This document discusses genetic transformation, which is the alteration of a cell by incorporating exogenous DNA. It describes natural transformation in bacteria, which occurs when competent bacteria take up exogenous DNA from their environment. The document also discusses artificial transformation techniques used in labs, such as treating bacteria with calcium chloride to make them competent, then exposing them to exogenous DNA. It explains how the DNA enters the cell and can become incorporated into the genome. Selection methods for transformed cells, such as antibiotic resistance markers, are also summarized.
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.
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.
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.
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
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
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
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
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
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
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
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
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Second ppt on endocrine system, describing hypothalamus, pituitary and thyroid glands.
This describes the hormones from these glands and their mode of action etc
This is on the basic details of the endocrine system including the different types of hormones. It describes the mechanisms of actions of hormones. The general control mechanisms of hormone production and release are also included.
This ppt is about the variations in metabolic processes between different types of cells in different organs of our body. The reasons for the variations are also descried. This is the first set of slides on the topic.
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The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
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V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
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high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
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and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
2. Transformation
• Genetic alteration of a cell by the
incorporation of exogenous genetic material
(exogenous DNA)
• Transformation occurs naturally in some
species of bacteria
• It can also be done by artificial means in
other cells
• For transformation we use competent
bacteria
3. Natural transformation
• It is a bacterial adaptation for DNA transfer
• It is a complex, energy requiring developmental process
• To take up and recombine exogenous DNA into its chromosome bacteria must be
in a competent state
• A special physiological state which requires expression of about 40 genes
• Competence for transformation is typically induced by high cell density and/or
nutritional limitation- conditions associated with the stationary phase of bacterial
growth
• In some bacteria transformation could occur at the end of exponential growth as
bacterial growth approaches stationary phase-eg Haemophilus influenzae
• In some transformation occurs at high cell density and is associated with biofilm
formation -eg Streptococcus mutans
• In some transformation occurs toward the end of logarithmic growth eg B.
subtilis
4. For the repair of DNA damage
• Competence is specifically induced by DNA damaging conditions
• DNA material can be transferred between different strains of
bacteria, in a process that is called horizontal gene transfer
• Some species upon cell death release their DNA to be taken up by
other cells
• Transformation works best with DNA from closely related species
• Naturally competent bacteria carry sets of genes that provide the
protein machinery to bring DNA across the cell membrane(s)
• Due to the differences in structure of the cell envelope
between Gram-positive and Gram-negative bacteria, there are
some differences in the mechanisms of DNA uptake in these cells,
however most of them share common features that involve related
proteins
5. Process of transformation
• The DNA first binds to the surface of the competent cells on
a DNA receptor, and passes through theplasma
membrane via DNA translocase
• Only single-stranded DNA may pass through, one strand is
therefore degraded by nucleases in the process
• The translocated single-stranded DNA may then be
integrated into the bacterial chromosomes by
• In Gram-negative cells, due to the presence of an extra
membrane, the DNA requires the presence of a channel
formed by secretins on the outer membrane
• The uptake of DNA is generally non-sequence specific,
although in some species the presence of specific DNA
uptake sequences may facilitate efficient DNA uptake
6. Artificial competence
• Make the cell passively permeable to DNA by exposing it to
conditions that do not normally occur in nature
• The cells are incubated in a solution containing
divalent cations (eg calcium chloride) under cold
conditions
• The surface of bacteria such as E. coli is negatively charged
due to phospholipids and lipopolysaccharides on its cell
surface
• The DNA is also negatively charged
• One function of the divalent cation is to shield the charges
by coordinating the phosphate groups and other negative
charges
• This allow a DNA molecule to adhere to the cell surface
7. Making competent bacteria
• Exposing the cells to divalent cations in cold condition may
also change or weaken the cell surface structure of the cells
making it more permeable to DNA
• The heat-pulse to create a thermal imbalance on either side of
the cell membrane, which forces the DNA to enter the cells
through either cell pores or the damaged cell wall
• Electroporation is another method of promoting competence
• The cells are briefly shocked with an electric field of 10-
20 kV/cm
• This create holes in the cell membrane through which the
plasmid DNA may enter
• After the electric shock the holes are rapidly closed by the
cell's membrane-repair mechanisms
8. Transformation in yeast
• Yeast cells treated with enzymes to degrade their cell walls
yielding spheroplasts
• These cells are very fragile but take up foreign DNA at a high
rate
• Exposing intact yeast cells to alkali cations such as those
of cesium or lithium allows the cells to take up plasmid DNA
• Electroporation: Formation of transient holes in the cell
membranes using electric shock; this allows DNA to enter as
described above for Bacteria
• Enzymatic digestion or agitation with glass beads may also be
used to transform yeast cells
• Different yeast genera and species take up foreign DNA with
different efficiencies
9. Selection and screening in plasmid
transformation
• Transformation usually produces a mixture of relatively few transformed cells and
an abundance of non-transformed cells
• Need to select the cells that have acquired the plasmid
• The plasmid therefore requires a selectable marker such that those cells without
the plasmid may be killed or have their growth arrested
• Antibiotic resistance is the most commonly used marker for prokaryotes
• The transforming plasmid contains a gene that confers resistance to an antibiotic
that the bacteria are otherwise sensitive to
• The mixture of treated cells is cultured on media that contain the antibiotic so that
only transformed cells are able to grow
• Another method of selection is the use of certain auxotrophic markers that can
compensate for an inability to metabolise certain amino acids, nucleotides, or
sugars
• This method requires the use of suitably mutated strains that are deficient in the
synthesis or utility of a particular biomolecule, and the transformed cells are
cultured in a medium that allows only cells containing the plasmid to grow
• β lactase gene
10. Use of reporter genes as markers
• Reporter genes such as the lacZ gene which codes for β-
galactosidase used in blue-white screening
• This method of screening relies on the principle of α-
complementation
• A fragment of the lacZ gene (lacZα) in the plasmid can
complement another mutant lacZ gene (lacZΔM15) in the
cell
• Both genes by themselves produce non-functional
peptides, however, when expressed together, as when a
plasmid containing lacZ-α is transformed into
a lacZΔM15cells, they form a functional β-galactosidase
• The presence of an active β-galactosidase may be detected
when cells are grown in plates containing X-gal
• Form characteristic blue colonies