Devices and Parts
Systems and Synthetic Biology
498A
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Basic Genetic Parts
1. Promoters and Operators
2. Ribosome Binding Sites (RBS)
3. Coding Sequences
4. Terminators
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Structure of a Genetic Unit
Operators
Start Codon
Stop Codon
Gene
Promoter
RBS
Terminator
5’- UTR
All these regions are potentially engineerable.
RBS = Ribosome Binding Site
UTR = Untranslated Region
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RNA Polymerase Binds to Promoters
mRNA
Changes in the promoter sequence
can change the efficiency of RNA
polymerase binding to the DNA.
The promoter is therefore a site
which can be engineered.
http://mgl.scripps.edu/people/goodsell/pdb/pdb40/pdb40_1.html
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Strong and Weak Promoters
The strength of a promoter is one of the factors which determines the
rate of transcription.
Strong Promoter. The recA promoter is a strong promoter.
TTGATA -- 16 -- TATAAT
TTGACA -- 17 -- TATAAT
Most common Promoter
(Consensus sequence)
It differs from the averaged promoter sequence by one nucleotide and
one base pair in the spacer region.
Weak Promoter. The araBAD promoter is a weak promoter.
CTGACG -- 18 -- TACTGT
TTGACA -- 17 -- TATAAT
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Operators – Regulating Expression
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Ribosome Binding Sites
Shine-Dalgarno sequence
Helps recruit the Ribosome to the mRNA, changes
to the RBS sequence can change the translation efficiency.
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E. coli Gene Structure
Stop codon
(TAG, TAA, TGA)
Start codon
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RNA Polymerase Stops at a
Terminator
Changes in the terminator sequence
can change the efficiency of RNA
polymerase stopping. If the gene is
part of an operon, terminators can
modulate relative expression levels of
the different genes in the operon.
The terminator is therefore a site
which can be engineered.
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Operon Structure: Use Terminators to
Control Polycistronic Genes.
Gene A
100%
Promoter
Gene B
60%
Gene C
30%
Terminator
Terminators have different efficiencies.
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Internal Devices
cI
Lambda Repressor Protein, tetramer but
has two binding sites. About 233 amino
acids long.
metJ
Methionine Repressor, dimer, binds to the
metR promoter and inhibits expression.
Requires methionine in the medium
(to make the metJ activator –
S-adenosyl methionine)
105 amino acids long.
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Lambda Phage
Lysogenic
Lytic – when times go bad
Lambda Phage
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Input Devices
LacI
Lactose Operon (IPTG, Lactose)
LuxR
The Luminescence, lux operon
AHL (3-oxohexanoyl-homoserine lactone)
TetR
Tetracycline Operon (Tetracycline)
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Lac Operon
LacI Promoter: BBa_R0011
Permease
Positive
Feedback
Loop
Lactose
(Internal)
Lactose
(External)
Lac Genes
Allolactose
(Internal)
Membrane
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Lux Operon
Quorum Sensing in V. fischeri
Quorum : A minimum population unit
Bobtail squid (~3cm long)
Vibrio fischeri
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Quorum Sensing in V. fischeri
• Cell-density dependent gene expression in prokaryotes
– Quorum = A minimum population unit
• Vibrio fiscehri is a marine bacterium found both as
– a free-living organism, and
– a symbiont of some marine fish and squid.
• As a free-living organism, it exists in low density is non-luminescent..
• As a symbiont, it lives in high density and is luminescent..
• The transcription of the lux genes in this organism controls this
luminescence.
• A single cell of V. fischeri can sense when a quorum of
bacteria is achieved—leading to bioluminescence…
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Quorum Sensing in V. fischeri
The bacteria live in a sack on the underside of
the squid. The squid provides a nutrient
solution containing glucose and amino acids, in
return, the bacteria glow.
The squid only allows the bacteria to grow
during the night, so that a predatory beneath
the squid, when looking up, will appear to see
star light, rather than a silhouetted squid.
Come morning, the squid flushes 95% of the
bacteria out of the sack.
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Bacterial Luciferase/Luciferin
Bacterial Luciferin is a light-emitting biological
molecule.
Bacterial Luciferin
Luciferase
Luciferin + ATP + O2 -> PPi + AMP + Oxyluciferin
This is the luciferin found in fireflies
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Lux Operon in Vibrio
Fatty acid reductase genes
R
luxR
I
C
D
A
B
G
E
luxI
Luciferase, α and β subunits
luxR Transcription Binding Site
lux Box
luxI codes for AHL synthase. This enzyme makes AHL
(3-oxohexanoyl-homoserine lactone ) from SAM (Sadenosylmethionine)
Flavin Reductase
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Quorum Sensing
• The lux region is organized in two transcriptional units:
– OL: containing luxR gene (encodes protein LuxR = a transcriptional
regulator)
– OR: containing 7 genes luxICDABEG.
• Transcription of luxI produces the protein LuxI, required for endogenous
production of the AHL.
• The genes luxA & luxB code for the luciferase subunits
• The genes luxC, luxD & luxE code for proteins of the fatty acid reductase,
needed for aldehyde substrate for luciferase.
• The gene luxG encodes a flavin reductase.
• Along with LuxR and LuxI, cAMP receptor protein (CRP) controls
luminescence.
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Lux Regulation
The Lux operon in V. fischeri responds to a small molecule called AHL (3oxohexanoyl-homoserine lactone), also sometimes called 3OC6HSL
AHL can bind to LuxR product to form an active dimer. This dimer binds to the
so-called lux box and activates the lux genes.
AHL
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Biochemical Network
Diffuses out of the cell
AHL
SAM
Low constitutive rate
At low AHL, the lux operon is off (Inhibition from luxR)
If sufficient AHL accumulates (eg due to population density), the active luxR activates
the operon and the lux genes are switched on.
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Biochemical Network
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Biochemical Network
LuxA, LuxB
AHL
LuxI
Lux Box
LuxR
luxICDABEG
LuxC, LuxD, LuxE
luxR
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BioBrick Parts
LuxR: BBa_C0062
LuxI: BBa_C0161
Activating LuxR Promoter: BBa_R0062
Repressing LuxR Promoter: BBa_R0063
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BioBrick Parts
LuxR: BBa_C0062
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Applications
Cell-Cell Communication
You L, Cox III RS, Weiss R, Arnold
FH (2004) Programmed
population control by cell–cell
communication and regulated
killing. Nature 428: 868–871
(If cell density increase, AHL
would trigger cell death)
Basu S, Gerchman Y, Collins CH,
Arnold FH, Weiss R (2005) A
synthetic multicellular system
for programmed pattern
formation. Nature 434: 1130–
1134
Synthetic biology: new engineering rules for an emerging
discipline. MSB, (2), 2006. Andrianantoandro, et al
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TetR
TetR stands for tetracycline repressor protein. Tetracycline is a family
of antibiotics that work by inhibiting the prokaryotic 30S ribosome unit.
With the widespread use of tetracycline over the last 40
years, bacteria have developed resistance in various ways to
tetracycline, including
1. Protecting the ribosome.
2. Actively pumping the antibiotic out of the cell
3. Chemically modifying the antibiotic.
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Actively Remove Tet From Cell
In gram-negative bacteria, one of the most common mechanisms for resistance is
by actively pumping (by using the proton gradient) the antibiotic out of the cell via
a TetA membrane protein.
The Tet operon is composed of only two genes, tetA and tetR
The expression of TetA is tightly regulated by a homodimer Tet repressor (TetR) which
binds specifically at a site upstream from the tetA gene.
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TetR Operon – Pumping Tet Out
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TetR Operon
+
=
An unusual aspect of the circuit is the way that TetR activates both the production
of the pump as well as itself. Presumably TetR is kept low in the cell until tetracycline is
present, at which point the circuit comes on.
However, it is known that if the concentration of the pump is too high, there can be a
significant drain on the proton gradient resulting in lower growth rates and even death.
Once can speculate therefore that the reason why TetR also regulates itself is that as the
concentration of the pump rises, so does TetR, which in turn will throttle back
the production of the pump.
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BioBrick TetR Parts
TetR: BBa_C0040
TetR Repressor Promoter: BBa_R0040
anhydrotetracycline (ATc) is a tetracycline analog, showing
increased (30x) affinity for tet repressor but has no antibiotic
activity.
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Output Devices
An entire family of fluorescent proteins, GFP, YFP, RFP
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Output Devices
An entire family of fluorescent proteins, GFP, YFP, RFP
Chromophore formed from amino acids side chains, require
oxygen to form the chromophore. A popular yellow protein is
called venus.
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