This document discusses gene cloning and summarizes key steps in the process: 1. DNA is extracted from a sample and cut into fragments using restriction enzymes. 2. Bacterial plasmids are also cut with the same restriction enzymes. 3. DNA fragments are inserted into the plasmids through recombination, creating recombinant plasmids. 4. The recombinant plasmids are introduced into bacteria through transformation. Transformed bacteria are selected by their ability to grow in antibiotic-containing media, as the plasmids contain antibiotic resistance genes.

2. All genes are made of the same molecule that is chemically quite
homogeneous (DNA – all As,Cs, Gs and Ts)
There is a huge amount of genetic material
E. coli >1000 genes (4,600,000 bp)
Humans – perhaps 100,000 genes
Single genes only make a tiny fraction of the whole genome
In E. coli, each gene is app. 0.05 % of the genome
In humans, each gene is app. .000005% of the genome
Finding a gene is not like like looking for a needle in a haystack, it is more
like looking for one particular piece of hay in a haystack! In this case all
the hay looks the same, and you can only tell the difference by analyzing
the chemistry of each piece!
What’s the trouble with isolating a gene?

3. Restriction enzymes - isolated from bacteria – probably involved in
protecting the cell from viral foreign DNA
Example: EcoRI – from E. coli
Cleaves six base pair site – E. coli DNA is protected by a
corresponding DNA methylase that adds methyl groups to the central
base pairs and prevents cleavage
These enzyme are purified and used as tools in vitro
Will cleave DNA at any GAATTC sequence - every genome has a
specific pattern of GAATTC sequences that occurs at random, but is
always the same
Break the genome into more manageable peices

4. Properties of plasmid
cloning vector
1. Small
2. Stable in the chosen
host – usually E. coli
3. High copy number
4. Easy to purifiy
5. Can accommodate
foriegn DNA
6. Single “cloning” sites
7. Selectable marker –
antibiotic resistance
8. Easily introduced into
host (transformation or
transduction
How do you stably maintain and replicate a foreign
DNA fragment? – by hitching a ride on a stable
replicon

5. α−complementation
LacZ+ - blue colony
LacZ- - while colony
-of you interrupt the lacZ
gene, the colony is white
α−complementation – relies on modular structure of β-
galactosidase
- basic idea is often used with cloning vectors – called insertional inactivation
Brock Biology of
Microorganisms,
vol. 9, Chapter 10

6. How is DNA cloned?
• DNA is extracted-
here from blood
• Restriction enzymes,
e.g. EcoRI, HindIII,
etc., cut the DNA
into small pieces
• Different DNA pieces
cut with the same
enzyme can join, or
recombine.
Blood sample
DNA
Restriction enzymes

7. The action of a restriction enzyme, EcoRI
Note: EcoRI gives a ‘sticky’ end

8. DNA Cloning, II
• Bacterial plasmids (small
circular DNA additional to
a bacteria’s regular DNA)
are cut with the same
restriction enzyme
• A chunk of DNA can thus
be inserted into the
plasmid DNA to form a
“recombinant”

9. DNA cloning, III
• The recombinant
plasmids are then
mixed with bacteria
which have been
treated to make them
“competent”, or
capable of taking in
the plasmids
• This insertion is called
transformation

10. DNA Cloning IV
• The plasmids have
naturally occurring genes
for antibiotic resistance
• Bacteria containing
plasmids with these
genes will grow on a
medium containing the
antibiotic- the others die,
so only transformed
bacteria survive

11. Antibiotic Resistance
• The medium in this petri
dish contains the
antibiotic Kanamycin
• The bacteria on the right
contain Kanr, a plasmid
that is resistant to
Kanamycin, while the one
on the left has no
resistance
• Note the difference in
growth

12. Screening I
Screening can involve:
1. Phenotypic screening-
the protein encoded
by the gene changes
the colour of the
colony
2. Using antibodies that
recognize the protein
produced by a
particular gene

13. Screening II
3. Detecting the DNA sequence of a cloned
gene with a probe (DNA hybridization)

14. Screening III
• Once colonies are
identified, they are
cultured in broth to
increase numbers
and therefore the
amount of DNA
• Samples are also
prepared for storage
at -80 degrees. They
can be kept for many
years this way.

15. 500 bp
1 Kb
1.5 Kb
2 Kb
3 Kb
10 Kb
M
S1
S2
Purification of fragment for
Purification of fragment for
1. Sequencing
1. Sequencing
2. T/A cloning
2. T/A cloning
Visualizing PCR product
Visualizing PCR product

16. NY-lipA 1 MGSSIIIIITAAAWCRGHHMMKGCR-VMFVLLGLWLVFGLSVPGGRAEAATSRANDAPIVLLHGFTGWGREEMFGFKYWG 79
B. stearothermophilus1 1 -------------------MMKGCR-VMVVLLGLCFVFGLSVPGGRTEAASLRANDAPIVLLHGFTGWGREEMFGFKYWG 60
B. stearothermophilus2 1 -------------------MMKGCR-VMVVLLGLWFVFGLSVPGGRTEAASPRANDAPIVLLHGFTGWGREEMLGFKYWG 60
B. thermoleovorans 1 --------------------MKCCR-VMFVLLGLWLVFGLSVSGGRAEAAASRANDAPIVLLHGFTGWGREEMFGFKYWG 59
B. thermocatenulatus 1 -------------------MMKGCR-VMVVLLGLWFVFGLSVPGGRTEAASPRANDAPIVLLHGFTGWGREEMLGFKYWG 60
Bacillus sp. TP10A.1 1 -------------------MMKCCRRVALVLLGLWLVFCLSVPGGRAEAAASRANDAPIVLLHGFTGWGREEMFGFKYWG 61
NY-lipA 80 GVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRIHIIA 159
B. stearothermophilus1 61 GVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRIHIIA 140
B. stearothermophilus2 61 GVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAANDGHARFGRTYPGLLPELKRGGRVHIIA 140
B. thermoleovorans 60 GVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRIHIIA 139
B. thermocatenulatus 61 GVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRVHIIA 140
Bacillus sp. TP10A.1 62 GVRGDIEQLLNAQGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRVHIIA 141
NY-lipA 160 HSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLEAA 239
B. stearothermophilus1 141 HSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLEAA 220
B. stearothermophilus2 141 HSQGGQTARMLVSLLENGSQEEREYAKEHNVSLSPLFEGGHRFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLEAA 220
B. thermoleovorans 140 HSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLEAA 219
B. thermocatenulatus 141 HSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLKAA 220
Bacillus sp. TP10A.1 142 HSQGGQTARMLVSLLENGSKEEREYAKAHNVSLSPLFEGGHNFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLKAA 221
NY-lipA 240 AVASNAPYTSEIYDFKLDQWGLRREPGESFDHYFERLKRSPVWTSTDTARYDLSVPGAETLNRWVKASPNTYYLSFSTER 319
B. stearothermophilus1 221 AVASNVPYTSQVYDFKLDQWGLRRQPGESFDHYFERLKRSPVWTSTDTARYDLSVSGAEKLNQWVQASPNTYYLSFSTER 300
B. stearothermophilus2 221 AVASNAPYTSEIYDFKLDQWGLRREPGESFDHYFERLKRSPVWTSTDTARYDLSVPGAETLNRWVKASPNTYYLSFSTER 300
B. thermoleovorans 220 AVASNVPYTSQVYDFKLDQWGLRRQPGESFDHYFERLKRSPVWTSTDTARYDLSVSGAEKLNQWVQASPNTYYLSFATER 299
B. thermocatenulatus 221 AVASNVPYTSQVYDFKLDQWGLRRQPGESFDHYFERLKRSPVWTSTDTARYDLSIPGAEKLNQWVQASPNTYYLSFSTER 300
Bacillus sp. TP10A.1 222 AVASNVPYTSQVYDFKLDQWGLRRQPGESFDQYFERLKQSPVWTSADTARYDLSVPGAEALNQWVQASPNTYYLSFATER 301
NY-lipA 320 TYRGALTGNYYPELGMNAFSAIVCAPFLGSYRNAALGIDSHWLENDGIVNTISMNGPKRGSSDRIVPYDGALKKGVWNDM 399
B. stearothermophilus1 301 TYRGALTGNHYPELGMNAFSAVVCAPFLGSYRNPTLGIDSHWLENDGIVNTISMNGPKRGSNDRIVPYDGTLKKGVWNDM 380
B. stearothermophilus2 301 TYRGALTGNYYPELGMNAFSAIVCAPFLGSYRNAALGIDSHWLGNDGIVNTISMNGPKRGSNDRIVPYDGTLKKGVWNDM 380
B. thermoleovorans 300 TYRGALTGNYYPELGMNAFSAVVCAPFLGSYRNPTLGIDDRWLENDGIVNTVSMNGPKRGSSDRIVPYDGALKKGVWNDM 379
B. thermocatenulatus 301 THRGALTGNYYPELGMNAFSAVVCAPFLGSYRNEALGIDDRWLENDGIVNTVSMNGPKRGSSDRIVPYDGTLKKGVWNDM 380
Bacillus sp. TP10A.1 302 TYRGALTGNYYPELGMNVFSAAVCAPFLGSYRNAALGIDDRWLENDGIVNTFSMNGPKRGSTDRIVPYDGTLKKGVWNDM 381
NY-lipA 400 GTYNVDHLEIIGVDPNPSFDIRAFYLRLAEQLASFGP 436
B. stearothermophilus1 381 GTYNVDHLEIIGVDPNPSFDIRAFYLRLAEQLASLQP 417
B. stearothermophilus2 381 GTYKVDHLEVIGVDPNPSFNIRAFYLRLAEQLASLRP 417
B. thermoleovorans 380 GTYNVDHLEIIGVDPNPSFDIRAFYLRLAEQLASLRP 416
B. thermocatenulatus 381 GTCNVDHLEVIGVDPNPSFDIRAFYLRLAEQLASLRP 417
Bacillus sp. TP10A.1 382 GTYNVDHLEVIGVDPNPLFDIHAFYLRLAEQLASLRP 418
Alignment of lipase gene with related lipases from other species
Alignment of lipase gene with related lipases from other species

17. Cloning into expression vector
Cloning into expression vector pET
pET 15b
15b
pET 15b cloning vector:
1. High expression rate (T7 promotor region, IPTG induced)
2. His. Tagged (for purification purposes)
Schematic digram of the TALON
Schematic digram of the TALONTM
TM IMAC
IMAC
System.
System.
Part A: TALON metal Affinity Resin, a
Part A: TALON metal Affinity Resin, a
sepharose bead bearing the tetradenate
sepharose bead bearing the tetradenate
chelator of the Co
chelator of the Co2+
2+ metal ion.
metal ion.
Part B: The polyhistidine
Part B: The polyhistidine-
-tagged recombinant
tagged recombinant
protein binds to the resin.
protein binds to the resin.

18. 1.
1. pCR
pCR plasmid isolation from clones showing
plasmid isolation from clones showing lipolytic
lipolytic activity
activity
2. Digestion with
2. Digestion with Nde
Nde I
I and
and BamH
BamH I
I of
of pCR
pCR plasmid and
plasmid and pET
pET 15b
15b
3.
3. Ligation
Ligation of the insert into
of the insert into pET
pET 15b
15b
4. Transformation into expression host (BL21 DE3
4. Transformation into expression host (BL21 DE3 Codon
Codon plus
plus-
-
Protease deficient)
Protease deficient)
Cloning steps:
Cloning steps:

19. Expression of the Lipase Encoded by
Expression of the Lipase Encoded by
ORF1
ORF1
kD M 1 2 3 4 1 2 3 4
In its native form: [using ORF1]
97
66
45
31
22
14
97
66
45
31
22
14
kD M 1 2 3 4 1 2 3 4
F Expression was
observed in all
fractions:
supernatant,
periplasm, soluble
cytoplasm and
insoluble
cytoplasm.

20. Characterization of the Lipase:
Characterization of the Lipase:
Effect of T and pH on the Activity
Effect of T and pH on the Activity
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Temperature °C
Residual
activity
[%]
Optimum
temperature
Thermostability
(A)
0
20
40
60
80
100
120
5 6 7 8 9 10 11
pH
Residual
activity
[%]
(A)
PNP-palmitate, 60°C
PNP-palmitate, T 60°C, pH 7.5
PNP-palmitate, 60°C,pH 7.5
0
50
100
150
0 20 40 60 80 100 120
Time (min)
Relative
activity
[%]
pH 5.0
pH 10.5
(B)
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Time (min)
Residual
activity
[%]
70 °C
(B)
PNP-palmitate, 60°C, pH 9.5
PNP-palmitate, pH 7.5 PNP-palmitate, T 60°C

21. 1.Grow cells in LB medium and compare between induced and
1.Grow cells in LB medium and compare between induced and
non
non-
-induced conditions (induction with IPTG)
induced conditions (induction with IPTG)
2. Measure enzyme activity
2. Measure enzyme activity spectrophotometrically
spectrophotometrically and compare
and compare
with wild type
with wild type
3. Bench
3. Bench-
-top scale fermentation study
top scale fermentation study
Fermentation study
Fermentation study

22. 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0
50
100
150
200
250
300
350
2xYT
SB
LBinduced
LBnon-induced
Lipase
activity
(U/l)
Time (h) after induction
Effect of different media
Effect of different media
2.0 2.5 3.0 3.5 4.0 4.5 5.0
50
100
150
200
250
300
350
400
zero time Ind
Ind.30
Ind.60
Ind. 120
Lipase
activity
(Ul
-1
)
Time (h) after induction
Effect of different induction time
Effect of different induction time