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Molecular tool kit

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.)

• 3 categories of restriction enzymes:


• type I, which recognize specific DNA sequences but make their cut at
seemingly random sites that can be as far as 1,000 base pairs away from the
recognition site (EcoB and EcoK)
• type II, which recognize and cut directly within the recognition site (HindII and
HindIII)
• type III, which recognize specific sequences but make their cut at a different
specific location that is usually within about 25 base pairs of the recognition site
Finding the transcription start site by CAGE
Full-length cDNA

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.

• Synthesis of double-stranded DNA from single-stranded


templates
• Filling in recessed 3' ends of DNA fragments
• Digesting away protruding 3' overhangs
• Preparation of radioactive DNA probes
Klenow: large protein fragment produced when DNA
polymerase I from E. coli is enzymatically cleaved by
the protease subtilisin.
Klenow: large protein fragment produced when DNA
polymerase I from E. coli is enzymatically cleaved by
the protease subtilisin.

exo- Klenow fragment


The 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for certain
applications  mutations in the gene that encodes Klenow  enzyme being expressed
that retain 5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' →
5').
T4 DNA Polymerase

T4 is a bacteriophage of E. coli: activities of T4 DNA


polymerase = Klenow fragment of DNA polymerase I - it
functions as a 5' -> 3' DNA polymerase and a 3' -> 5'
exonuclease, but does not have 5' -> 3' exonuclease activity.

Difference with Klenow DNA polymerase I


• The 3' -> 5' exonuclease activity of T4 DNA polymerase is
roughly 200 times that of Klenow fragment, making it preferred
by many investigators for blunting DNAs with 3' overhangs.

• While Klenow fragment will displace downstream


oligonucleotides as it polymerizes, T4 DNA polymerase will not.
This attribute makes T4 DNA polymerase the more efficient
choice for certain types of oligonucleotide mutagenesis reactions.
Reverse transcriptase
PCR
Overview

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

proximal promoter core promoter


Upstream sequences (~500 bp – 1 kb)
• Augment the level of expression or choice of tissue specificity
• Separation from enhancers?
(negative elements, e.g. Cooper et al., Genome Res., 2006: 16, 1)

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

GFP (green fluorescent protein


from) jelly fish Aequorea victoria

lacZ gene of bacteria


•Puromycin resistant gene (puro): Puromycin is an aminonucleoside antibiotic
produced by Streptomyces alboniger. It specifically inhibits peptidyl transfer on
ribosomes, therefore it inhibits the growth of various insect and animal cells. The
expression of the pac gene (puromycin N-acetyltransferase from S. alboninger)
confers puromycin resistance to transfected cells.

•The hygromycin resistance gene (hygro): Hygromycin B is an aminoglycosidic


antibiotic produced by Stretomyces hygroscopicus. It is used for the selection and
maintenance of prokaryotic and eukaryotic cells transfected with the hygromycin
resistance gene of E. coli origin. Hygormycin B kills cells by inhibiting protein
synthesis. The resistance gene encodes for a kinase that inactivates hygromycin B
through phosphorylation.
Yeast transformation

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

• not metabolic engineering, not cancer cell engineering, not


bioprocess engineering
• not a particular application, but it is the method one uses to realize
a particular application (means to an end)
• Builds on genetic engineering:
• Writing DNA
• Recombinant DNA (restriction enzymes,…)
• PCR
• Reading it out: sequencer
• 3 more layers are added:
• Automated construction of DNA (DNA synthesis)
• Standards (defining how we put things together): “standard
biobrick biological part”
Drew Endy:
Standard biobrick biological part

What’s a part?
Level in an abstraction hierarchy:
Basic biological function that can be encoded as genetic material,
e.g. ribosome binding site

What’s a standard part?


A part whose properties have been limited in such a way that the
standard may support something
e.g. reliable physical and functional composition
(not the latest bit of sequence information from a new organism,
rather a biobrick is a part that is being refined and reengineered to
support reliable physical and functional composition)
Drew Endy:
Synthetic biology is an approach to engineering biology
• not metabolic engineering, not cancer cell engineering, not
bioprocess engineering
• not a particular application, but it is the method ones uses to
realize a particular application (means to an end)
• Builds on genetic engineering:
• Writing DNA
• Recombinant DNA (restriction enymes,…)
• PCR
• Reading it out: sequencer
• 3 more layers are added:
• Automated construction of DNA (DNA synthesis)
• Standards (defining how we put things together): “standard
biobrick biological part”
• Abstraction (how can we formulize complexity, “no need to
memorize DNA”) (http://openwetware.org/wiki/Synthetic_Biology:Abstraction_hierarchy)
Abstraction hierarchy

• human invention designed to assist people in engineering very


complex systems by ignoring unnecessary details
(e.g. writing down the string of nucleotides required for the design of a
biological system is unattainable)

• Similarities found in other disciplines, e.g. computer engineering


• C++ or Java designed for ease of writing and reading (e.g. these
programs translated in lower sets of instructions that are more easily
translatable in strings that are machine interpretable and
implementable)

• What is a biological abstraction hierarchy?


• Loosely defined, work in progress!
Biological abstraction hierarchy: examples

Drew
Endy

Layer name Definition Example


DNA sequence of nucleotides ATGGATCATGATG
a finite sequence of
RBS, CDS, promoter,
Part nucleotides with a
terminator
specific function
multiple parts with a
Device inverter
higher level function
multiple devices hooked
System ring oscillator
together
Biological abstraction hierarchy: examples

Screenability model
Layer name Definition Example

DNA sequence of nucleotides ATGGATCATGATG

a finite sequence of
RBS, CDS, promoter,
Part nucleotides with a specific
terminator
function

one or more parts which


promoter, terminator,
Device can be screened for
inverter
functionality

multiple devices which


System cannot be screened for ring oscillator
functionality
Biological abstraction hierarchy: examples

Composition model

Layer name Definition Example


DNA sequence of nucleotides ATGGATCATGATG
a sequence of DNA with a
specific function that can be
RBS, CDS, promoter,
Part physically combined with other
terminator
parts via an assembly
standard
a set of parts that can be
functionally combined with
Device other devices via a common, inverter
standard signal carrier (i.e.
PoPS, RiPS, PhPS)
a set of devices that cannot be
functionally combined with
System ring oscillator
other devices via a common,
standard signal
Parts, devices, systems….
http://parts.mit.edu/registry/index.php/Part_Types
Parts
A terminator is a stretch of DNA
which halts the otherwise
continuous process of transcription
(e.g. stem loop, polyA,…)
"conjugative plasmid“: transfer
engineered plasmids from cell to cell

DNA parts act as DNA itself. This includes


restriction site complexes, DNA secondary
structure, and spacers.

These parts code for RNA segments with novel


structures or functions, such as stem-loop
riboregulators.

Binding region for RNA polymerase, i.e. a promoter


Devices

RFP = red flourescent protein


CFP = cyan flourescent protein
GFP = green flourescent protein
YFP = yellow flourescent protein
Devices
produce proteins in response to
some stimuli

ordered list of basic parts or other


composite parts

To enable communication between an individual cell and its


neighbors in culture or on a plate (Sender device and a Receiver
device based on the Lux system of V. Fischeri or its analogs)
Application: synchronization

a genetic inverter receives as input the concentration of repressor A and,


via gene expression, sends as output the concentration of repressor B
(HIGH input produces LOW output and vice versa; an inverter can function
like a Boolean NOT, e.g. lacI repressor)

RFP = red flourescent protein


CFP = cyan flourescent protein
GFP = green flourescent protein
YFP = yellow flourescent protein
Systems

Measurement of the relative strength of a promoter when it


is not being repressed (external validation via plasmid copy number
and cell metabolism)

Polymerase Per Second (or PoPS) = number of times that an RNA


polymerase molecule passes a specific point on DNA per unit time (e.g.,
from the 3' end of a promoter part into the 5' end of a downstream part
such as a ribosome binding site).
(Note: PoPS are measured indirectly by introducing a test gene with the
same regulatory region but with a CFP protein coding region and
measuring the fluorescence.)

Other measurement concept: RiPS (Ribosomes Per Second; RiPSmc


= RiPS per mRNA copy) = level of translation as the number of
ribosome molecules that pass a point on mRNA each second, on a per
mRNA copy basis.
Biological abstraction hierarchy: examples

Drew
Endy

Layer name Definition Example


DNA sequence of nucleotides ATGGATCATGATG
a finite sequence of
RBS, CDS, promoter,
Part nucleotides with a
terminator
specific function
multiple parts with a
Device inverter
higher level function
multiple devices hooked
System ring oscillator
together
How to construct a standard biobrick biological part

Approach depends on fabrication method:


1) PCR
2) Direct synthesis

on type of part that is being constructed


1) Standard part
2) Protein coding 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:
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' GTTTCTT C GAATTC GCGGCCGC T TCTAGA G [part] 3'
PREFIX: 3' CAAAGAA G CTTAAG CGCCGGCG A AGATCT C [part] 5'
(1) (2) (3) (4) (5) (6) (7) (8)

(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) 20 bp of sequence matching 3’ end of part sequence


(2) SpeI site
(3) Extra base to prevent creation of EcoBI or EcoKI methylation sites (could inhibit
restriction enyzme digest)
(4) NotI site
(5) PstI site
(6) Random extra spacer base
(7) Extra bases to prevent cutting of PCR product with 1) PstI, 2) A for TA cloning
efficiency
How to construct a standard biobrick biological part
2) PCR and protein coding sequence

Specific sequences corresponding to the BioBrick ends must be included on the


end of each of the two PCR primers:
PREFIX: 5' GTTTCTT C GAATTC GCGGCCGC T TCTAGA [ATG remaining CDS] 3'
3' CAAAGAA G CTTAAG CGCCGGCG A AGATCT [TAC remaining CDS] 5'
(1) (2) (3) (4) (5) (6) (7) (8)

(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

Specific sequences corresponding to the BioBrick ends must be included on the


end of each of the two PCR primers:
5' [part] TAATAA T ACTAGT A GCGGCCG CTGCAG G AAGAAAC 3'
SUFFIX: 3' [part] ATTATT A TGATCA T CGCCGGC GACGTC C TTCTTTG 5'
(1) (2) (3) (4) (5) (6) (7) (8)

(1) 20 bp of sequence matching 3’ end of part sequence


(2) Two sequential stop codons (TAA is default)
(3) SpeI site
(4) Extra base to prevent creation of EcoBI or EcoKI methylation sites (could inhibit
restriction enyzme digest)
(5) NotI site
(6) PstI site
(7) Random extra spacer base
(8) Extra bases to prevent cutting of PCR product with 1) PstI, 2) A for TA cloning
efficiency
How to construct a standard biobrick biological part
3) Direct synthesis

These sites MUST be present:


EcoRI, SpeI, XbaI, PstI, NotI

These sites are preferentially absent:


ApoI, AvrII, NheI, NsiI, SbflI

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

P 5’T ACTAGT AGCGGCCG CTGCAG3’


3’A TGATCA TCGCCGGC GACGTC5’

SpeI PstI
E X S P

Prefix:
5’GAATTC GCGGCCGCT TCTAGA G3’ S P
3’CTTAAG CGCCGGCGA AGATCT C5’

XbaI
Suffix

E X 5’T ACTAGT AGCGGCCG CTGCAG3’


3’A TGATCA TCGCCGGC GACGTC5’

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)

electron-carrying redox mediator


Summary: example
Summary: example
Glasgow project: parts submitted to the registry
Adding and documenting a biobrick

http://parts.mit.edu/igem07/index.php/Podcasts
BrickiT!
http://brickit.wiki.sourceforge.net/

• portable web-based registry that helps synthetic biologists


to plan, organize and track their local biobrick samples

• Open-source project

• A database-backed Django web server


(Django is a popular modular and object-oriented web framework
written in the Python programming language)

• http://openwetware.org/wiki/Protocols --> the do’s and don’t’s in


molecular biology
Biofusion (Phillips & Silver) aka Biobrick 2.0

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

P 5’T ACTAGT AGCGGCCG CTGCAG3’


3’A TGATCA TCGCCGGC GACGTC5’

SpeI PstI
Biobrick 3.0 (Mueller & iGem Freiberg)

P S
3

Prefix:
GAATTC GCGGCCGCT TCTAGATG GCCGGC3’
S
5’
3’CTTAAG CGCCGGCGA AGATCTAC CGGCCG5’

EcoRI XbaI NgoMIV


Suffix

P 5’ACCGGT TAAT ACTAGT AGCGGCCG CTGCAG3’


3’AGGCCA ATTA TGATCA TCGCCGGC GACGTC5’

AgeI SpeI PstI


Biobrick 3.0 (Mueller & iGem Freiberg)

E X N A S P
3

E X N ACCGGC A S P
TGGCCG
MAG Thr Gly TG*Y**