23 Recombinant DNA Technology Clones -> Cells or organisms
with identical DNA
Slide 24
24 Restrictionendonucleases 5-> 3 3
38 Ligation conditions Temperature: 4-10C -> takes long time
16C -> good temperature, but maybe inconvenient RT (room temp)
-> much faster, compromise Concentration of DNA : high ->
intermolecular (between different molecules) ligation fevered low
-> intramolecular (within one molecule) ligation fevered Optimal
vector-insert ratio: from 1:3 to 3:1 (molar ratio -> vector:
insert) depending on size e.g.: vector: 5kb + insert: 500 bp ->
molar ratio of 1:1 -> 500ng vector + 50 ng insert vector: 6kb +
insert: 50kb -> 1:1 -> 500ng vector + 5ug insert -> W V /S
V :W I /S I
Slide 39
39
Slide 40
40 Plasmid Cloning Vectors
Slide 41
41 Plasmid Cloning Vectors
Slide 42
42 Insertional inactivation Gene in cloning site: LacZ ->
pUC18 (lacZ complements the host defect in lacZ) -> pUC18 into
host organism -> active lacZ (-galactosidase) from plasmid->
cleavage of X-gal (blue colonies) -> gene cloned into polylinker
-> lacZ gene disrupted -> no cleavage of X-gal (white
colonies)
Slide 43
43 Insertional inactivation Gene in cloning site: Resistance
marker -> pBR322 (cloning sites within antibiotica resistence
marker) -> plasmid into host -> resistance against 2
antibiotica -> gene cloned within one resistance marker ->
gene for antibiotica resistance marker disrupted -> sensitive
against one antibioticum
Slide 44
44
Slide 45
45 Transformation and Selection
Slide 46
46 Horizontal gene transfer - Transformation -> uptake of
naked DNA (chemical transformation, electroporation) - Conjugation
-> DNA transfer by cell cell contact - Transduction -> DNA
transfer by bacteriopage infection Other methods of Gene transfer
-> used with fungi, animal and plant cells: - Microinjection -
protoplasts
Slide 47
47 Electron micrograph of bacteriophage . Page 107 Electron
micrograph of the filamentous bacteriophage M13.
Bacteriophages
Slide 48
48 Bacteriophage T2 injecting its DNA into an E. coli Page
84
Slide 49
49 Life Cycle of Bacteriophage
Slide 50
50
Slide 51
51
Slide 52
52 A simplified genetic map of lambda: For regulation of the
growth cycle of the phage, the structural genes encoding the head
and tail components of the virus capsid can be ignored. The
pertinent components involved in genetic control are: P L is the
promoter responsible for transcription of the left-hand side of the
lambda genome, including N and cIII. O L is a short non-coding
region of the phage genome (approximately 50bp) which lies between
the cI and N genes next to P L. P R is the promoter responsible for
transcription of the right-hand side of the lambda genome,
including cro, cII and the genes encoding the structural proteins.
O R is a short non-coding region of the phage genome (approximately
50bp) which lies between the cI and cro genes next to P R. cI is
transcribed from its own promoter and encodes a repressor protein
of 236 amino acids which binds to O R, preventing transcription of
cro but allowing transcription of cI, and to O L, preventing
transcription of N and the other genes in the left-hand end of the
genome. cII and cIII encode activator proteins which bind to the
genome, enhancing the transcription of the cI gene. cro encodes a
66 amino acid protein which binds to O R, blocking binding of the
repressor to this site. N encodes an antiterminator protein which
acts as an alternative rho factor for host cell RNA polymerase,
modifying its activity and permitting extensive transcription from
P L and P R. Q is an antiterminator similar to N, but only permits
extended transcription from P R.
Slide 53
53 In a newly infected cell, N and cro are transcribed from P L
and P R respectively:
Slide 54
54 This autoregulation of cI synthesis keeps the cell in a
stable state of lysogeny. If this is the case, how do such cells
ever leave this state and enter a productive, lytic replication
cycle? Physiological stress and particularly ultraviolet
irradiation of cells results in the induction of a host cell
protein, RecA. This protein, whose normal function is to induce the
expression of cellular genes which permit the cell to adapt to and
survive in altered environmental conditions, cleaves the cI
repressor protein. In itself, this would not be sufficient to
prevent the cell re-entering the lysogenic state. However, when
repressor protein is not bound to OR, cro is transcribed from PR.
This protein also binds to OR, but unlike cI, which preferentially
binds to the right-hand end of OR, the cro protein binds
preferentially to the left-hand end of OR, preventing the
transcription of cI and enhancing its own transcription in a
positive feedback loop. Thus, the phage is locked into a lytic
cycle and cannot return to the lysogenic state.
Slide 55
55 Page 107 Replication of bacteriophage upon infection of a
cell
Slide 56
56
Slide 57
57
Slide 58
58
Slide 59
59 Cloning of foreign DNA in phages. Page 110
Slide 60
60 What is a gene library ?
Slide 61
61 Creation of Libraries
Slide 62
62 Creation of Libraries
Slide 63
63 Sizes of Some DNA Molecules. Page 92
Slide 64
64
Slide 65
65 Cosmid = Cos - Plasmid
Slide 66
66
Slide 67
67
Slide 68
68
Slide 69
69 Fragmentation of genomic DNA
Slide 70
70
Slide 71
71 cDNA synthesis
Slide 72
72 DNA Library Clones -> genetically identical
Slide 73
73 Genomic phage library
Slide 74
74 Evaluation of library
Slide 75
75 Evaluation of library
Slide 76
76 Ordered library Microarrays
Slide 77
77 Ordered library Chromosome Walking -> also used in Human
Genome Project