A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Codon optimization is one of the key steps in achieving the high level expression of target gene. There are some key factors for consideration including transcription efficiency, translation efficiency, gene synthesis and protein folding.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Codon optimization is one of the key steps in achieving the high level expression of target gene. There are some key factors for consideration including transcription efficiency, translation efficiency, gene synthesis and protein folding.
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
S1 Mapping is a laboratory method used for locating the start and end points of
transcripts and for mapping introns.
This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.
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
S1 Mapping is a laboratory method used for locating the start and end points of
transcripts and for mapping introns.
This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.
Biotechnology is the utilization of biology to figure out problems and develop beneficial products. The most important area of biotechnology is the manufacturing of therapeutic proteins and other drugs via genetic engineering.
Central dogma of molecular biology with video slide.Asheesh Pandey
Central dogma of Molecular Biology with video of DNA replication, Transcription and Translation. It help to understand the basic of DNA structure and its function.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
Normal Labour/ Stages of Labour/ Mechanism of LabourWasim Ak
Normal labor is also termed spontaneous labor, defined as the natural physiological process through which the fetus, placenta, and membranes are expelled from the uterus through the birth canal at term (37 to 42 weeks
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
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.
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
Honest Reviews of Tim Han LMA Course Program.pptxtimhan337
Personal development courses are widely available today, with each one promising life-changing outcomes. Tim Han’s Life Mastery Achievers (LMA) Course has drawn a lot of interest. In addition to offering my frank assessment of Success Insider’s LMA Course, this piece examines the course’s effects via a variety of Tim Han LMA course reviews and Success Insider comments.
1. What Does It Mean: “To Clone”?What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an
original molecule or cell
To "clone a gene" is to make many copies of it - for example, by
replicating it in a culture of bacteria.
Cloned gene can be a normal copy of a gene (= “wild type”).
Cloned gene can be an altered version of a gene (= “mutant”).
Recombinant DNA technology makes manipulating genes
possible.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
6. Genetic engineering produces proteins
that offer advantages over proteins
isolated from other biological sources.
These advantages include:
◦ High purity
◦ High specific activity
◦ Steady supply
◦ Batch-to-batch consistency
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
7. Case Study: The Use of RecombinantCase Study: The Use of Recombinant
DNA to Produce Human InsulinDNA to Produce Human Insulin
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
8. Why synthesize human insulin?Why synthesize human insulin?
Patients’ immune systems do not
produce antibodies against human insulin
as they do with bovine or porcine insulin
Projected decline in the production of
animal-derived insulin
Need for a more reliable and sustainable
method of obtaining the product
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
9. Why is insulin needed?Why is insulin needed?
Protein hormone produced by beta cells
of islets of Langerhans in the pancreas
Regulates blood sugar by allowing uptake
of glucose from bloodstream into body
cells
Patients with diabetes have insufficient
or impaired production of insulin
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
10. Structure of InsulinStructure of Insulin
Two polypeptide chains; one with 21
amino acids and the second with 30
amino acids
Chains are linked via a disulfide bond
Gene encoding the insulin protein is
found on chromosome 11
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
11. Recombinant DNA TechniqueRecombinant DNA Technique
Restriction enzymes
used to cut out
insulin gene and to
cut a bacterial
(E. coli) plasmid at
the same “sticky
ends”
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
12. Recombinant DNA TechniqueRecombinant DNA Technique
Mutant strains of E. coli used to avoid
bacteria attacking “foreign” genes
Insert insulin gene next to E. coli
B-galactosidase gene which controls
transcription
Bacterial cells replicate and make copies
of insulin gene
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
13. Recombinant DNA TechniqueRecombinant DNA Technique
Insulin protein is purified (B-galactosidase
removed)
Chains are mixed and disulfide bridges
form
Yeast cells provide a sterile growth
medium
Final product is Humulin - chemically
identical to human insulin
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
14. Possible Complications of UsingPossible Complications of Using
Human InsulinHuman Insulin
hypoglycemia (low blood sugar) tends to
be more common than with animal
insulin
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
15. The Role of RestrictionThe Role of Restriction
EndonucleasesEndonucleases
Restriction endonucleases, first
discovered in the late 1960s, are named
for preventing invasion by foreign DNA by
cutting it into pieces
These enzymes cut at sites within the
foreign DNA instead of chewing from the
ends
By cutting DNA at specific sites they
function as finely honed molecular knives
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
16. Naming Restriction EndonucleasesNaming Restriction Endonucleases
Restriction endonucleases are named using the 1st
three letters of their name from the Latin name of
their source microorganism Hind III
◦ First letter is from the genus H from Haemophilus
◦ Next two letters are the 1st
two letters of the species
name in from influenzae
◦ Sometimes the strain designation is included
“d” from strain Rd
◦ If microorganism produces only 1 restriction enzyme,
end the name with Roman numeral I Hind I
◦ If more than one restriction enzyme is produced, the
others are numbered sequentially II, III, IV, etc.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
17. Restriction Endonuclease SpecificityRestriction Endonuclease Specificity
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY at that
sequence
◦ These enzymes can recognize
4-bp, 6-bp, 8-bp sequences
◦ The frequency of cuts lessens
when the recognition
sequence is longer
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
18. Restriction Enzyme TerminologyRestriction Enzyme Terminology
A 6-bp cutter will yield DNA fragments
averaging 4000-bp or 4 kilobases (4kb) in
length
Heteroschizomers recognize the same
DNA sequence but use a different cutting
site – they are also called isochizomers
These enzymes cut DNA strands
reproducibly in the same place, which is
extremely useful in gene analysis
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
19. Use of Restriction EndonucleasesUse of Restriction Endonucleases
Many restriction endonucleases make
staggered cuts in the 2 DNA strands
◦ This leaves single-stranded overhangs, called
sticky ends that can base-pair together briefly
◦ This makes joining 2 different DNA molecules
together much easier
Staggered cuts occur when the
recognition sequence usually displays
twofold symmetry, palindromes
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
20. Restriction-Modification SystemRestriction-Modification System
What prevents these enzymes
from cutting up the host
DNA?
◦ They are paired with methylases
◦ Theses enzymes recognize,
methylate the same site
Together they are called a
restriction-modification
system, R-M system
Methylation protects DNA,
after replication the parental
strand is already methylated
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
21. Basics of type II Restriction EnzymesBasics of type II Restriction Enzymes
No ATP requirement.
Recognition sites in double stranded DNA have a 2-fold axis
of symmetry – a “palindrome”.
Cleavage can leave staggered or "sticky" ends or can produce
"blunt” ends.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
23. An Experiment Using RestrictionAn Experiment Using Restriction
EndonucleaseEndonuclease
An early experiment used EcoRI to
cut 2 plasmids, small circular pieces
of DNA independent of the host
chromosome
Each plasmid had 1 site for EcoRI
◦ Cutting converted circular plasmids
into linear DNA with the same
sticky ends
◦ The ends base pair
Some ends re-close
Others join the 2 pieces
DNA ligase joins 2 pieces with
covalent bonds
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
24. Plasmids – vehicles for cloningPlasmids – vehicles for cloning
Plasmids are naturally occurring
extrachromosomal DNA molecules.
Plasmids are circular, double-stranded
DNA.
Plasmids are the means by which
antibiotic resistance is often
transferred from one bacteria to
another.
Plasmids can be cleaved by restriction
enzymes, leaving sticky or blunt ends.
Artificial plasmids can be constructed
by linking new DNA fragments to the
sticky ends of plasmid.
02/20/15
Tetr
Ampr
Ori
pBR322
4361bp
Ori
pUC18
Ampr
MCS
LacZ
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
25. Cloning VectorsCloning Vectors
A cloning vector is a plasmid that can be
modified to carry new genes.
Plasmids useful as cloning vectors must
have:
An origin of replication.
A selectable marker (antibiotic
resistance gene, such as ampr
and
tetr
).
Multiple cloning site (MCS) (site
where insertion of foreign DNA will
not disrupt replication or inactivate
essential markers).
Easy to purify away from host DNA.
02/20/15
Tetr
Ampr
Ori
pBR322
4361bp
Ori
pUC18
Ampr
MCS
LacZ
Older cloning vector
Newer cloning vector
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
26. Insert – Target DNA
2. Restriction Enzymes
1. PCR product
RE1
RE2
RE1
RE1
RE2
RE2
T
T
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
27. VectorsVectors
Vectors function as DNA carriers to allow
replication of recombinant DNAs
Typical experiment uses 1 vector plus a piece
of foreign DNA
◦ Depends on the vector for its replication
◦ Foreign DNA has no origin of replication, the site
where DNA replication begins
There are 2 major classes of vectors:
◦ Plasmids
◦ Phages
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
28. Vector requirementsVector requirements
Selectable marker
◦ Resistance to ampicillin, kanamycin, hygromycin, herbicide, etc.
◦ Allows only recombinant clones to survive
Multiple cloning site (mcs)
◦ many (unique) restriction sites
For vectors that amplify in E. coli cells:
◦ Origin of replication (Ori): required for plasmid to replicate in
bacterium
◦ Control of copy number (F factor plasmid): reduces copy
number of plasmids in a given cell, lessens problems of
rearrangements (chimerism)
For vectors that amplify in yeast cells:
◦ Autonomous replication sequence (ARS): similar to Ori
◦ CEN: centromere
◦ TEL: telomere
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
29. Plasmids As VectorsPlasmids As Vectors
pBR plasmids were developed early but
are rarely used today
pUC series is similar to pBR
◦ 40% of the DNA, including tetracycline
resistance has been deleted
◦ Cloning sites are clustered together into one
area called the multiple cloning site (MCS)
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
30. pBR322 PlasmidpBR322 Plasmid
pBR322 illustrates
cloning methods simply
◦ Resistance for 2 antibiotics
Tetracycline
Ampicillin
◦ Origin of replication
between the 2 resistance
genes
◦ Only 1 site for several
restriction enzymes
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
31. pBR322 CloningpBR322 Cloning
Clone a foreign DNA into the
PstI site of pBR322
Cut the vector to generate
the sticky ends
Cut foreign DNA with PstI
also – compatible ends
Combine vector and
foreign DNA with DNA ligase
to seal sticky ends
Now transform the
plasmid into E. coli
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
32. Bacterial TransformationBacterial Transformation
Traditional method involves incubating
bacterial cells in concentrated calcium
salt solution
◦ The solution makes the cell membrane leaky,
permeable to the plasmid DNA
Newer method uses high voltage to drive
the DNA into the cells in process called
electroporation
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
33. Transformation
Two main methods:
1. Chemical transformation – Chilling cells in the presence
of Ca2+ prepares the cell walls to become permeable
to plasmid DNA. Cells are briefly heat shocked which
causes the DNA to enter the cell
2. Electoporation- making holes in bacterial cells, by
briefly shocking them with an electric field of 10-
20kV/cm. Plasmid DNA can enter the cell through
these holes.
Use of bacterial cells to amplify
the DNA of interest
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
34. Screening TransformantsScreening Transformants
Transformation produces bacteria with:
◦ Religated plasmid
◦ Religated insert
◦ Recombinants
Identify the recombinants using the antibiotic
resistance
◦ Grow cells with tetracycline so only cells with plasmid
grow, not foreign DNA only
◦ Next, grow copies of the original colonies with ampicillin
which kills cells with plasmid including foreign DNA
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
35. Screening With Replica PlatingScreening With Replica Plating
Replica plating transfers clone
copies from original
tetracycline plate to a plate
containing ampicillin
A sterile velvet transfer tool
can be used to transfer copies
of the original colonies
Desired colonies are those
that do NOT grow on the new
ampicillin plate
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
36. pUC andpUC and ββ-galactosidase-galactosidase
Newer pUC plasmids have:
◦ Ampicillin resistance gene
◦ Multiple cloning site inserted into the gene lacZ’
coding for the enzyme β-galactosidase
Clones with foreign DNA in the MCS disrupt the ability of
the cells to make β-galactosidase
Plate on media with a β-galactosidase indicator (X-gal)
and clones with intact β-galactosidase enzyme will
produce blue colonies
Colorless colonies should contain the plasmid with
foreign DNA
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
37. Lac Z gene
LacZ genepromotor
RNA
pol.
Gene expression dogma
DNA
LacZ mRNA
Ribosome
β-galactosidase
RNA
Protein
X-gal BLUE coloniesBLUE colonies
WHITEWHITE coloniescoloniesX-gal
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
38. Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG)
b. Heat-inducible
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
39. Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG or arabinose)
b. Heat-inducible
3) Expression vectors with a fusion tag for affinity purification
Facilitate the purification of the expressed protein
1) 6 Histidine tag
2) Glutathione transferase tag (GST)
3) Maltose-binding protein tag
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
40. LacZpromotor operator
Repressor
Lac Z gene
LacZpromotor
RNA
pol.
RNA
pol.
IPTG
IPTG
IPTG
LacZpromotor operator
IPTG
IPTG
RNA
pol.
IPTG
X-gal
Β-galactosidase
X + galactose
Cells which produce ß-galactosidase form BLUE coloniesBLUE colonies.
Cells without ß-galactosidase production form WHITEWHITE coloniescolonies.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
41. X
XX
X
X
Plasmid without Insert
Plasmid +Insert
without plasmid
Screening
LacZ
pGEM
Insert
WHITE coloniesWHITE colonies BLUEBLUE coloniescolonies
prom
otor
operat
or
T
T
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
42. An Expression Vector for Making a LacZ Fusion Protein
LacZ can be replaced with:
1) Glutathione S-transferase (GST)
2) Maltose-binding protein (MBP)
Affinity Ligand:
Glutathione
Starch (amylose)02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
43. A plasmid DNA will be purified from the bacteria cells.
Insert
Vector
Confirmation by digestion with restriction
enzyme and separation of the digestion
products on agarose gel
EcoRI
EcoRI
Plasmid DNA will be digested with EcoRI, and analyzed by gel
electrophoresis for identification of the clone containing insert.
pGEM
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
44. Directional CloningDirectional Cloning
Cut a plasmid with 2 restriction enzymes
from the MCS
Clone in a piece of foreign DNA with 1
sticky end recognizing each enzyme
The insert DNA is placed into the vector
in only 1 orientation
Vector religation is also prevented as the
two restriction sites are incompatible
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
45. SummarySummary
First generation plasmid cloning vectors include
pBR322 and the pUC plasmids
pBR322 has
◦ 2 antibiotic resistance genes
◦ Variety of unique restriction sites for inserting foreign DNA
◦ Most of these sites interrupt antibiotic resistance, making
screening straightforward
pUC has
◦ Ampicillin resistance gene
◦ MCS that interrupts a β-galactosidase gene
MCS facilitates directional cloning into 2 different
restriction sites
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
46. Relaxed, cccDNA
(covalently, closed, circular)
Supercoiled
DNA
Open (nicked) circular
Gyrase
Topoisomerase
Endonuclease
DNA ligase
Endonuclease
Interconversions of
different forms of plasmids
Relaxed Plasmid (multiple copies per cell)
Stringent Plasmid (limited copies per cell)
Non-integrative
Episome (Integrated)
tra genes (conjugative / non-conjugative)
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
47. Phages As VectorsPhages As Vectors
Bacteriophages are natural vectors that
transduce bacterial DNA from one cell to
another
Phage vectors infect cells much more
efficiently than plasmids transform cells
Clones are not colonies of cells using
phage vectors, but rather plaques, a
clearing of the bacterial lawn due to
phage killing the bacteria in that area
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
48. Vectors Host - Features Insert Size
Plasmid E.coli- Introduced by transformation 1-5 kb
(electroporation or heat shock)
Phage E.coli- Virus that infects bacteria 10-15 kb
Introduced by transfection
Cosmid E.coli- Plasmid with “cos” sites for 30-45 kb
packaging into lambda phage particles.
Introduced by infection into E. coli
02/20/15
DNA molecule originating from a virus, a plasmid, or the cell of a higher
organism into which another DNA fragment of appropriate size can be
integrated without loss of the vector’s capacity for self-replication
Vectors introduce foreign DNA into host cells, where the DNA can be
reproduced in large quantities
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
49. RNase H
Pol I
RNA II primes
DNA synthesis
Replication
RNA II
(555 bp)
RNA I (108
bp)
5’
5’3’
3’
Origin
Infrequentl
y
Frequently
RNA I/ RNA II hybrid +
Rop dimer (initial
pairing of duplex)
Rop dimer
RNA II inactivated
for primer function
No Replication
Plasmid copy number (regulation of
replication of Col E1-derived plasmids
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
50. Partitioning and segregative stability of
plasmids
par gene
Incompatibility of plasmids
Inability of two different plasmids to
coexist in the same cell in the absence of
selection pressure (same mechanism of
replication control)
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
51. λλ Phage VectorsPhage Vectors
First phage vectors were constructed by
Fred Blattner and colleagues
◦ Removed middle region
◦ Retained genes needed for phage replication
◦ Could replace removed phage genes with
foreign DNA
Originally named Charon phage
More general term, replacement vectors
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
52. Phage Vector AdvantagesPhage Vector Advantages
Phage vectors can receive larger amounts
of foreign DNA
◦ Charon 4 can accept up to 20kb of DNA
◦ Traditional plasmid vectors take much less
Phage vectors require a minimum size
foreign DNA piece (12 kb) inserted to
package into a phage particle
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
53. Cloning Using a Phage VectorCloning Using a Phage Vector
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
54. Genomic LibrariesGenomic Libraries
A genomic library contains clones of all
the genes from a species genome
Restriction fragments of a genome can be
packaged into phage using about 16 – 20
kb per fragment
This fragment size will include the
entirety of most eukaryotic genes
Once a library is established, it can be
used to search for any gene of interest
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
55. Plaque HybridizationPlaque Hybridization
Searching a genomic
library requires probe
showing which clone
contains desired gene
Ideal probe – labeled
nucleic acid with
sequence matching the
gene of interest
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
56. CosmidsCosmids
Cosmids are designed for cloning large DNA fragments
◦ Behave as plasmid and phage
◦ Contain
cos sites, cohesive ends of phage DNA that allow the DNA to be packaged
into a λ phage head
Plasmid origin of replication permitting replication as plasmid in bacteria
◦ Nearly all λ genome removed so there is room for large inserts
(40-50 kb)
◦ So little phage DNA can’t replicate, but they are infectious
carrying recombinant DNA into bacterial cells
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
57. M13 Phage VectorsM13 Phage Vectors
Long, thin, filamentous phage M13
Contains:
◦ Gene fragment with β-galactosidase
◦ Multiple cloning site like the pUC family
Advantage
◦ This phage’s genome is single-stranded DNA
◦ Fragments cloned into it will be recovered in
single-stranded form
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
58. M13 Cloning to Recover Single-M13 Cloning to Recover Single-
stranded DNA Productstranded DNA Product
After infecting E. coli cells, single-
stranded phage DNA is converted
to double-stranded replicative
form
Use the replicative form for cloning
foreign DNA into MCS
Recombinant DNA infects host cells
resulting in single-stranded
recombinant DNA
Phage particles, containing single-
stranded phage DNA is secreted
from transformed cells and can be
collected from media
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
59. PhagemidsPhagemids
Phagemids are also vectors
◦ Like cosmids have aspects of
both phages and plasmids
◦ Has a MCS inserted into lacZ’
gene to screen blue staining /
white colonies
◦ Has origin of replication of
single-stranded phage f1 to
permit recovery of single-
stranded recombinant DNA
◦ MCS has 2 phage RNA
polymerase promoters, 1 on
each side of MCS
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
60. Bacterial Artificial Chromosomes(BACs) andBacterial Artificial Chromosomes(BACs) and
Yeast Artificial Chromosomes(YACs)Yeast Artificial Chromosomes(YACs)
BACs can hold up to 300 kbs.
The F factor of E.coli is capable of
handling large segments of DNA.
Recombinant BACs are introduced
into E.coli by electroportation ( a brief
high-voltage current). Once in the
cell, the rBAC replicates like an F
factor.
Example: pBAC108L
Has a set of regulatory genes, OriS,
and repE which control F-factor
replication, and parA and parB which
limit the number of copies to one or
two.
A chloramphenicol resistance gene,
and a cloning segment.
YACs can hold up to 500 kbs.
YACs are designed to replicate as
plasmids in bacteria when no foreign
DNA is present. Once a fragment is
inserted, YACs are transferred to cells,
they then replicate as eukaryotic
chromosomes.
YACs contain: a yeast centromere, two
yeast telomeres, a bacterial origin of
replication, and bacterial selectable
markers.
YAC plasmidYeast chromosome
DNA is inserted to a unique restriction
site, and cleaves the plasmid with another
restriction endonuclease that removes a
fragment of DNA and causes the YAC to
become linear. Once in the cell, the rYAC
replicates as a chromosome, also
replicating the foreign DNA.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
61. PROPERTY P1 pBAC pucBAC pCYPAC YAC
Vector Size (kb) 31 6.5 7.2 19.3 11.5
Vector Copy # single single multiple multiple multiple
Insert Size (kb) 75-95 0-300 0-300 0-300 0-2000
Cloning Strategy 2 arms single digest single digest single digest double digest
BamHI/ScaI Bam or Hind Bam or Hind Bam/Sca link Bam/EcoRI
Cloning Method Packaging Electroporate Electroporate Electroporate Spheroplast
Maintenance (copy #) single single single single single
Chimeric Clones (%) 0 2 2 0 (24/24) 20-60
Positive Selection yes no no yes yes
Copy # Induction yes no no yes no
Large Fragment Cloning VectorsLarge Fragment Cloning Vectors
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
62. Selected Protein Expression Systems
Living Hosts:
Escherichia coli
Saccharomyces cerevisiae
Picchia pastoris
Baculovirus
Mammalian tissue culture
Transgenic animals (flies, mice, barnyard animals)
Specialized Systems:
Xenopus oocytes
In vitro transcription-translation
Choosing a System:
-- yield
-- product purification
-- post-translational modifications
-- genetic manipulation of gene/cDNA
-- cost
-- experimental difficulty
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
64. SummarySummary
Two kinds of phage are popular cloning vectors
‑ λ phage
‑ Has nonessential genes removed making room for inserts
- Cosmids accept DNA up to 50 kb
- M13 phage
- Has MCS
- Produces single-stranded recombinant DNA
Plasmids called phagemids also produce single-
stranded DNA in presence of helper phage
Engineered phage can accommodate inserts up to 20
kb, useful for building genomic libraries
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
65. Eukaryotic Vectors and VeryEukaryotic Vectors and Very
High Capacity VectorsHigh Capacity Vectors
There are vectors designed for cloning genes
into eukaryotic cells
Other vectors are based on the Ti plasmid to
carry genes into plant cells
Yeast artificial chromosomes (YAC) and
bacterial artificial chromosomes (BAC) are
used for cloning huge pieces of DNA
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
66. Identifying a Specific Clone WithIdentifying a Specific Clone With
a Specific Probea Specific Probe
Probes are used to identify a desired clone
from among the thousands of irrelevant ones
Two types are widely used
◦ Polynucleotides also called oligonucleotides
◦ Antibodies
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
67. Polynucleotide ProbesPolynucleotide Probes
Looking for a gene you want, might use
homologous gene from another organism
◦ If already cloned
◦ Hope enough sequence similarity to permit
hybridization
◦ Need to lower stringency of hybridization
conditions to tolerate some mismatches
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
68. Control of HybridizationControl of Hybridization
StringencyStringency
Factors that promote separation of two strands in
a DNA double helix:
◦ High temperature
◦ High organic solvent concentration
◦ Low salt concentration
Adjust conditions until only perfectly matched DNA
strands form a duplex = high stringency
Lowering these conditions lowers stringency until
DNA strands with a few mismatches can hybridize
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
69. SummarySummary
Specific clones can be identified using
polynucleotide probes binding to the
gene itself
Knowing the amino acid sequence of the
a gene product permits design of a set of
oligonucleotides that encode part of the
amino acid sequence
Can be a very quick and accurate means
of identifying a particular clone
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
70. cDNA CloningcDNA Cloning
cDNA is the abbreviation for
complementary DNA or copy DNA
A cDNA library is a set of clones
representing as many as possible of the
mRNAs in a given cell type at a given time
◦ Such a library can contain tens of thousands
of different clones
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
71. Making a cDNA LibraryMaking a cDNA Library
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
72. Reverse Transcriptase PrimerReverse Transcriptase Primer
Central to successful cloning is the
synthesis of cDNA from an mRNA
template using reverse transcriptase (RT),
RNA-dependent DNA polymerase
◦ RT cannot initiate DNA synthesis without a
primer
◦ Use the poly(A) tail at 3’ end of most
eukaryotic mRNA so that oligo(dT) may serve
as primer
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
73. Ribonuclease HRibonuclease H
RT with oligo(dT) primer has made a
single-stranded DNA from mRNA
Need to start to remove the mRNA
Partially degrade the mRNA using
ribonuclease H (RNase H)
◦ Enzyme degrades RNA strand of an RNA-DNA
hybrid
◦ Remaining RNA fragments serve as primers
for “second strand” DNA using nick
translation
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
74. Nick TranslationNick Translation
The nick translation process
simultaneously:
◦ Removes DNA ahead of a nick
◦ Synthesizes DNA behind nick
◦ Net result moves or translates the
nick in the 5’ to 3’ direction
Enzyme often used is E. coli
DNA polymerase I
◦ Has a 5’ to 3’ exonuclease activity
◦ Allows enzyme to degrade DNA
ahead of the nick
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
75. Trailing Terminal TransferaseTrailing Terminal Transferase
Don’t have the sticky ends of genomic DNA cleaved
with restriction enzymes
Blunt ends will ligate, but inefficient
Generate sticky ends using terminal
deoxynucleotidyl transferase (TdT), terminal
transferase with one dNTP
◦ If use dCTP with the enzyme
◦ dCMPs are added one at a time to 3’ ends of the cDNA
◦ Same technique adds oligo(dG) ends to vector
◦ Generate ligation product ready for transformation
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
76. Vector ChoiceVector Choice
Choice based on method used to detect
positive clones
Plasmid or phagemid like pUC or pBS will
be used with colony hybridization and a
labeled DNA probe
If λ phage like λgt11, cloned cDNA under
control of lac promoter for transcription
and translation of the cloned gene and
antibody screening
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
77. Rapid Amplification of cDNARapid Amplification of cDNA
EndsEnds
If generated cDNA is not full-length,
missing pieces can be filled in using rapid
amplification of cDNA ends (RACE)
Technique can be used to fill in either the
missing portion at the 5’-end (usual
problem)
Analogous technique can be used to fill in
a missing 3’-end
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
78. 5’-RACE5’-RACE
Use RNA prep containing
mRNA of interest and the
partial cDNA
Anneal mRNA with the
incomplete cDNA
Reverse transcriptase will
copy rest of the mRNA
Tail the completed cDNA
with terminal transferase
using oligo(dC)
Second strand synthesis
primed with oligo(dG)
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
79. SummarySummary
Make cDNA library with synthesis of cDNAs one strand at
a time
◦ Use mRNAs from a cell as templates for 1st
strands, then 1st
strand as template for 2nd
◦ Reverse transcriptase generates 1st
strand
◦ DNA polymerase I generates the second strands
Give cDNAs oligonucleotide tails that base-pair with
complementary tails on a cloning vector
Use these recombinant DNAs to transform bacteria
Detect clones with:
◦ Colony hybridization using labeled probes
◦ Antibodies if gene product translated
Incomplete cDNA can be filled in with 5’- or 3’-RACE
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
80. Methods of Expressing ClonedMethods of Expressing Cloned
GenesGenes
Cloning a gene permits
Production of large quantities of a
particular DNA sequence for detailed
study
Large quantities of the gene’s product
can also be obtained for further use
◦ Study
◦ Commerce
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
81. Expression VectorsExpression Vectors
Vectors discussed so far are used to first put a
foreign DNA into a bacterium to replicate and
screen
Expression vectors are those that can yield
protein products of the cloned genes
◦ For high level expression of a cloned gene best
results often with specialized expression vectors
◦ Bacterial vectors have a strong promoter and a
ribosome binding site near ATG codon
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
82. Fusion ProteinsFusion Proteins
Some cloning vectors, pUC
and pBS, can work as
expression vectors using
lac promoter
If inserted DNA is in the
same reading frame as
interrupted gene, a fusion
protein results
◦ These have a partial β-
galactosidase sequence at
amino end
◦ Inserted cDNA protein
sequence at carboxyl end
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
83. Inducible Expression VectorsInducible Expression Vectors
Main function of expression vector is to yield the
product of a gene – usually more is better
For this reason, expression vectors have very strong
promoters
Prefer keep a cloned gene repressed until time to
express
◦ Large quantities of eukaryotic protein in bacteria are
usually toxic
◦ Can accumulate to levels that interfere with bacterial
growth
◦ Expressed protein may form insoluble aggregates, inclusion
bodies
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
84. Controlling theControlling the laclac PromoterPromoter
lac promoter is somewhat inducible
◦ Stays off until stimulated
◦ Actually repression is incomplete or leaky
◦ Some expression will still occur
To avoid this problem, express using a
plasmid or phagemid carrying its own lacI
repressor gene, such as pBS
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
85. Arabinose PromoterArabinose Promoter
The hybrid trc promoter combines
strength of the trp (tryptophan operon)
promoter with inducibility of lac
promoter
Promoter from ara operon, PBAD, allow fine
control of transcription
◦ Inducible by arabinose, a sugar
◦ Transcription rate varies with arabinose
concentration
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
86. Tightly Controlled PromoterTightly Controlled Promoter
Lambda (λ) phage promoter, PL, is tightly
controlled
Expression vectors with this promoter-
operator system are used in host cells
with temperature-sensitive λ repressor
gene
◦ Repressor functions are low temperatures
◦ Raise temperature to nonpermissive
temperature, the repressor doesn’t function
and cloned gene is expressed
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
88. cI repressor
Bacterial chromosome
Bacterial chromosome
Po
Ptrp λcI repressor
Transcription
No Transcription
Po PL ATG Gene of Interest
Vector
Vector
Tryptophan
trp repressor
No Transcription
λcI repressor
Transcription
ATG Gene of Interest
Po
Po
Ptrp
PL
+ Tryptophan
- Tryptophan
Regulation of gene
expression
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
89. SummarySummary
Expression vectors are designed to yield
the protein product of a cloned gene
When a lac inducer is added, cell begins
to make T7 polymerase which transcribes
the gene of interest
Many molecules of T7 polymerase are
made, so gene is turned on to a very high
level with abundant amount of protein
product made
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
90. Expression Vectors That ProduceExpression Vectors That Produce
Fusion ProteinsFusion Proteins
Most vectors express fusion proteins
◦ The actual natural product of the gene isn’t made
◦ Extra amino acids help in purifying the protein product
Oligohistidine expression vector has a short sequence
just upstream of MCS encoding 6 His
◦ Oligohistidine has a high affinity for divalent metal ions like
Ni2+
◦ Permits purification by nickel affinity chromatography
◦ His tag can be removed using enzyme enterokinase without
damage to the protein product
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
92. Fusion Proteins inFusion Proteins in λλgt11gt11
This phage contains
lac control region and
lacZ gene
Products of gene
correctly inserted will
be fusion proteins
with a β-galactosidase
leader
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
93. Antibody Screening WithAntibody Screening With λλgt11gt11
Lambda phages with cDNA
inserts are plated
Protein released are
blotted onto a support
Probe with antibody to
protein
Antibody bound to protein
from plaque is detected
with labeled protein A
Partial cDNAs can be
completed with RACE
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
94. SummarySummary
Expression vectors frequently produce fusion
proteins
◦ One part of the protein comes from coding
sequences in the vector
◦ Other part from sequences in the cloned gene
Many fusion proteins have advantage of
being simple to isolate by affinity
chromatography
Vector lgt11 produces fusion proteins that
can be detected in plaques with a specific
antiserum
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
95. Bacterial Expression SystemBacterial Expression System
ShortcomingsShortcomings
There are problems with expression of
eukaryotic proteins in a bacterial system
◦ Bacteria may recognize the proteins as foreign and
destroy them
◦ Posttranslational modifications are different in
bacteria
◦ Bacterial environment may not permit correct
protein folding
Very high levels of cloned eukaryotic proteins
can be expressed in useless, insoluble form
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
96. Eukaryotic Expression SystemsEukaryotic Expression Systems
Avoid bacterial expression problems by
expressing the protein in eukaryotic cell
Initial cloning done in E. coli using a shuttle
vector, able to replicate in both bacterial and
eukaryotic cells
Yeast is suited for this purpose
◦ Rapid growth and ease of culture
◦ Still a eukaryote with more appropriate
posttranslational modification
◦ Secretes protein in growth medium so easy
purification
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
97. Use of Baculovirus As ExpressionUse of Baculovirus As Expression
VectorVector
Viruses in this class have a large circular DNA
genome, 130 kb
Major viral structural protein is made in huge
amounts in infected cells
◦ Promoter for this protein, polyhedrin, is very active
◦ These vectors can produce up to 0.5 g of protein
per liter of medium
◦ Nonrecombinant viral DNA entering cells cannot
result in infectious virus as it lacks an essential
gene supplied by the vector
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
98. Animal Cell TransfectionAnimal Cell Transfection
Calcium phosphate
◦ Mix cells with DNA in a phosphate buffer
◦ Then solution of calcium salt added to form a
precipitate
◦ Cells take up the calcium phosphate crystals which
include some DNA
Liposomes
◦ DNA mixed with lipid to form liposomes, small vesicles
with some of the DNA inside
◦ DNA-bearing liposomes fuse with cell membrane
carrying DNA inside the cell
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
99. Choosing a Mammalian Cell Expression System
Advantages:
Cell biology (ie. Localization, cell cycle etc.)
Post-translational modification (mammalian)
Improving expression vectors and transfection methods
Disadvantages:
Low yield of expressed proteins
Relatively expensive (tissue culture costs)
Must use shuttle vectors
Key terms:
Transient transfection: introduction of episomal expression vector into
mammalian tissue culture cells for short term expression experiments.
Stable transfection: introduction and integration of vector into the host cell
chromosome for long-term expression studies.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
103. SummarySummary
Foreign genes can be expressed in eukaryotic
cells
These eukaryotic systems have advantages
over prokaryotic ones
◦ Made in eukaryotic cells tend to fold properly and
are then soluble rather than aggregated into
insoluble inclusion bodies
◦ Posttranslational modifications are made in a
eukaryotic manner
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
104. Using the Ti Plasmid to TransferUsing the Ti Plasmid to Transfer
Genes to PlantsGenes to Plants
Genes can be introduced into plants with
vectors that can replicate in plant cells
Common bacterial vector promoters and
replication origins are not recognized by plant
cells
Plasmids are used containing T-DNA
◦ T-DNA is derived from a plasmid known as tumor-
inducing (Ti)
◦ Ti plasmid comes from bacteria that cause plant
tumors called crown galls
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
105. Ti Plasmid InfectionTi Plasmid Infection
Bacterium infects plant, transfers Ti
plasmid to host cells
T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells
T-DNA genes direct the synthesis of
unusual organic acids, opines which can
serve as an energy source to the infecting
bacteria but are useless to the plant
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
106. Ti Plasmid Transfers Crown GallTi Plasmid Transfers Crown Gall
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)
107. Use of the T-DNA PlasmidUse of the T-DNA Plasmid
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)