomes involved in the replication and stability, which are ends of chromosomes involved in the replication and stability
of linear DNA.of linear DNA.
Origin of replicationOrigin of replication sequences necessary for the replication in yeast cells.sequences necessary for the replication in yeast cells.
AA yeast centromereyeast centromere, which is a specialized chromosomal region where spindle, which is a specialized chromosomal region where spindle
fibers attach during mitosis.fibers attach during mitosis.
A selectable marker for identification in yeast cells.A selectable marker for identification in yeast cells.
Ampicillin resistanceAmpicillin resistance gene for selective amplification.gene for selective amplification.
Recognition sites for restriction enzymes.Recognition sites for restriction enzymes.
.
20. THE PROCEDURE FORTHE PROCEDURE FOR
MAKING YAC VECTORS IS ASMAKING YAC VECTORS IS AS
FOLLOWSFOLLOWS
1. The target DNA is partially digested by a1. The target DNA is partially digested by a
restriction endonuclease, and the YAC vector isrestriction endonuclease, and the YAC vector is
cleaved by restriction enzymes.cleaved by restriction enzymes.
2. The cleaved vector segments are ligated with a2. The cleaved vector segments are ligated with a
digested DNA fragment to form an artificialdigested DNA fragment to form an artificial
chromosome.chromosome.
3. Yeast cells are transformed to make a large3. Yeast cells are transformed to make a large
number of copies.number of copies.
They are the largest of the cloning vectors, with aThey are the largest of the cloning vectors, with a
cloning limit of 100-1000 kbcloning limit of 100-1000 kb, however they have, however they have
very low efficiency.very low efficiency.
.
21. YEAST/YAC VECTORSYEAST/YAC VECTORS
CEN4CEN4 is the centromere ofis the centromere of
chromosome 4 ofchromosome 4 of YeastYeast. The. The
centromere will segregate thecentromere will segregate the
daughter chromosomes.daughter chromosomes.
ARSARS is autonomously replicatingis autonomously replicating
sequence, its function is as a yeastsequence, its function is as a yeast
origin of replication.origin of replication.
TRP1TRP1 andand URA3URA3 are yeast selectableare yeast selectable
markers, one for each end, to ensuremarkers, one for each end, to ensure
the right reconstituted YACs survivethe right reconstituted YACs survive
in the yeast cells.in the yeast cells.
TELTEL is the telomeric DNA sequence,is the telomeric DNA sequence,
which is extended by the telomerasewhich is extended by the telomerase
enzyme inside theenzyme inside the yeast cell.yeast cell.
SUP4SUP4 is a gene, which is insertionallyis a gene, which is insertionally
inactivated, for a red-white color test,inactivated, for a red-white color test,
like blue-white screening inlike blue-white screening in E. coliE. coli..
Function: YAC vectors can accept genomic DNA fragments of more
than 1 Mb, and hence can be used to clone entire human genes.
B B
S
pYAC3
SnaBI
BamHI
.
23. SHUTTLE VECTORSSHUTTLE VECTORS
DefinitionDefinition: They are the vectors that can: They are the vectors that can
shuttle between more than one host,shuttle between more than one host,
for example, one isfor example, one is E. coliE. coli and theand the
other is yeast.other is yeast.
Structure and functionStructure and function: Most of the: Most of the
vectors for use in eukaryotic cells arevectors for use in eukaryotic cells are
constructed as shuttle vectors.constructed as shuttle vectors.
• InIn E. coliE. coli::
• This means that they can surviveThis means that they can survive
and have the genes (ori and ampand have the genes (ori and amprr
))
required for replication andrequired for replication and
selection inselection in E. coliE. coli..
• In theIn the desired eukaryotic cellsdesired eukaryotic cells::
• They can also survive in theThey can also survive in the desireddesired
host cellshost cells, and let the target insert, and let the target insert
sequences take effects.sequences take effects.
E.coli
Yeast
.
24. YEAST EPISOMAL PLASMIDSYEAST EPISOMAL PLASMIDS
StructureStructure of YEpsof YEps
aa oriori: for replication in: for replication in E.coliE.coli
aa ampamprr
: for selection in: for selection in E. coliE. coli
aa 22µµ origin: for replication inorigin: for replication in
yestyest
LEU2LEU2: is homologous gene: is homologous gene
and a selectable marker inand a selectable marker in
yeast, involved in leucineyeast, involved in leucine
synthesis.synthesis.
X geneX gene: a shuttle sequence.: a shuttle sequence.
ori
ampr
2µ origin
LEU2X gene
Function of YEps
• It replicates as plasmids
• It integrates into a yeast
chromosome by homologous
recombination.
YEps
.
25. EXPRESSION VECTORSEXPRESSION VECTORS
• In DNA cloning experiments all the genes cloned areIn DNA cloning experiments all the genes cloned are not expressednot expressed fully because offully because of
weak promotersweak promoters in vector DNA.in vector DNA.
• This can be dramatically improved by placing such genesThis can be dramatically improved by placing such genes downstream of strongdownstream of strong
promoters.promoters.
• An additional problem in maximizing expression of cloned genes in E. coli which isAn additional problem in maximizing expression of cloned genes in E. coli which is
frequently encountered with genes from a heterologous source is that the genefrequently encountered with genes from a heterologous source is that the gene carriescarries
no translation start signalno translation start signal which can be efficiently recognized by the E. coli translationwhich can be efficiently recognized by the E. coli translation
system.system.
• This problem may arise for heterologous genes cloned into any host. Thus, evenThis problem may arise for heterologous genes cloned into any host. Thus, even
though the gene can be transcribed from a promoter within the vector, the resultingthough the gene can be transcribed from a promoter within the vector, the resulting
mRNA is poorly translated and little or no protein product will be synthesized.mRNA is poorly translated and little or no protein product will be synthesized.
• In such cases alternative strategies available areIn such cases alternative strategies available are fusing the gene to amino terminalfusing the gene to amino terminal
region of vector gene that is efficiently translated in the hostregion of vector gene that is efficiently translated in the host or coupling the gene to aor coupling the gene to a
DNA fragment carrying both strong promoter and a ribosomal binding site.DNA fragment carrying both strong promoter and a ribosomal binding site.
• Vectors with this additional feature are calledVectors with this additional feature are called expression vectorsexpression vectors..
.
26. .
T7
expressional
vector
E.COLIE.COLI/T7 EXPRESSION/T7 EXPRESSION
VECTORSVECTORS
• Definition ofDefinition of expression vectorsexpression vectors::
Cloned geneCloned gene→→expression vectorexpression vector→→
hosthost→→fusionfusion proteinprotein..
• StructureStructure
• T7 promoterT7 promoter: a strong promoter;: a strong promoter;
• RBSRBS: ribosome binding site;: ribosome binding site;
• ATGATG: translation initiation condon: translation initiation condon
• MCSMCS: Multiple cloning sites: Multiple cloning sites
• TTTT: transcription terminator.: transcription terminator.
• ampamprr
,,.. ori,ori,
• His-tagHis-tag: Some expression vectors are: Some expression vectors are
designed to have six histidine codonsdesigned to have six histidine codons
that encode a hexahistidine tag at thethat encode a hexahistidine tag at the
N terminus of the expressed protein,N terminus of the expressed protein,
which allows one-step purification onwhich allows one-step purification on
an affinity column containing Nian affinity column containing Ni2+2+
..
T7
RBS MCS
TT
ATG
27. INSECT CELL/BACULOVIRUSINSECT CELL/BACULOVIRUS
DefinitionDefinition: Baculovirus is an: Baculovirus is an ♣♣ insect virusinsect virus whichwhich
can be used for the overexpression ofcan be used for the overexpression of ♣♣ animalanimal
proteinsproteins ♣♣ inin insect cell cultureinsect cell culture..
MechanismMechanism::
• Viral promoterViral promoter: This viral gene has an: This viral gene has an extremelyextremely
active promoter.active promoter.
• Insect cell cultureInsect cell culture: The same promoter can be: The same promoter can be
used to drive the over-expression of a foreignused to drive the over-expression of a foreign
gene engineered into the baculovirus genome.gene engineered into the baculovirus genome.
FunctionFunction: This method is being used increasingly: This method is being used increasingly
for large-scale culture of proteins offor large-scale culture of proteins of animalanimal
originorigin, since the insect cells can produce many, since the insect cells can produce many
of the post-translational modifications of animalof the post-translational modifications of animal
proteins, which a bacterial expression systemproteins, which a bacterial expression system
cannotcannot..
Baculovirus-infected SF21 cells.
28. .
MAMMALIAN CELL/VIRAL VECTORSMAMMALIAN CELL/VIRAL VECTORS
• SV40SV40: This virus can infect a: This virus can infect a
number of mammaliannumber of mammalian
species. The SV40 genome isspecies. The SV40 genome is
only 5.2 kb in size.only 5.2 kb in size.
• Since it has packagingSince it has packaging
constraints similar to phageconstraints similar to phage λλ,,
so it can be not used forso it can be not used for
transferringtransferring largelarge fragments.fragments.
29. .
MAMMALIAN CELL/VIRALMAMMALIAN CELL/VIRAL
VECTORSVECTORS
• RetrovirusesRetroviruses: They have a ssRNA: They have a ssRNA
genome, which is copied into dsDNAgenome, which is copied into dsDNA
after infection. The DNA is then stablyafter infection. The DNA is then stably
integrated into the host genome by aintegrated into the host genome by a
transposition mechanism.transposition mechanism.
• They have some strong promoters, andThey have some strong promoters, and
they have been considered as vectorsthey have been considered as vectors
forfor gene therapygene therapy, since the foreign DNA, since the foreign DNA
will be incorporated into thewill be incorporated into the hosthost
genomegenome in ain a stable mannerstable manner..
Editor's Notes
Wild type phage DNA itself cannot be used as a vector since it contains too many
restriction sites. Further, these sites are often located within the essential regions for
phage's growth and development. From these wild phages, derivatives with single target
sites and two target sites have been synthesized. Phage vectors which contain single
site for the insertion of foreign DNA have been designated as insertional vectors;
vectors with two cleavage sites, which allow foreign DNA to be substituted for the DNA
sequences between those sites, are known as replacement vectors.