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Restriction enzymes
"When I come to the laboratory of my father, I usually see some plates lying on the tables. These plates contain colonies
of bacteria. These colonies remind me of a city with many inhabitants. In each bacterium there is a king. He is very long,
but skinny. The king has many servants. These are thick and short, almost like balls. My father calls the king DNA, and
the servants-enzymes... My father has discovered a servant who serves as a pair of scissors. If a foreign king invades a
bacterium, this servant can cut him in small fragments, but he does not do any harm to his own king."
-Sylvia (10 years old), daughter of Werner Arber
Prior to Arber's work, researchers Salvador Luria and Mary Human had shown that various phages were host specific,
with each phage surviving and flourishing only in one host bacterial strain and growing poorly in others
Those phages that grew poorly were said to be "restricted" by their host. Arber wanted to know why.
Restriction digest Electrophoresis
Types of cuts
5’ overhang
3’ overhang
blunt
Restriction enzymes
• Scientists have isolated more than 800 different restriction enzymes from bacteria,
which altogether recognize and cut more than 100 different restriction sites.
• Most restriction sites are 4 to 6 bases long, and most are palindromic
• HindIII, for example, is an H. influenzae restriction enzyme (The first three letters of
a restriction enzyme's name are abbreviations of the bacterial species from which the
enzyme has been isolated (e.g., Eco- for E. coli and Hin- for H. influenzae), and the
fourth letter represents the particular bacterial strain.)
20 bp
18 bp
Cut and paste exercise
Klenow: large protein fragment produced when DNA
polymerase I from E. coli is enzymatically cleaved by
the protease subtilisin.
retains the 5’ → 3’
polymerase activity and
the 3’ → 5’ exonuclease
activity for removal of
precoding nucleotides
and proofreading, but
loses its 5' → 3'
exonuclease activity.
E. coli DNA
M-MuLV
E. coli DNA polymerase I T4 DNA T7 DNA Taq DNA
Reverse
polymerase I - Klenow polymerase polymerase polymerase
Transcriptase
Fragment
5'3'
exonuclease + - - - +
activity
3'5'
exonuclease + + + + -
activity
Error Rate (x10-
6) 9 40 <1 15 285
Strand
- + - - -
Displacement
Heat
+ + + + -
Inactivation
Site-specific mutagenesis
Bacterial transformation
Regulatory elements
Proximal Promoter
1 c2 3
Average
activity of 45
promoters
across 7 cell
lines
Promoter bashing
Firefly gene
ecoding luciferase
lacZ gene
GFP (green
fluorescent
protein from
jelly fish
Firefly gene ecoding luciferase
Frequently used reporter genes
Use of auxotrophic
markers instead of
antibiotics
Synthetic Biology
&
Biobricks
Drew Endy:
Synthetic biology is an approach to engineering biology
http://parts.mit.edu/igem07/index.php/Podcasts
What’s a part?
Level in an abstraction hierarchy:
Basic biological function that can be encoded as genetic material,
e.g. ribosome binding site
Drew
Endy
Screenability model
Layer name Definition Example
a finite sequence of
RBS, CDS, promoter,
Part nucleotides with a specific
terminator
function
Composition model
Drew
Endy
(1) Permit EcoRI cutting by providing extra “spacer” bases + addition of A base on the
opposite strand by Taq pol for high efficiency TA cloning if desired
(2) Random extra spacer base
(3) EcoRI site
(4) NotI site
(5) Extra base to prevent creation of EcoBI or EcoKI methylation sites (could inhibit
restriction enyzme digest)
(6) XbaI site
(7) Extra G to prevent creation of a) GATC site (methylation), b) ATG start codon
(8) 20 bp of sequence matching 5’ end of part sequence
How to construct a standard biobrick biological part
1) PCR and standard part
(promoters, RBS’s, terminators and others)
PCR if template DNA exists and part is short enough for creation via
primer annealing and extension
Specific sequences corresponding to the BioBrick ends must be included on the
end of each of the two PCR primers:
5' [part] T ACTAGT A GCGGCCG CTGCAG G AAGAAAC 3'
SUFFIX: 3' [part] A TGATCA T CGCCGGC GACGTC C TTCTTTG 5'
(1) (2) (3) (4) (5) (6) (7)
(1) Permit EcoRI cutting by providing extra “spacer” bases + addition of A base on the
opposite strand by Taq pol for high efficiency TA cloning if desired
(2) Random extra spacer base
(3) EcoRI site
(4) NotI site
(5) Extra base to prevent creation of EcoBI or EcoKI methylation sites (could inhibit
restriction enyzme digest)
(6) XbaI site
(7) ATG start codon
(8) 20 bp of sequence matching 5’ end of part sequence
How to construct a standard biobrick biological part
2) PCR and protein coding sequence
Sites to include:
GATC (plasmid purified DNA can be cut (via DpnI)
whereas PCR product cannot
How is all this information useful for our project?
Goal:
P S
+
P S
P S
P S
Prefix:
GAATTC GCGGCCGCT TCTAGA G3’
S
5’
3’CTTAAG CGCCGGCGA AGATCT C5’
EcoRI XbaI
Suffix
SpeI PstI
E X S P
Prefix:
5’GAATTC GCGGCCGCT TCTAGA G3’ S P
3’CTTAAG CGCCGGCGA AGATCT C5’
XbaI
Suffix
SpeI
Biobrick puzzles: 2 pieces
P S
+
P S
P S
http://parts.mit.edu/registry/index.php/Assembly:Standard_assembly
Biobrick puzzles: 3 pieces
http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly
Summary: example
Glasgow project last year http://parts.mit.edu/igem07/index.php/Presentations
Summary: example (Glasgow)
Summary: example (Glasgow)
Summary: example (Glasgow)
http://parts.mit.edu/igem07/index.php/Podcasts
BrickiT!
http://brickit.wiki.sourceforge.net/
• Open-source project
E X S P
3
5’T A
E 5’GAATTC
X GCGGCCGCT TCTAGA G3’ S P
3’CTTAAG CGCCGGCGA AGATCT C5’
3’A TGATC
Thr Arg
http://openwetware.org/wiki/Silver:_BB_Strategy
Biobrick 3.0 (Mueller & iGem Freiberg)
P S
3
Prefix:
GAATTC GCGGCCGCT TCTAGA G3’
S
5’
3’CTTAAG CGCCGGCGA AGATCT C5’
EcoRI XbaI
Suffix
SpeI PstI
Biobrick 3.0 (Mueller & iGem Freiberg)
P S
3
Prefix:
GAATTC GCGGCCGCT TCTAGATG GCCGGC3’
S
5’
3’CTTAAG CGCCGGCGA AGATCTAC CGGCCG5’
E X N A S P
3
E X N ACCGGC A S P
TGGCCG
MAG Thr Gly TG*Y**