This document discusses key applications of plant metabolic engineering, including enabling plants to fix their own nitrogen, altering nutrient content of crop plants, enhancing photosynthetic efficiency, and using plants for biofuel production. Some of the challenges discussed are expressing the large number of genes required for nitrogen fixation in plants, developing efficient nutrient exchange in plant-microbe symbiosis, and modifying lignin in plants to more easily access cellulose for biofuels while avoiding negative impacts on growth.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
The different types of external stresses that influence the plant growth and development.
These stresses are grouped based on their characters
Biotic
Abiotic
Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress in plants.
This damages the cellular constituents of plants which are associated with a reduction in plant yield.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
Agrobacterium mediated Transformation-Mechanism of gene transfer,Virulence induction in presence of Plant secondary metabolites,Chromosomal genes and Vir genes,Agrobacterium tumefaciens – pathogen and useful tool for genetic engineering
Introduction
Definition of an Insect Resistant Plant
What is the Bt gene?
History
The crystal ( cry)Proteins
Definition of cry protein
How does Bt work?
Mechanism of Bt toxicity
Mode of Action of Insecticidal Crystal Protein
Bt Technology
The Insect Resistance Problem
Advantages
Limitations
Conclusion
References
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
Synthetic Biology for Plant ScientistsSachin Rawat
Tools of synthetic biology can be utilised to engineer metabolic pathways to optimize production of secondary metabolites and ligno-cellulose. The presentation describes an approach to develop an artificial positive feedback loop to increase accumulation of cell wall polysaccharides. These will decrease the cost of production of plant-based biofuels, paper and other plant products.
The different types of external stresses that influence the plant growth and development.
These stresses are grouped based on their characters
Biotic
Abiotic
Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress in plants.
This damages the cellular constituents of plants which are associated with a reduction in plant yield.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
Agrobacterium mediated Transformation-Mechanism of gene transfer,Virulence induction in presence of Plant secondary metabolites,Chromosomal genes and Vir genes,Agrobacterium tumefaciens – pathogen and useful tool for genetic engineering
Introduction
Definition of an Insect Resistant Plant
What is the Bt gene?
History
The crystal ( cry)Proteins
Definition of cry protein
How does Bt work?
Mechanism of Bt toxicity
Mode of Action of Insecticidal Crystal Protein
Bt Technology
The Insect Resistance Problem
Advantages
Limitations
Conclusion
References
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
Synthetic Biology for Plant ScientistsSachin Rawat
Tools of synthetic biology can be utilised to engineer metabolic pathways to optimize production of secondary metabolites and ligno-cellulose. The presentation describes an approach to develop an artificial positive feedback loop to increase accumulation of cell wall polysaccharides. These will decrease the cost of production of plant-based biofuels, paper and other plant products.
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http://sandymillin.wordpress.com/iateflwebinar2024
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Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
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key application of plant metabolic engineering
1. Key Applications of Plant
Metabolic Engineering
Mirza Faisal Qaseem
08-arid-876
PhD scholar (Bot)
PMAS- Arid Agriculture
University
Rawalpindi, Pakistan
2. Contents
• Challenges in plant metabolic engineering
• Plants That Can Fix Their Own Nitrogen
• Crop Plants with Altered Nutrient Content
• Enhancing Photosynthetic Efficiency
• TALENs
• CRISPRs
3. Plants
• Derive energy entirely from the sun
• Carbon from CO2
• Defend themselves from pests and predators
• Complex symbioses
• Can survive extremes of
– temperature
– nutrient
– water availability
4.
5. Challenges in plant
metabolic engineering
• Four long-standing grand challenges in plant
metabolic engineering
• Two deal with important applications in food
and energy
• Two are of general utility in improving plant
fitness
• Solution
– Subjecting plant genome to deletion editing and
insertions
6. Plants That Can Fix Their Own
Nitrogen
• 180 million tons of nitrogen fertilizer is used
every year in industrial farming
• Disadvantages
– Substantial cost
– Deleterious effects
• Soil
• Surrounding environment
• Water
• Plants able to make their own nitrogen
Transform agriculture by reducing or eliminating
this enormous dependence on fertilizer
7. Plant engineering using Bacteria
• First method takes advantage of the fact that
some bacteria carry out their own version of
the Haber-Bosch process—reducing
atmospheric N2 into a more bio available form,
NH3—using the enzyme nitrogenase
8. Nitrogenase
• Complex enzyme
– multiple metalloclusters
• Require
– large quantity of biochemical energy
• Transfer the electrons needed to activate the
exceptionally stable N2 triple bond.
• By expressing nitrogenase, plants would be
able to fix their own nitrogen.
9. • Nitrogen fixed by a plant could be used
immediately to generate amino acid and
nucleic acid monomers
• Transport them to neighboring cells
• Disadvantage
– Process induce a metabolic cost
• Can be regulated by the endogenous level of
nitrogen to maximize its efficiency
10. Challenges
• Eighteen gene products (in Klebsiella oxytoca)
are necessary and sufficient for the
production of nitrogenase and its complex
iron-molybdenum cofactor.
• Biosynthetic gene cluster for nitrogenase has
been refactored—taken apart, recoded, and
put back together using known components—
and shown to be active in its new host
11. The successful transfer of other large gene
clusters from one microbe to another suggests
that the process of functionalizing microbes is
undergoing a dramatic improvement
12. Challenges
• 18 components of the nitrogenase
biosynthetic apparatus would need to be
expressed simultaneously in plants and
function in concert
• Since plants are eukaryotic and multicellular,
where in the plant cell should the genes be
expressed and in which cell types of the plant
13. • Nitrogenase which is poisoned by oxygen and
must therefore be expressed under anaerobic
conditions.
• Tools that enable organelle and cell-type
specific expression will be of great utility here
and in other plant engineering efforts.
14. Second method of increasing N uptake
• Engineer a rhizosphere symbiosis between a
nitrogen-fixing microorganism and a plant
host
• Primary advantages
i. It uncouples the difficulties of utilizing
nitrogenase from the biology of the plant host
ii. Outsources the demanding chemistry involved
to a bacterial strain better suited to the task.
15. ii. The well-studied symbioses between legumes
and their nitrogen-fixing, rootnodulating
bacterial symbionts prove that a bacterial
mutualist can satisfy the nitrogen needs of a
plant host
• Nitrogen-fixing bacteria in the rhizosphere opens
the possibility that symbioses of this sort are a
much more widely distributed phenomenon
16.
17. Challenges
• Enabling efficient nutrient exchange
• Maintaining specificity of the host-microbe
pair
– Both could take years to develop and are likely to
require not just plant but also microbial metabolic
engineering.
– Advanced molecular breeding tools that enable
access to natural variation in a plant’s wild
ancestors are a promising alternative approach to
increasing crop plant yields
18. Crop Plants with Altered
Nutrient Content
• Golden rice proves the concept that the
nutrient content of a crop plant can be
improved by metabolic engineering
• Nutrient levels of certain plants was enhanced
using breeding
– rice
– maize
– wheat
– Tomatoes
19. • Golden rice
– adding the beta-carotene pathway to rice, to
produce rice with higher levels of vitamin A
• Targets for nutrient engineering
– Metabolic pathways
– alter the level of a nutrient in its native host
20. Engineering of Metabolic pathways
• Those pathways that produce
– Phytoalexins
– Flavonoids and other molecules
• Play a role in the chemopreventive properties
of vegetables and fruits.
21. Alteration of a nutrient level
• Not require knowing the genes in its
biosynthetic pathway
• Example
• Expressing two transcription factors
from snapdragon in tomato
• Levels of the flavonoid anthocyanin have been
increased 3-fold
• Flavonoid confer improved chemopreventive
properties in cancer susceptible mice
22. Expression of Health promoting
molecule in a new host.
• Golden rice is example of this strategy
• Only two genes were required for the production
of beta-carotene
• A more ambitious prospect would be to transfer
the 13-gene glucoraphanin pathway to a widely
consumed crop such as rice, wheat, or maize
• Require
– New approaches for discovering the genes
– new tools for site-selective genome editing
23.
24. • Introducing or increasing the level of a
nutrient compound could also alter the taste
of a plant, potentially impacting its
palatability.
– since small molecule metabolites make an
important contribution to flavor.
• Notable targets in this area include the steviol
glycosides and the mixed esters that give
strawberry plants their distinctive flavor
25. Engineering Crops for Biofuel
Production
• Plants are ideal invention
• Combat the dual challenges
– Rising greenhouse gases
– Used for green energy
• They capture CO2 from the air and turn it into
sugar, the ideal substrate for biofuel production.
• Plants protect this energy rich polymer
• Most of the carbon is stored as dehydrated
crystalline cellulose, wrapped in a meshwork of
crosslinked phenylpropanoids, lignin
26. • Cellulose presents a challenge in itself.
• The beta-1,4-linked chains of cellulose can pack
tightly together, excluding water in a way that
prevents glycosidic enzymes from releasing its
constituent sugar monomers
• Cellulose co purifies with lignin which inhabit the
action of cellulases
• So there is need for costly and energy intensive
pretreatment to separate the cellulose from
lignin
27. • Lignin could be degraded into valuable
aromatic monomers either
• Chemically
• Enzymes found in
– White rot fungi
– Microorganisms
• Neither has been shown to work in a real-
world setting
28. • Lignin biosynthesis can be genetically
modified to change its chemical composition
or to reduce its content in plant tissues to
improve the processing of polysaccharides.
• Arabidopsis thaliana knockout mutant of
caffeoyl shikimate esterase
– Gave a 4-fold increase in the efficiency of
saccharification
29. • In Lignin modified plants Discernible effects
on plant growth and development
– Transgenics contained 25% less cellulose content
– 40% lighter and smaller than wild-type
• Protein engineering and expression of a 4-O-
methyltransferase in A. thaliana substantially
reduced lignin content
• Interestingly, no significant changes in growth
phenotype were observed and
saccharification yields improved by 25%.
30. • Ideal scenario would be for plants to degrade
their own lignin, releasing pure cellulose that
could be more easily degraded into glucose
• For that matter, the plant could be engineered
to break down its own cellulose on demand,
releasing fermentation- ready sugars for
biofuel production.
31. Challenges
• Feasibility of enzymatically degrading lignin to
liberate cellulose
• Although this would undoubtedly be a difficult
task, the ability of white rot fungi to degrade
lignin proves the concept that there exist
enzymes (e.g., lignin peroxidases) that can
cleave the lignin meshwork into monomers
and smaller polymers
32. • Heavily crosslink (and consolidate) the lignin
– causing it to precipitate and making it easier to
separate from cellulose.
• Process likely to be carried out by suites of
degrading enzymes in rot fungi, a critical step
would be to first identify sets of enzymes that
could be coexpressed to make the necessary
modifications to lignin.
33. Enhancing Photosynthetic
Efficiency
• Rubisco is the enzyme that catalyzes the first
key step in CO2 fixation as part of the Calvin
Cycle
• Its low turnover rate and ability to also use
oxygen as a substrate in photorespiration
make it notoriously inefficient.
• Plants make more Rubisco than any other
protein
34. Alternative carbon fixation systems
• Improve the efficiency of photosynthesis by
actively concentrating CO2 and reducing the
oxygenase activity of Rubisco
• Plants have evolved two systems to improve
photosynthesis efficiency
• C4
• Crassulacean acid metabolism (CAM)
35. C3, C4 and CAM plants
• C3
• the process of fixing carbon into C3 acids
occurs in the same cell type
• C4
• Evolved to separate the Calvin cycle and
carbon capture into different cell types.
– CO2 is first captured within mesophyll cells to
produce C4 acids
36. – Which diffuse to bundle sheath cells where they
are decarboxylated and concentrated to maximize
Rubisco’s carboxylating efficiency.
• CAM plants
• Photosynthesis and carbon capture are
separated temporally
– capture CO2 at night
– Close their stomata during the day
– C4 acids generated by CAM photosynthesis are
decarboxylated and concentrated to enhance
Rubisco’s efficiency
37. • All the enzymes of the C4 cycle are known and
already exist in C3 plants.
• However, expressing the enzymes of the C4 cycle
alone will not be enough, as the plant’s anatomy
is crucial for the success of the pathway
• Genes that are responsible for controlling C4 leaf
anatomy remain largely unknown and are being
identified by mutant populations of model C4
plants like Sorghum
38. • Cell-specific promoters will need to be
identified to enable cell-type-specific
expression in bundle-sheath or mesophyll
cells.
• > 20 genes needed for the installation of C4
photosynthesis in C3 plant
• Sophisticated transformation
• Genome editing technologies
39. Technologies
• Transcriptomic and metabolomic analyses
• Genome editing tools
– TALENs
– CRISPR/Cas9
• Synthetic biology parts list specific to plants
– tissue-specific promoters,
– Transporters
– multi-gene expression constructs
– biosynthetic enzymes
40. Transcription activator-like effector
nucleases TALENs
• Artificial restriction enzymes
• Generated by fusing a
• TAL effector DNA binding domain
• DNA cleavage domain.
• TAL effectors
– are proteins secreted by Xanthomonas bacteria
when they infect various plant species.
41. • TALEs can be quickly engineered to bind
practically any desired DNA sequence
• Combining such an engineered TALE with a DNA
cleavage domain (which cuts DNA strands), one
can engineer restriction enzymes that are specific
for any desired DNA sequence.
• When these restriction enzymes are introduced
into cells, they can be used for genome editing in
situ
42. Applications
– Elucidating basic function and regulating a gene
– Study of metabolic pathways
– Embryonic stem cell research
– Research on disease model
– Provide therapeutic avenues for genetic disorders
including monogenic diseases
– Method of the Year” for 2011 by Nature Methods
43. Problems
• Non specific
• off-target cleavage may result
• lead to the production of enough double-
strand breaks to overwhelm the repair
machinery.
• consequently yield chromosomal
rearrangements and/or cell death.
44.
45. CRISPRs (clustered regularly interspaced
short palindromic repeats)
• DNA loci containing short repetitions of base
sequences
• Repetition is followed by short segments of
"spacer DNA“
• CRISPR loci range in size from 24 to 48 base pairs
• Spacer DNA are regions of non-coding
DNA between tandemly repeated genes.
• CRISPRs are found in approximately 40% of
sequenced bacteria genomes and 90% of
sequenced archaea
46. • CRISPR/Cas system is a prokaryotic immune
system
• Confers resistance to foreign genetic elements
such as plasmid and phages and provides a
form of acquired immunity.
• CRISPR spacers recognize and cut these
exogenous genetic elements
• Since 2013, the CRISPR/Cas system has been
used for gene editing and gene regulation
47. Applications
• Artificial immunization against phage by introduction
of engineered CRISPR loci in industrially important
bacteria, including those used in food production and
large-scale fermentation
• Genome engineering at cellular or organismic level by
reprogramming a CRISPR/Cas system to achieve RNA-
guided genome engineering.
• Discrimination of bacterial strains by comparison of
spacer sequences
48.
49.
50. Reference
• Lau W, Fischbach MA, Osbourn A, Sattely ES
(2014) Key Applications of Plant Metabolic
Engineering. PLoS Biol 12(6): e1001879.
doi:10.1371/journal.pbio.1001879