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

1. Success of DNA Ligation
• Generally, ligation reactions are designed to promote the formation of
recombinant DNA but problems can arise due to;
- Vector cyclization
- Vector-vector concatemers and
- Target DNA-target DNA ligation
Vector molecules are often treated to minimize their ability to undergo cyclization
Two common ways of achieving this;
a.Cutting of vectors with two different restriction endonucleases:
Vector cut with two restriction endonucleases which do not produce
complementary overhanging ends (e.g. EcoRI and BamHI).
A small vector fragment between cut ends removed, resulting vector molecule
cannot religate
b. Vector dephosphorylation:
- 5’ phosphate groups at both ends of vector DNA removed by alkaline phosphatase.
- Foreign DNA with intact 5’-PO4
-
can bond with 3’- OH of vector.

2. Some possible ligation reaction products:

3. Getting DNA into Cells:
Transformation
• New genes can be inserted into plant, animal and bacterial cells.
• However, transforming non-bacterial cells is difficult.
• Animal cells have only a cell membrane and can be easily transformed.
• Plants cells have a rigid cell wall barrier are either transformed
- directly with Agrobacterium tumefaciens as a carrier of the new gene, or
- cell wall removed enzymatically to produce protoplast.
Various methods are used for genetic transformation.

4. Host Cell Types

5. Host Cell Types:
Host cells, used for cloning, are specialized cells whose genotype has been
selected to optimize their use in DNA cloning
Major
Group
Prokaryotic/
Eukaryotic
Type Examples
Bacteria Prokaryotic Gram –ive
Gram +ive
Escherichia coli
Bacillus subtilis
Streptomyces spp.
Fungi Eukaryotic Microbial
Filamentous
Saccharomyces cerevisiae
Aspergilius nidulans
Plants Eukaryotic Protoplasts
Intact cells
Whole organism
Various types
Various types
Various types
Animals Eukaryotic Insect cells
Mammalian cells
Oocytes
Whole organism
Drosophila melanogaster
Various types
Various types
Various types
Ref: Table 5.1 An Introduction to genetic Engineering by
Desmond S. T. Nicholl
Table 5.1 Types of host cell types used for genetic engineering

6. Prokaryotic Hosts:
• Bacterial cells are widely used because of their capacity for;
- rapid cell division
 replication of extra-chromosomal vectors is not restricted by its cell
division.
 many vectors go through several cycles of replication during the
cell cycle and can reach high copy numbers.
- there is no post-translational modification of primary transcript.
many eukaryotic gene inserts may;
- be not functional in a prokaryotic host so difficult to isolate or
- may not transcribe into a fully functional protein.

7. Bacterial cells are used for large scale production of recombinant proteins (fusion
proteins or tagged proteins).
Problems with over-expression in bacteria include;
- toxicity of large amounts of the recombinant protein
- lack of posttranslational processing
- inability to synthesize large mammalian proteins, protein folding & solubility.

8. Eukaryotic Hosts:
• They are complex multicellular systems.
• Eukaryotic microbial hosts have many properties of bacteria and so easy to use
rather than complex animal & plant systems.
• Normally cell cultures of higher eukaryotes are used.
• Although some DNA cloning systems involve human and other mammalian cells as
hosts, the great bulk of cell-based DNA cloning has used modified bacterial or
fungal host cells.
Expression in Eukaryotic cells
• Many proteins need specific modifications to work properly… expression in
bacterial cells is not sufficient.
• Plasmid based eukaryotic expression systems which work after transient
transfection into mammalian cell lines have been produced.
• Viral based system are also popular.

9. Transformation

10. Transformation: uptake of naked DNA (generally < 15 kb linear DNA)
Competence = ability to take up DNA
- Chemically induced: i.e. E. coli trated with 10 mM Ca2+
at 4°C
- Electroporation: msec electrical pulse  pores in membrane
- Heat shift:  membrane disruption

11. Chemically Induced Transformation
Introduction of recombinant plasmid into
Host Cell
Calcium chloride Method: Host bacterial
cells are made competent by placing
in an ice-cold calcium chloride
solution which changes cell wall &
allows easy entry of new DNA.

12. Electroporation:
• Electroporation, or electropermeabilization, is a significant increase in
the electrical conductivity and permeability of the cell plasma membrane
caused by an externally applied electrical field.
• The cells are placed in a solution with the insert DNA & subjected to a
high voltage electric shock - usually between 4,000 and 8,000 V/cm - for a
fraction of a second.
• This causes small holes to form in the cell membrane through which the
DNA enters the cells.

13. Microinjection:
• The most commonly used method to transfer DNA directly into animal cells
such as egg cells is to inject the DNA directly into a newly-fertilized egg cell
using a glass capillary tube.
– Uses fine glass needles to inject the foreign DNA directly into the host
cell
– Developed to inject DNA into protoplasts, cultured embryonic cell
suspensions and multicellular structures
– Time consuming

14. Lipofection:
• It is used to transform all cell types.
• DNA to be transferred is placed into liposomes which
are small lipid vesicles.
• The liposome fuse with part of the cell membrane of the
host cells and the contents - the new DNA - enters the
cells.
– Targeted DNA encapsulated in a spherical lipid
bilayer termed a liposome.
– In the presence of PEG, endocytosis occurs.
– After endocytosis, the DNA is free to recombine
and integrate with the host genome.

15. Biolistics:
• It is used widely in the production of genetically
modified corn, and also in the genetic
immunization of animals.
• Tiny tungsten or gold particles, about 0.004 of
a millimeter in diameter, are coated with the
DNA to be transferred.
A blast of high-pressure helium gas or
gunpowder shoots the particles carrying
the DNA into the cells.

16. Transfection
• Introduction of foreign DNA into eukaryotic cells using a virus vector.
• Transfection of animal cells typically involves opening transient pores or
'holes' in cell plasma membrane, to allow uptake of material.
• Transfection can also be carried out by mixing a cationic lipid with the
material to produce liposomes, which fuse with the cell plasma
membrane and deposit their cargo inside.
• Infection by lambda is much more efficient than plasmid transformation –
….. ~ 109
plaques per μg of DNA vs ~ 106
colonies per μg of plasmid DNA

17. Transformation success
Transformation Efficiency:
Quality of a given preparation of competent cells may be measured..
Defined as number of colonies formed (on selective plate) per μg of
input DNA (pure plasmid vector used for cloning)
• T. efficiencies range from 103
per μg (for crude preparation)
• 109
per μg (for carefully prepared competent cells
• 105
per μg is adequate for simple cloning experiment
Frequency of transformation = No. of Transformed cells
Total # of cells in the culture
Transformation efficiency = No. of Transformed cells
Amount of DNA in μg

18. Recombinants Selection &
Screening

19. Positive Selection:
• Used to identify bacteria that contain a plasmid
• Common markers are antibiotic resistance genes carried
Antibiotic resistance genes:
• A host cell strain is chosen that is sensitive to a particular antibiotic, often
ampicillin, tetracycline or chloramphenicol.
• The corresponding vector has been engineered to contain a gene which
confers resistance to the antibiotic.
• After transformation, cells are plated on agar containing the antibiotic to
rescue cells transformed by the vector e.g. Amp and Tet resistance
• Shuttle vectors also carry antibiotic resistance genes which function in
eukaryotic cells (ie neomycin resistance, hygromycin resistance,
methotrexate resistance etc).

20. A vector carries both an ampicllin and a tetracycline resistance gene.
The phenotype of bacteria containing the intact plasmid is Ampr
Tetr
.
• Insertion of foreign DNA into the Pst I site located in the Ampr
gene results in an
Amps
Tetr
phenotype.
• Conversely, insertion of foreign DNA into the EcoRI, Hind III or Sal I sites located in
the Tetr
gene results in an Ampr
Tets
phenotype.
a. Insertional inactivation of antibiotic resistance:
Negative Selection
A second selection system to distinguish between recombinant and normal
plasmids
Site of insertion is chosen such that it disrupts a selectable marker - a
phenomenon called Insertional inactivation.
Two types of selectable markers are used for negative
selection;

21. Replica plating

22. Insertional Inactivation of Enzymatic Activity:
• System is based on beta-galactosidase
gene of the E coli lac operon.
• Plasmid vector contains a second gene for
an enzyme activity.
• Coding sequence of this gene contains
restriction site for DNA insertion.
• Insertion of the foreign DNA at this site
interrupts reading frame of gene resulting
in insertional mutagenesis.
• Media containing XGAL (a synthetic
chromagenic enzyme substrate is used for
recombinant selection.

23. Recombinant Identification
Screening: blue and white selection
• The transformation culture is plated on special media to help identify which cells
have received the recombinant plasmid.
• Two types of media: LB + X-gal, & LB+ X-gal + amp
Selection:
– Cells with the plasmid can grow on ampicillin media.
– Cells without the plasmid cannot grow on ampicillin media.
Screening:
– Cells with a functional LacZ gene can convert X-gal to X + gal
X-gal -------------------------------> X + galactose
Colorless
ß- galactosidase
Blue
• Cells which produce ß- galactosidase form BLUE coloniesBLUE colonies
• Cells able to grow on ampicillin without ß- galactosidase production form
WHITE coloniesWHITE colonies.

24. Screening: blue and white
selection

25. Other systems used for selection: Gene Complementation
One fragment of marker gene is present in host cell and other in
vector
Full gene expression only achieved when host transformed with
vector
β-galactosidase gene complementation:
• Host cell, a mutant, contains a fragment of the β-galactosidase gene
(no full gene) …… cannot make any functional β-galactosidase.
• Vector is engineered to contain a different fragment of the β-
galactosidase gene.
• After transformation by the vector, functional complementation
occurs resulting in active β-galactosidase .
• Can be assayed by blue-white colony selection as previously.

26. Inducible or tissue-specific promoters:
• Present in some vectors, such markers permit controlled expression of introduced
genes in transfected cells or transgenic animals.
• Reporter genes encode an enzyme activity not found in organism being studied.
……. number of genes used, most popular is the E. coli ß glucuronidase.
Gus Gene Assay in Transformed Tissues:
• GUS gene (encoding β-glucuronidase enzyme), initially used as a gene fusion
marker in E. coli and nematode C. elegans, but more recently in plants.
• Substrate used for histochemical localization of β-glucuronidase activity in tissues
and cells is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)
• Selection is based on blue precipitate at the site of enzyme activity.
Amino-acid or Nitrogenous-base biosynthesis enzymes:
They are used for positive selection in yeast (HIS, LEU and ADE genes).

27. Plaque Formation:
• E. coli containing a lambda provirus (a lambda lysogen) are immune to
subsequent phage infection and so can grow in the presence of virus.
• This results in 'cloudy plaque' morphology (cloudy appearance is due to
the presence of lysogenic bacteria that continue to grow within the plaque).
• Recombinant phages are unable to lysogenize and have a 'clear plaque'
morphology (no lysogenic hosts growing within the plaque).