1. Cloning Overview of cloning Plasmid-based vectors
Intro to phage lambda
Genomic and cDNA libraries

2. Prior knowledge!
Restriction enzymes: cut DNA at specific sequences often leave 5’ or 3’ overhangs (“sticky ends”)
DNA ligase: joins DNA strands via 5’ phosphate and 3’ OH
Alkaline phosphatase: removes 5’ phosphate from DNA preventing ligation of that strand
DNA polymerases: extend a growing DNA strand from the 3’ OH, using a template strand to determine the order of base addition
G’GATCC
CCTAG.G G
CCTAGp
pGATCC G
pGATCC G G
CCTAGp
HOGATCC G G
CCTAGOH
..GTAGCA
..CATCGTACTAGCTAG..

3. Overview of cloning strategy
plasmid
vector
Cloning: joining a given piece of DNA to functions that will enable replication of that piece of DNA = replication origin
Choice of vector: depends on the DNA to be cloned (size, availability, screening...) +
insert
bacterial cell
host
chromosomal DNA
clone

4. Choice of vector depends on purpose of cloning and size of insert
Small fragments: 0.1 – 1 kb (1 kb = 1000 bp) small genes for production of protein in E. coli promoters or control elements to study regulation
Intermediate fragments: 1 kb – 10 kb larger genes for expression cDNAs (cDNA library?) small operons
Larger fragments: 10 – 100 kb genomic fragments for genomic library** genome (organism) reconstruction experiments
As well as the size of insert fragment, we must also consider the functions of the vector DNA

5. For gene expression (and promoter-probing) tend to use plasmid-based vectors
Expression of genes: identification of promoter regions; expression of cloned DNA sequences as proteins; gene therapy
promoter
“reporter” gene
$$$
bacterial promoter
“useful” gene
Which cloning vector do we need?

6. Protein production

7. Promoter probing
The expression of individual genes is being studied using reporter fusions. When (or where) a gene is expressed is largely determined at the level of its transcription initiation

8. Promoter probing Spore-specific expression
Mycelium-specific expression

9. Plasmids not vector of choice for cloning gene clusters or making genomic libraries
Characterisation of the gene/gene cluster: coding sequences; non-coding/regulatory sequences e Gg Ag d b
50 kb
exon 1
exon 2
exon 3
intron 1
intron 2
1.6 kb
ORGANISATION OF HUMAN b-LIKE GLOBIN GENE CLUSTER
Which cloning vector do we need?

10. Types of vectors
VECTOR - name given to the DNA molecule into which foreign DNA is inserted for subsequent propagation in host cell
plasmid
bacteriophage lambda
bacteriophage M13
cosmid
phagemid
ESSENTIAL FEATURES OF ALL VECTORS
ability to replicate in host cell
ability to be readily introduced into host cell
ability to readily insert foreign DNA into vector
You will be expected to know the properties of all of these vectors

11. What about the bacterial host?
1. We need efficient transformation by plasmid DNA
Use a host deficient in natural restriction modification systems
Example: E. coli strain K contains the K restriction-modification system encoded by hsdRMS, so delete the hsdR gene
2. We need stable maintenance of the transformed DNA
Avoid rearrangements by using mutants in recombination genes such as recA, recF
3. Disablement of the host for safety reasons
Use an auxotroph: host cannot synthesise a metabolite/amino acid;
Host can only grow on growth medium supplied in the laboratory
Strains carrying recombinant plasmids therefore unlikely to escape and propagate outside laboratory

12. Plasmid-based vectors
Natural plasmids: <1 kb to >100 kb in size From 1 to 1000 copies per cell Replicate independently of chromosome Carry e.g. antibiotic resistance genes
Use in cloning: Synthetic plasmids constructed from: - replication origin - selectable marker - cloning site(s)

13. Engineered plasmids used as cloning vectors
ESSENTIAL FEATURES FOR USE AS VECTOR
ori (replication) copies per cell
selectable marker (drug resistance gene)
suitable region for insertion of foreign DNA (restriction enzyme sites)
suitable size
pBR322
4361 bp
BamHI
PstI
EcoRI
tet
ori
amp

14. pUC19 – a typical plasmid cloning vector
2686 bp
amp
lacZa
mcs
ori
2686 bp
>500 copies per bacterial cell
POLYLINKER
(multiple restriction enzyme sites)
EcoRI
SmaI
XbaI
PstI
HindIII
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTT
KpnI
BamHI
SalI

15. Cloning DNA with plasmid vectors
1. GENERATION OF RECOMBINANT MOLECULE
ampr 5’ P P
linearise with
restriction enzyme
(e.g. at site within polylinker)
vector DNA 5’
dephosphorylate with calf intestinal phosphatase (prevents vector religation) +
T4 DNA ligase P
DNA fragment P
ligate vector with fragment to be cloned (cDNA, genomic DNA etc; fragment has ends that are “compatible” with ends of cut vector, and is not de-phosphorylated)

16. + 2. INTRODUCTION OF RECOMBINANT PLASMID INTO HOST BACTERIA
ampr
recombinant DNA molecule
introduce DNA into host bacteria
chemical transformation (treat with CaCl2 to
make competent + heat shock)
electroporation
size limitation for DNA uptake
inefficient process
bacterial chromosome
plasmid +
most bacteria do NOT take up plasmid - need selection

17. 3. SELECTION OF BACTERIA CONTAINING PLASMID
plasmid replication + bacterial cell division
spread on ampicillin plates
cells that have not taken up plasmid cannot grow
cells that have taken up plasmid (ampr) form colonies

18. Plasmids – uses and limitations
Isolation of DNA Centrifuge liquid culture to collect cell pellet Break open cells Remove protein, degrade RNA Precipitate plasmid DNA (see previous lecture)
Yields improved with high copy number plasmids
Limitation on transforming host bacteria Chemical (CaCl2) Electroporation (2 – 3 kV)
Insert size range 0 – 10 kbp
Not really good enough for making a genomic library

19. Bacteriophage lambda (λ)
genomic
DNA
head
tail
Efficient injection of λ DNA into the host
Can we use this property for a cloning experiment?

20. Genomic organisation of lambda
genome = 49 kb linear double-stranded DNA
DNA present in phage particles as linear double-stranded molecule with single-stranded complementary termini of 12 nt (“cohesive ends”)
right cohesive end
cccgccgctgga 5′ 3′ 3′ 5′
gggcggcgacct
left cohesive end
(not to scale!)

21. Properties of the lambda cos site
right cohesive end
cccgccgctgga
gggcggcgacct
left cohesive end
CIRCULARISATION
cos
nick

22. Lambda life cycles LYSIS or LYSOGENY
phage particle attaches to E.coli cell and injects its DNA
Efficient!
lambda DNA cirularises (via cohesive ends)
bacterial chromosome
LYSIS
DNA replication, synthesis of phage products (capsid proteins), particle assembly, cell lysis and phage release or
LYSOGENY
(integration of  into host genome)
 = prophage

23. What does growth of lambda look like?
LIQUID CULTURE ASSAY
lysogenisation
(sometimes)
phage stock
bacterial culture
bacterial lysis
PLAQUE ASSAY
low m.o.i.
phage stock
bacterial culture
add aliquot to “top agar”
+ pour onto agar plate
lysis + lysogeny
(turbid plaques)

24. Lambda replication – the lytic pathway
cos site
circularisation
replication via “rolling circle” mechanism
catenane “rolled off” the  DNA molecule
cos sites
ori
lambda head and tail proteins synthesised
- heads and tails assembled
separately

25. Packaging of lambda in vivo
1. Packaging of concatenate into prehead
concatenate is cleaved at cos site during packaging into prehead (phage encoded endonuclease)
fixed length is always packaged (packaging constraint)
2. Filled heads associate with preformed tails +
mature lambda particle

26. Supplement – Methods of Selection
How to distinguish an “empty” vector (i.e. no insert present) from a recombinant molecule (containing a cloned insert)?
Differential antibiotic resistance Possible with plasmids such as pBR322
LacZ complementation (“blue-white selection”) will work with plasmids (pUC19 is a good example) and also phage (typically M13; also used in variants of lambda)
Spi selection Specific for lambda replacement vectors
cI selection Can be adapted for both insertion and replacement vectors

27. Useful features – lacZ complementation (1)
Visual identification of recombinant clones using lacZ
lacZ gene encodes -galactosidase
portion of the lacZ gene (LacZ’, N terminal 146 aa) is incorporated into cloning vector (encodes promoter and -peptide) - this region of the protein is by itself inactive
vector also contains regulatory sequences that bind Lac repressor (which keeps the lacZ gene repressed except in presence of inducer IPTG)
lacz promoter region (also binds the LacI repressor)
LacZ’ (-peptide)
pUC19
2686 bp
amp
lacZa
mcs
ori

28. Useful features – lacZ complementation (2)
E. coli host
plasmid
lacZa
mcs
-gal N terminus
(peptide):
engineered to express only the -gal C terminus:
transform E.coli with plasmid, add IPTG (inducer of LacZ’) and X-gal (chromogenic substrate)
active -gal (complementation) turns substrate BLUE - observe blue colonies (or plaques)
lacZa

29. Useful features – lacZ complementation (3)
polylinker cloning site is positioned within the LacZ’ region of plasmid or phage (does NOT itself interupt reading frame or synthesis of LacZ -peptide)
insertion of foreign DNA within polylinker usually disrupts LacZ’ reading frame - -peptide not made and hence no -gal formed on introduction into host
foreign DNA
peptide reading frame disrupted, no gal formed; WHITE colonies or plaques
recombinant
parent vector
polylinker
lacZa (LacZ’)
-peptide; -gal formed
blue colonies or plaques
in appropriate host E. coli

30. Genomic clones and libraries+
vector DNA
(e.g. cosmid)
genomic DNA fragments
genomic DNA
ligate and introduce into host bacteria
- each colony contains a recombinant that contains a different genomic DNA fragment
GENOMIC CLONE: a recombinant DNA containing a DNA fragment derived from the nuclear DNA (genome) of the organism
GENOMIC LIBRARY: collection of clones which together contains copies of all the nuclear DNA (genome) of the organism

31. Note differences between cDNA and genomic libraries
1. Genomic libraries represent all sequences present in the nuclear genome
nearly all genes are cloned with equal probability (genome contains equal number of copies of most genes, therefore all are equally represented in starting genomic DNA preparation)
clones from eukaryotes may contain introns and flanking regions of genes
many clones will contain sequences that are not expressed as mRNA
may be specifically used for cloning sequences from nuclear DNA other than those coding for mRNA (i.e. other than genes ) e.g. cloning of telomeres (ends of chromosomes) or repetitive DNA.

32. Genomic libraries – ‘Size is important’
1. Preparation of insert genomic DNA
important to have pure, high MW DNA, free of nicks
2. Fragment genomic DNA to suitable size for ligation into vector
want kb for replacement vector; 40 kb for cosmid vector
complete digestion with restriction enzyme would generate many fragments that are too small to clone in or cosmid vectors and would not therefore be represented in library
e.g. EcoRI sites: E
4 kb
1 kb
20 kb
2 kb
- only the 20 kb clone would be represented in the library
(site size and frequency)

33. Genomic libraries – Partial digests
partially digest genomic DNA with frequently cutting restriction enzyme e.g. Sau3A - get cleavage of random subset of sites and generates random collection of fragments
purify fragments within desired size range (e.g kb) gel electrophoresis or sucrose gradient centrifugation
EcoRI: G`AATTC On average: once per 4096 bp
Sau3A: `GATC
On average:
once per 256 bp
BamHI: G`GATCC On average: once per 4096 bp

34. Genomic libraries – cosmid vector as example
ligate purified insert DNA into lambda arms or cosmid vector
random genomic
fragments
Sau3A +
cos
left arm
right arm
lambda replacement vector arms purified away from stuffer
BamHI
Follow it through (you should be familiar with the approach by now).
recombinant concatemer
B/S
S/B

35. Genomic libraries – Plaques are clonal!
package recombinant concatemer into phage particles in vitro
each phage contains
a distinct piece
of genomic DNA
infect E. coli
each plaque contains many identical phage
different plaques contain different phage, each carrying a
distinct fragment of genomic DNA

36. How many clones do I need for a genomic library?
want ALL genomic sequences to be represented
mammalian genome = 3 x 109 bp DNA
if average insert size is 15 kb, approx 200,000 phage carry one genome’s worth of DNA
But it’s very unlikely we’ll get just one clone for each piece of DNA. We’ll need much more, perhaps to 2 x 106 phage to ensure all sequences are represented

37. Clones required for libraries of different organisms
no. clones required
library insert size:
species genome size 17 kb 35 kb
E. coli 4.8x
S. cerevisiae 1.4x107 2,500 1,200
D. melanogaster 1.7x108 30,000 14,500
Tomato 7x ,500 59,000
Human 3x , ,000
Frog 2.3x1010 4,053,000 1,969,000
(p = 95%)

38. Ordered, overlapping libraries used to be required for genome sequencing

39. Ordered, overlapping libraries used to be required for genome sequencing
From ordered library
“Shotgun” approach

40. Building up whole genome sequences
>File 1023
GGATCCGGCCACCACGACGGTGCACGACGTACTGCAACGGCTCAACCACGGCCTCATCCGCGGCGACAGCTCGTCCAGCAGCGTCGAGGAGTACCGCGCGGAGATCGAGGAGGCCGGCTTCCGGGTGCGGCAGGCGGAGCGCATCGACAACCTCCTCGGTGACTGGCTGATAGTCGCCGTCAAGCCCTGACCCTGCGTGGCCGCCCTTGCTACCGTGATCATGCCCGCGGGTGTGTTCCCGCCGGGCGGATCACAGCGACGTCAGAGAGGGCGGAACGATGCGGCAGGCGACCACGAATCCGGCAGGAAAGGTCCGCATCACGACGCTAAGGAATCCGAACCCCCTCCATGAACAAGCTGAATCTGGGCATCCTGGCCCACGTTGACGCCGGCAAGACCAGCCTCACCGAGCGCCTGCTGCACCGCACCGGTGTGATCGACGAGGTCGGCAGCGTGGACGCCGGCACCACGACGACCGACTCGATGGAGCTGGAGCGGCAGCGCGGCATCACCATCCGGTCCGCCGTGGCCACGTTCGTCCTGGACGATCTCAAGGTCAACCTCATCGACACCCCGGGCCACTCCGACTTCATCTCCGAGGTCGAGCGGGCGCTCGGGGTGCTCGACGGCGCGGTCCTGGTGGTCTCGGCCGTCGAGGGCGTCCAGCCGCAGACCCGCATCCTGATGCGGACCCTGCGCAGGCTGGGCATTCCCACGCTGGTCTTCGTCAACAAGATCGACCGGGGCGGCGCGCGTCCCGACGGTGTGCTGCGGGAGATCCGCGACCGGCTCACCCCCGCCGCGGTGGCACTGTC
>file 6492
GAGATCCGCGACCGGCTCACCCCCGCCGCGGTGGCACTGTCCGCCGTGGCGGACGCCGGCACGCCGCGGGCCCGCGCGATCGCGCTCGGCCCGGACACCGACCCGGACTTCGCCGTCCGGGTCGGTGAGCTGCTGGCCGACCACGACGACGCGTTCCTCACCGCCTACCTGGACGAGGAACACGTACTGACCGAGAAGGAGTACGCGGAGGAACTGGCCGCGCAGACCGCGCGCGGTCTGGTGCACCCGGTGTACTTCGGGTCCGCGCTGACCGGCGAGGGCCTGGACCATCTGGTGCACGGCATCCGGGAGTTGCTGCCGTCCGTGCACGCGTCGCAGGACGCGCCGCTGCGGGCCACCGTGTTCAAGGTGGACCGTGGCGCGCGCGGCGAGGCCGTCGCGTACCTGCGGCTGGTCTCCGGCACGCTGGGCACCCGCGATTCGGTGACGCTGCACCGCGTCGACCACACCGGCCGGGTCACCGAGCACGCCGGACGCATCACCGCGCTGCGGGTCTTCGAGCACGGGTCGGCCACCAGCGAGACCCGGGCGACCGCCGGGGACATCGCGCAGGCGTGGGGCCTGAAGGACGTACGGGTCGGTGACCGGGCCGGGCACCTCGACGGTCCCCCGCCGCGCAACTTCTTCGCGCCGCCCAGCCTGGAGACCGTGATCAGGCCGGAGCGCCCGGAGGAAGCGGGACGGCTGCACGCCGCGCTGCGCATGCTGGACGAGCAGGACCCCTCGATCGACCTGCGGCAGGACGAGGAGAACGCGGCCGGCGCGGTGGTCCGCCTCTACGGGGAGGTGCAGAAGGAGATCCTCGGCAGCACGCTCGCGGAGTCCTTCGGCGTACGGGT

41. Building up whole genome sequences
>Contig c34
GGATCCGGCCACCACGACGGTGCACGACGTACTGCAACGGCTCAACCACGGCCTCATCCGCGGCGACAGCTCGTCCAGCAGCGTCGAGGAGTACCGCGCGGAGATCGAGGAGGCCGGCTTCCGGGTGCGGCAGGCGGAGCGCATCGACAACCTCCTCGGTGACTGGCTGATAGTCGCCGTCAAGCCCTGACCCTGCGTGGCCGCCCTTGCTACCGTGATCATGCCCGCGGGTGTGTTCCCGCCGGGCGGATCACAGCGACGTCAGAGAGGGCGGAACGATGCGGCAGGCGACCACGAATCCGGCAGGAAAGGTCCGCATCACGACGCTAAGGAATCCGAACCCCCTCCATGAACAAGCTGAATCTGGGCATCCTGGCCCACGTTGACGCCGGCAAGACCAGCCTCACCGAGCGCCTGCTGCACCGCACCGGTGTGATCGACGAGGTCGGCAGCGTGGACGCCGGCACCACGACGACCGACTCGATGGAGCTGGAGCGGCAGCGCGGCATCACCATCCGGTCCGCCGTGGCCACGTTCGTCCTGGACGATCTCAAGGTCAACCTCATCGACACCCCGGGCCACTCCGACTTCATCTCCGAGGTCGAGCGGGCGCTCGGGGTGCTCGACGGCGCGGTCCTGGTGGTCTCGGCCGTCGAGGGCGTCCAGCCGCAGACCCGCATCCTGATGCGGACCCTGCGCAGGCTGGGCATTCCCACGCTGGTCTTCGTCAACAAGATCGACCGGGGCGGCGCGCGTCCCGACGGTGTGCTGCGG
GAGATCCGCGACCGGCTCACCCCCGCCGCGGTGGCACTGTCCGCCGTGGCGGACGCCGGCACGCCGCGGGCCCGCGCGATCGCGCTCGGCCCGGACACCGACCCGGACTTCGCCGTCCGGGTCGGTGAGCTGCTGGCCGACCACGACGACGCGTTCCTCACCGCCTACCTGGACGAGGAACACGTACTGACCGAGAAGGAGTACGCGGAGGAACTGGCCGCGCAGACCGCGCGCGGTCTGGTGCACCCGGTGTACTTCGGGTCCGCGCTGACCGGCGAGGGCCTGGACCATCTGGTGCACGGCATCCGGGAGTTGCTGCCGTCCGTGCACGCGTCGCAGGACGCGCCGCTGCGGGCCACCGTGTTCAAGGTGGACCGTGGCGCGCGCGGCGAGGCCGTCGCGTACCTGCGGCTGGTCTCCGGCACGCTGGGCACCCGCGATTCGGTGACGCTGCACCGCGTCGACCACACCGGCCGGGTCACCGAGCACGCCGGACGCATCACCGCGCTGCGGGTCTTCGAGCACGGGTCGGCCACCAGCGAGACCCGGGCGACCGCCGGGGACATCGCGCAGGCGTGGGGCCTGAAGGACGTACGGGTCGGTGACCGGGCCGGGCACCTCGACGGTCCCCCGCCGCGCAACTTCTTCGCGCCGCCCAGCCTGGAGACCGTGATCAGGCCGGAGCGCCCGGAGGAAGCGGGACGGCTGCACGCCGCGCTGCGCATGCTGGACGAGCAGGACCCCTCGATCGACCTGCGGCAGGACGAGGAGAACGCGGCCGGCGCGGTGGTCCGCCTCTACGGGGAGGTGCAGAAGGAGATCCTCGGCAGCACGCTCGCGGAGTCCTTCGGCGTACGGGT

42. Current uses of gene libraries
Many cosmid libraries were the substrates for genome sequencing projects, so now obtain desired clone by analysing the sequence data. More often than not clones can be ordered by ;
While genomic clones are good substrates for PCR, genomic DNA can also be used for most purposes. Direct PCR amplification becomes less reliable the bigger the genome - use libraries when you can!
Genetic tests, e.g. functional complementation;
Although construction of genomic libraries no longer critical for genome sequencing project, still valuable for functional genomics. The ‘foreign’ DNA can be manipulated in E. coli (i.e. creation of gene modifications/disruption) and then transferred into original organism – functional genomics.