The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
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
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
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
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
DNA cloning is a technique for reproducing DNA fragments.
It can be achieved by two different approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
a vector is required to carry the DNA fragment of interest into the host cell.
This presentation deals with the introduction of Recombinant DNA Technology. The role of different enzymes. Specifically Restriction endonucleases and roles of various vectors.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
Restriction fragment length polymorphism (abbreviated RFLP) refers to differences (or variations) among people in their DNA sequences at sites recognized by restriction enzymes. Such variation results in different sized (or length) DNA fragments produced by digesting the DNA with a restriction enzyme.
Mapping and quantifying transcripts:
Northern blots
S1 mapping of 5’ and 3’ end transcripts
Primer extension
Runoff transcription and G –less cassette transcription
Nuclear Runon transcription
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
1. SCREENING AND SELECTION FOR RECOMBINANTS
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077
2. SCREENING AND SELECTION FOR RECOMBINANTS
The art of cloning is to find the one particular transformed cell/recombinant cell that contains the cloning vector with the
gene of interest (referred to as a recombinant cell).
It is thus most important to be able to select recombinant cells from transformed cells (containing the vector without
insert DNA).
IDENTIFICATION OF RECOMBINANTS:
After the introduction of rDNA into suitable host cells, it is essential to identify those cells which have received the rDNA
molecules. This process is called screening or selection.
Generally, the selection methods are based on the expression or non-expression of certain traits such as antibiotic
resistance, expression of an enzyme such as β- galactosidase or protein such as GFP (Green Fluorescent Protein) and
dependence or independence of a nutritional requirement such as the amino acid leucine.
Direct selection, which means that the cloning experiment is designed in such a way that the clones obtained are the
clones containing the required gene. Almost invariably, selection occurs at the plating-out stage. Direct selection is the
method of choice, as it is quick and usually unambiguous. However, it is not applicable to all genes.
Identification of the clone from a gene library, which entails an initial “shotgun” cloning experiment, to produce a clone
library representing all or most of the genes present in the cell, followed by analysis of the individual clones to identify
the correct one.
3. I) DIRECT SELECTION OF RECOMBINANTS:
Two examples will be discussed:
(i) direct antibiotic resistance screening, and
(ii) blue-white color screening.
II) INDIRECT SELECTION OF RECOMBINANTS:
(i) Screening by Nucleic acid hybridization
(ii) Screening by Colony hybridization
(iii) Screening by Immunological assay
(iv) Screening by Protein/Enzyme activity
4. 1.) Direct antibiotic resistance screening
Most cloning vectors are designed so that the insertion
of a DNA fragment into the vector destroys the integrity
of one of the genes present on the molecule (usually an
antibiotic resistance gene).
INSERTIONAL SELECTION INACTIVATION METHOD:
In this approach, one of the genetic trait is disrupted by
inserting foreign DNA. Antibiotic resistance genes act as
a good insertion inactivation system.
Plasmid pBR322 contains two antibiotic resistance
genes, one for ampicillin (amprgene), and the other for
tetracycline (tetr gene).
If the target DNA is inserted into tetr gene using Bam HI,
the property of resistance to tetracycline will be lost.
Such recombinants would be tetssensitive.
When such recombinants (containing target DNA in tetr
gene) are grown into medium containing tetracycline,
they will not grow because their tetr gene has been
inactivated. But they are resistant to ampicillin because
ampr gene is functional.
On the other hand, the self-ligated recombinants will show
resistance to ampicillin and tetracycline. Therefore, they will
grow on medium containing both the antibiotics.
6. Blue white selection-
2. Visual Screening :Blue-white colour screening
A more sophisticated procedure for screening for the presence of recombinant plasmids,
which can be carried out on a single transformation plate, is called blue-white screening.
This method also involves the insertional inactivation of a gene and uses the production of a
blue compound as an indicator. The gene is lacZ, which encodes the enzyme ß-galactosidase,
and is under the control of the lac promoter.
If the host E. coli strain is expressing the lac repressor, expression of a lacZ gene on the
vector may be induced using IPTG (isopropyl- ß-D-thiogalactopyranoside), and the expressed
enzyme can utilize the synthetic substrate X-gal (5-bromo-4-chloro-3-indolyl- ß-D-
galactopyranoside) to yield a blue product.
7. pUC plasmids:
• pUC plasmids are small, high copy number plasmids of size 2686bp.
• This series of cloning vectors were developed by Messing and co-workers in the University of California. The p in
its name stands for plasmid and UC represents the University of California.
• pUC18 and pUC19 vectors are identical apart from the fact that the MCS is arranged in opposite orientation.
• pUC vectors consists of following elements:
► pMB1 “rep ” replicon region derived from plasmid pBR322 with single point mutation (to increase copy
number).
► “ bla ” gene encoding β lactamase which provide ampicillin resistance which is derived from pBR322. This site is
different from pBR322 by two point mutations.
► LacZ gene from E.coli lac operon system, which contains MCS restriction sites.
• pUC vectors contain a lacZ sequence and multiple cloning site (MCS) within lacZ. This helps in use of broad
spectrum of restriction endonucleases and permits rapid visual detection of an insert.
• “rop ” gene is removed from this vector which leads to an increase in copy number.
Copy number:
500-600 copies per cell
8. Alpha complementation:
The lac operon consists of structural and regulatory genes. The lac Z gene is a structural gene and is required
for galactoside metabolism. The lac-Z gene product (β-galactosidase) is a tetramer, and each monomer is made
of two parts - lacZ-alpha, and lacZ omega.
1. The pBSKS+ / pUC18 plasmid carries the alpha fragment of the lac Z gene. The alpha fragment is the amino-
terminus of the protein and is not functional by itself. The alpha fragment is denoted by lac Z'.
2. Typically, the E. coli host strain used for transformation is a mutant strain that has a deletion of the alpha
fragment of lac Z. The E. coli chromosome carries only the omega fragment, which is the carboxyl-terminus
of the protein. The omega fragment alone is also non-functional.
When both the alpha and omega fragments interacts, a functionally intact -galactosidase protein are produced.
This interaction is called alpha complementation.
It is determined that if the alpha fragment was deleted, the omega fragment is non-functional; however, alpha
fragment functionality can be restored in-trans via plasmid.
The substrates for the beta-galactosidase enzyme are galactosides, such as lactose. Lactose is hydrolyzed by
this enzyme into galactose and glucose. Artificial galactosides, such as 5-Bromo-4-Chloro-3-Indolyl-beta-D-
galactoside (X-Gal), are also substrates for -galactosidase. Isopropyl thiogalactoside (IPTG) acts as an inducer
of lacZ gene expression.
10. 1. Multiple cloning site is inserted into the gene lacZ’ coding for the alpha peptide of enzyme β-galactosidase.
(An MCS is a short DNA sequence consisting of restriction sites for many different restriction endonucleases).
2. Insertion of the MCS into the lacZ’ fragment does not affect the ability of the α-peptide, while cloning DNA
fragments into the MCS does.
3. Clones with foreign DNA in the MCS disrupt the ability of the cells to make β-galactosidase.
4. Plate on media with a Substrate X-gal (β-galactosidase indicator) and clones with intact β-galactosidase
enzyme indicate that cells containing empty vector without insert, will produce blue colonies. Colorless
(desirable) colonies indicates that cells contain the plasmid with foreign DNA
5. Therefore, recombinants can be detected by blue/white screening on growth medium containing X gal in
presence of IPTG as an inducer.
Cloning of insert DNA using β - galactosidase: as reporter gene
11.
12. II) Indirect methods:
Identification of the clone from a gene library:
Many different techniques are available for screening a library.
The most important approaches involve
screening by nucleic acid hybridization and
screening by Colony hybridization
screening by functional analysis- Immunological assay
- Protein Assay
13. Screening by Nucleic acid hybridization
1. The first step of a hybridization screening experiment
involves DNA from the clones are isolated and
separated on the agarose gel electrophoresis. The
transfer of the DNA from the gel to a nylon or
nitrocellulose membrane is carried out.
2. The DNA is denatured with alkali to produce single
strands that are bounded to the membrane by heat
treatment or UV irradiation.
3. The membrane is then immersed in a solution
containing a nucleic acid probe (usually radioactively
labeled) and incubated to allow the probe to hybridize
to its complementary sequence.
4. After hybridization, the membrane is washed to
remove unhybridized probe, and regions where the
probe has hybridized are visualized with
autoradiography.
14. DNA hybridization.
(1) The DNA of samples containing the putative target DNA is
denatured, and the single strands are kept apart, usually
by binding them to a solid support, such as a
nitrocellulose or nylon membrane.
(2) (2) The probe, which is often 100 to 1,000 bp in length, is
labeled, denatured, and mixed with the denatured
putative target DNA under hybridization conditions.
(3) (3) After the hybridization reaction, the membrane is
washed to remove nonhybridized probe DNA and
assayed for the presence of any hybridized labeled tag.
(4) If the probe does not hybridize, no label is detected.
(5) The asterisks denote the labeled tags (signal) of the
probe DNA.
15. Screening by Colony hybridization:
Screening a library with a labeled DNA probe (colony
hybridization).
Cells from the transformation reaction are plated onto solid agar
medium under conditions that permit transformed, but not
nontransformed, cells to grow.
(1) From each discrete colony formed on the master plate, a sample
is transferred to a solid matrix, such as a nitrocellulose or nylon
membrane. The pattern of the colonies on the master plate is
retained on the matrix.
(2) The cells on the matrix are lysed, and the released DNA is
denatured, deproteinized, and irreversibly bound to the matrix.
(3) A labeled DNA probe is added to the matrix under hybridization
conditions. After the nonhybridized probe molecules are washed
away, the matrix is processed by autoradiography to determine
which cells have bound labeled DNA.
(4) A colony on the master plate that corresponds to the region of
positive response on the X-ray film is identified. Cells from the
positive colony on the master plate are subcultured because they
may carry the desired plasmid-cloned DNA construct.
16. Screening by Immunological assay
The desired gene can also be identified by the activities of
the encoded gene product.
1. The cells are grown as colonies on master plates which
are transferred to a solid matrix (i.e., nitrocellulose).
2. The colonies are then subjected to lysis and the released
proteins bound to the matrix. These proteins are then
treated with a primary antibody which specifically binds
to the protein (acts as an antigen), encoded by the target
DNA.
3. After removing the unbound antibody by washings, a
second antibody is added which specifically binds to the
first antibody.
4. Again the unbound antibodies are removed by washings.
The second antibody carries an enzyme label (e.g., horse
reddish peroxidase or alkaline phosphatase) bound to it.
The detection process is so devised that as a colourless
substrate it is acted upon by this enzyme, a coloured
product is formed.
5. The colonies which give positive result (i.e., coloured
spots) are identified. The cells of a specific colony can be
sub-cultured from the master plate.
17. Screening by Protein activity
DNA hybridization and immunological assays work well for
many kinds of genes and gene products.
1. If the target gene produces an enzyme that is not
normally made by the host cell, a direct (in situ) plate
assay can be devised to identify members of a library
that carry the particular gene encoding that enzyme.
2. The genes for α-amylase, endoglucanase, β-glucosidase,
and many other enzymes from various organisms have
been isolated in this way.
3. This approach has proven effective for isolating genes
encoding biotechnologically useful enzymes from
microorganisms present in environmental samples.
4. This technique has enabled the isolation of many novel
proteins with interesting properties without the need to
first culture the natural host microorganism.
Screening a genomic library for enzyme
activity.
1. Cells of a genomic library are plated onto solid
medium containing the substrate for the
enzyme of interest.
2. If a functional enzyme is produced by a colony
that carries a cloned gene encoding the
enzyme, the substrate is converted to a
colored product that can be easily detected.
3. Note that other, noncolored colonies on the
medium also contain fragments of the genomic
library, but they do not carry the gene for the
enzyme of interest.