Week 1 of IGEM

SYNTHETIC
BIOLOGY
X.DU
INTRODUCTION
• Synthetic biology
contributes to our world.
OUTLINE
SYNTHETIC BIOLOGY VS BIOTECHNOLOGY
• Engineers design a new car by improving all sorts of individual components (engine,
chassis, wheels, bodywork) and combining them into better cars, and in synthetic biology
we combine knowledge of proteins, DNA, micro-organisms and cells into new
applications.' At first glance, synthetic biology has much in common with biotechnology.
'The difference is that the purpose of biotechnology is to improve existing life forms. In
synthetic biology we take this a step further: instead of looking for new applications for
existing organisms, we design new, synthetic life forms.'
OLD vs. NEW

INTRODUCTION
• In week 1, we are going to talk about the basic concept of synthetic biology
CENTRAL DOGMA
• Biology is a fascinating science that is the source of the wonders of synthetic biology.
Fortunately, to get started, we only need to know a few key processes in biology, outlined
by the central dogma: DNA is transcribed to RNA, which is translated into protein.
PROKARYOTES VS. EUKARYOTES
• Many core processes in biology differ between prokaryotes (cells with no nucleus) and
eukaryotes (cells having a nucleus containing DNA). The following problems will help
you learn or recall which processes happen in which types of cells.
• In prokaryotes
– Transcription and translation occur together
DNA
TRANSCRIPTION
mRNA
Ribosome
TRANSLATION
Polypeptide
(a) Prokaryotic cell. In a cell lacking a nucleus, mRNA

produced by transcription is immediately translated
without additional processing.
Figure 17.3a
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• In eukaryotes
– RNA transcripts are modified before becoming true
mRNA
Nuclear
envelope
TRANSCRIPTION DNA
Pre-mRNA
RNA PROCESSING
mRNA
Ribosome
TRANSLATION
(b) Eukaryotic cell. The nucleus provides a separate
Polypeptide compartment for transcription. The original RNA
transcript, called pre-mRNA, is processed in various
ways before leaving the nucleus as mRNA.
Figure 17.3b

• During transcription
– The gene determines the sequence of bases
along the length of an mRNA molecule
DNA Gene 2
molecule
Gene 1
Gene 3
DNA strand 3 5
(template) A C C A A A C C G A G T
TRANSCRIPTION
U G G U U U G G C U C A
mRNA 5 3
Codon
TRANSLATION
Protein Trp Phe Gly Ser

Figure 17.4 Amino acid
Cracking the Code
• A codon in messenger RNA
– Is either translated into an amino acid or serves as
a translational stop signal
Second mRNA base
U C A G
UUU UCU UAU UGU U
Phe Tyr Cys
UAC
U UUC UCC
Ser
UGC C
UUA UCA UAA Stop UGA Stop A
UUG Leu UCG UAG Stop UGG Trp G
Third mRNA base (3 end)

First mRNA base (5 end) CUU CCU CAU CGU U
His
CUC CCC CAC CGC C
C Leu Pro Arg
CUA CCA CAA CGA A
Gln
CUG CCG CAG CGG G
AUU ACU AAU AGU U
Asn
A
AUC lle ACC AAC AGC Ser C
Thr
AUA ACA AAA AGA A
Lys
AGG Arg G
Met or
AUG start ACG AAG
GUU GCU GAU GGU U
G GUC GCC GAC Asp GGC C
Val Ala Gly
GUA GCA GAA GGA A
Figure 17.5 GUG GCG GAG Glu GGG G
• Codons must be read in the correct reading
frame
– For the specified polypeptide to be produced

Evolution of the Genetic Code
• The genetic code is nearly universal
– Shared by organisms from the simplest
bacteria to the most complex animals

• In laboratory experiments
– Genes can be transcribed and translated after
being transplanted from one species to
another
Figure 17.6
• Concept 17.2: Transcription is the DNA-
directed synthesis of RNA: a closer look

Molecular Components of Transcription
• RNA synthesis
– Is catalyzed by RNA polymerase, which pries
the DNA strands apart and hooks together the
RNA nucleotides
– Follows the same base-pairing rules as DNA,
except that in RNA, uracil substitutes for
thymine

Synthesis of an RNA Transcript
• The stages of transcription are
Promoter
– Initiation 5
3
Transcription unit
3
5
DNA
Start point
– Elongation RNA polymerase 1 Initiation. After RNA polymerase binds to

the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
start point on the template strand.
– Termination 5
3
Template strand of
3
5
Unwound RNA DNA

DNA transcript
2 Elongation. The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5  3 . In the wake of
Rewound transcription, the DNA strands re-form a double helix.
RNA
5 3
3 3 5
5
RNA
transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.
5 3
3 5
5 3
Completed RNA
Figure 17.7 transcript

Elongation Non-template
strand of DNA
RNA nucleotides
RNA
polymerase
T C C A A
A T
3 C T U
3 end
T
G
A U
G
G
A
C A EG C A C
5 A
T A
A G G T T
Direction of transcription
5 Template
(“downstream”)
strand of DNA
Newly made
RNA

RNA Polymerase Binding and Initiation of Transcription
• Promoters signal the initiation of RNA synthesis
• Transcription factors
– Help eukaryotic RNA polymerase recognize
promoter sequences
TRANSCRIPTION DNA 1 Eukaryotic promoters
RNA PROCESSING Pre-mRNA
mRNA
TRANSLATION Ribosome
Polypeptide
Promoter
5 T A T A A AA 3
AT A T T T T
3 5
TATA box Start point Template
DNA strand
2 Several transcription
factors
Transcription
factors
5 3
3 5
3 Additional transcription
factors
RNA polymerase II
Transcription factors
5 3
3 5 5
RNA transcript
Figure 17.8 Transcription initiation complex

Elongation of the RNA Strand
• As RNA polymerase moves along the DNA
– It continues to untwist the double helix,
exposing about 10 to 20 DNA bases at a time
for pairing with RNA nucleotides

Termination of Transcription
• The mechanisms of termination
– Are different in prokaryotes and eukaryotes

• Eukaryotic cells modify RNA after transcription
• Enzymes in the eukaryotic nucleus
– Modify pre-mRNA in specific ways before the
genetic messages are dispatched to the
cytoplasm

Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is modified
in a particular way
– The 5 end receives a modified nucleotide cap
– The 3 end gets a poly-A tail
A modified guanine nucleotide 50 to 250 adenine nucleotides

added to the 5 end added to the 3 end
TRANSCRIPTION DNA
RNA PROCESSING Pre-mRNA Protein-coding segment Polyadenylation signal

5 3
mRNA
G P P P AAUAAA AAA…AAA
Ribosome
TRANSLATION
Start codon Stop codon
5 Cap 5 UTR 3 UTR Poly-A tail
Polypeptide
Figure 17.9
Split Genes and RNA Splicing
• RNA splicing
– Removes introns and joins exons
5 Exon Intron Exon Intron Exon 3

TRANSCRIPTION DNA Pre-mRNA 5 Cap Poly-A tail
1 30 31 104 105 146
RNA PROCESSING Pre-mRNA
mRNA Coding Introns cut out and

Ribosome segment exons spliced together
TRANSLATION
Polypeptide
mRNA 5 Cap Poly-A tail
1 146
3 UTR 3 UTR
Figure 17.10
• Is carried out by spliceosomes in some cases
RNA transcript (pre-mRNA)
5
Exon 1 Intron Exon 2
Protein
1 Other proteins
snRNA
snRNPs
Spliceosome
2 5
Spliceosome
components
Cut-out
intron
3
mRNA
5
Figure 17.11 Exon 1 Exon 2

Ribozymes
• Ribozymes
– Are catalytic RNA molecules that function as
enzymes and can splice RNA

The Functional and Evolutionary Importance of Introns
• The presence of introns

– Allows for alternative RNA splicing

• Proteins often have a modular architecture
– Consisting of discrete structural and functional
regions called domains
• In many cases
– Different exons code for the different domains in a
protein
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Figure 17.12 Polypeptide

• Translation is the RNA-directed synthesis of a
polypeptide: a closer look

Molecular Components of Translation
• A cell translates an mRNA message into
protein
– With the help of transfer RNA (tRNA)

• Translation: the basic concept
TRANSCRIPTION DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Amino
Polypeptide acids
tRNA with
amino acid
Ribosome attached
Trp
Ph e Gly
tRNA
C
C
GC G
A
Anticodon
A A A
U G G U U U G G C
5 Codons 3
mRNA
Figure 17.13
• Molecules of tRNA are not all identical
– Each carries a specific amino acid on one end
– Each has an anticodon on the other end

The Structure and Function of Transfer RNA
• A tRNA molecule
– Consists of a single RNA strand Athat is only
C
about 80 nucleotides long C
– Is roughly L-shaped Amino acid

3
A
C
attachment site C
A 5
C G
G C
C G
U G
U A
A U
U C A U
* C A C AG UA A G *
G * C U C
*
C G U G U * C G A G G
* * U C * A G G
* G AG C
(a) Two-dimensional structure. The four base-paired regions and three G C Hydrogen
loops are characteristic of all tRNAs, as is the base sequence of the U A bonds
amino acid attachment site at the 3 end. The anticodon triplet is * G
A
unique to each tRNA type. (The asterisks mark bases that have been A* C
chemically modified, a characteristic of tRNA.) * U
A G
A
Figure 17.14a Anticodon

Amino acid
5
attachment site
3
Hydrogen
bonds
A AG
3 5
Anticodon
Anticodon
(c) Symbol used
(b) Three-dimensional structure in this book
Figure 17.14b
• A specific enzyme called an aminoacyl-tRNA
synthetase
– Joins each amino acid to the correct tRNA
Amino acid Aminoacyl-tRNA
synthetase (enzyme)
P P P Adenosine
1 Active site binds the
amino acid and ATP.
ATP
2 ATP loses two P groups

and joins amino acid as AMP.
P Adenosine
Pyrophosphate P Pi
Pi
Pi
Phosphates
tRNA
3 Appropriate
tRNA covalently
Bonds to amino
Acid, displacing P Adenosine
AMP. AMP
4 Activated amino acid
is released by the enzyme.
Aminoacyl tRNA
(an “activated
Figure 17.15 amino acid”)

Ribosomes
• Ribosomes
– Facilitate the specific coupling of tRNA
anticodons with mRNA codons during protein
synthesis

• The ribosomal subunits
– Are constructed of proteins and RNA
molecules named ribosomal RNA or rRNA
TRANSCRIPTION DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Exit tunnel
Growing
polypeptide
tRNA
molecules
Large
subunit
E
P A
Small
subunit
5
mRNA 3
(a) Computer model of functioning ribosome. This is a model of a bacterial

ribosome, showing its overall shape. The eukaryotic ribosome is roughly
similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules
Figure 17.16a and proteins.
• The ribosome has three binding sites for tRNA
– The P site
– The A site P site (Peptidyl-tRNA
binding site)
A site (Aminoacyl-
tRNA binding site)
– The E site E site
(Exit site)
Large
subunit
E P A
mRNA
binding site Small
subunit
(b) Schematic model showing binding sites. A ribosome has an mRNA

binding site and three tRNA binding sites, known as the A, P, and E
Figure 17.16b sites. This schematic ribosome will appear in later diagrams.

Amino end Growing polypeptide
Next amino acid

to be added to
polypeptide chain
tRNA
mRNA 3
Codons
5
(c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon
base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing
polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the
polypeptide chain. Discharged tRNA leaves via the E site.
Figure 17.16c

Building a Polypeptide
• We can divide translation into three stages
– Initiation
– Elongation
– Termination

Ribosome Association and Initiation of Translation
• The initiation stage of translation
– Brings together mRNA, tRNA bearing the first
amino acid of the polypeptide, and two
subunits of a ribosome
Large
ribosomal
P site subunit
3 U A C 5
t t
Me 5 A U G 3 Me
Initiator tRNA
GTP GDP
E A
mRNA
5 3 5 3
Start codon
mRNA binding site Small Translation initiation complex

ribosomal
subunit
1 A small ribosomal subunit binds to a molecule of 2 The arrival of a large ribosomal subunit completes
mRNA. In a prokaryotic cell, the mRNA binding site the initiation complex. Proteins called initiation
on this subunit recognizes a specific nucleotide factors (not shown) are required to bring all the
sequence on the mRNA just upstream of the start translation components together. GTP provides
codon. An initiator tRNA, with the anticodon UAC, the energy for the assembly. The initiator tRNA is
base-pairs with the start codon, AUG. This tRNA in the P site; the A site is available to the tRNA
carries the amino acid methionine (Met). bearing the next amino acid.
Figure 17.17
Elongation of the Polypeptide Chain
• In the elongation stage of translation
– Amino acids are added one by one to the
preceding amino acid
1 Codon recognition. The anticodon
TRANSCRIPTION DNA
Amino end of an incoming aminoacyl tRNA
mRNA
of polypeptide base-pairs with the complementary
Ribosome
TRANSLATION mRNA codon in the A site. Hydrolysis
Polypeptide
of GTP increases the accuracy and
E efficiency of this step.
mRNA 3
Ribosome ready for P A
next aminoacyl tRNA 5 site site
2 GTP
2 GDP
E E
P A P A
2 Peptide bond formation. An

GDP rRNA molecule of the large
3 Translocation. The ribosome GTP
subunit catalyzes the formation
translocates the tRNA in the A
of a peptide bond between the
site to the P site. The empty tRNA
new amino acid in the A site and
in the P site is moved to the E site, E
the carboxyl end of the growing
where it is released. The mRNA
polypeptide in the P site. This step
moves along with its bound tRNAs, P A attaches the polypeptide to the
bringing the next codon to be
tRNA in the A site.
Figure 17.18 translated into the A site.
Termination of Translation
• The final stage of translation is termination
– When the ribosome reaches a stop codon in
the mRNA
Release
factor
Free
polypeptide
5
3 3
3
5 5
Stop codon
(UAG, UAA, or UGA)
1 When a ribosome reaches a stop 2 The release factor hydrolyzes 3 The two ribosomal subunits
codon on mRNA, the A site of the the bond between the tRNA in and the other components of
ribosome accepts a protein called the P site and the last amino the assembly dissociate.
a release factor instead of tRNA. acid of the polypeptide chain.
The polypeptide is thus freed
from the ribosome.
Figure 17.19
Polyribosomes
• A number of ribosomes can translate a single
mRNA molecule simultaneously
– Forming a polyribosome
Completed
Growing polypeptide
polypeptides
Incoming
ribosomal
subunits
Start of Polyribosom
e End of
mRNA mRNA
(5 end) (3 end)
(a) An mRNA molecule is generally translated simultaneously
by several ribosomes in clusters called polyribosomes.
Ribosomes
mRNA
0.1 µm
(b) This micrograph shows a large polyribosome in a prokaryotic
Figure 17.20a, b cell (TEM).
Completing and Targeting the Functional Protein
• Polypeptide chains
– Undergo modifications after the translation
process

Protein Folding and Post-Translational Modifications
• After translation
– Proteins may be modified in ways that affect
their three-dimensional shape

SYNTHETIC BIOLOGY CHOSE BACTERIA.
• Firstly, bacteria are relatively simple organisms that are easy and inexpensive to culture in
the laboratory. This allows researchers to manipulate their genetic material and study how
changes to their DNA affect their behavior and function.
• Secondly, bacteria have a rapid growth rate, which means that experiments can be
conducted quickly and results obtained relatively quickly.
SYNTHETIC BIOLOGY CHOSE BACTERIA.
• Thirdly, bacteria have a wide range of metabolic pathways and are capable of producing a
wide range of useful chemicals and proteins, which makes them attractive sources for
bioengineering.
• Additionally, because bacteria can be easily engineered and controlled, they are good
candidates for creating new technologies, such as gene editing tools and biosensors.
HOW TO STUDY BACTERIA
HOW TO STUDY BACTERIA
• Or from genetic dissection

• Common terms you need to know
PROMOTER
• A promoter is a region of DNA that is one element involved in regulating the expression
of a gene. This can happen in a few ways. For example, the promoter region might
bind RNA polymerase (RNAP, transcribes RNA molecules from DNA instructions)
really well, in which case we say it has a high affinity to RNAP and is a strong promoter.
On the other hand, it might bind RNAP poorly, have low affinity and be a weak promoter.
The promoter might also bind other proteins which can recruit RNAP, occlude it from
binding, or recruit other proteins that do this. These proteins are in general
called transcription factors and can enhance or repress gene expression.
INDUCER
• Furthermore, many repressor proteins can be activated or inactivated by small molecules

called inducers, as these proteins commonly originally evolved to activate clusters of
metabolism-related genes (e.g. LacI inhibits genes coding for proteins related to the
metabolism of lactose and is inactivated by the presence of allolactose, a product of
lactose metabolism, or IPTG, an allolactose mimic which is used in experiments).
Fig. 18-4
Regulatory Promoter
gene
Operator
DNA lacI lacZ

No
RNA
made
3
mRNA RNA
5 polymerase
Active
Protein repressor
(a) Lactose absent, repressor active, operon off
lac operon
DNA lacI lacZ lacY lacA
RNA
polymerase
3
mRNA
mRNA
5 5
Protein -Galactosidase Permease Transacetylase
Allolactose Inactive
(inducer) repressor
(b) Lactose present, repressor inactive, operon on

REPRESSORS AND OPERATOR SITES
• TetR and LacI are repressors from bacteria. They work by binding sequences of DNA
called operator sites at promoters, sterically blocking RNA polymerase from initiating
transcription. Fortunately, this mechanism also works in eukaryotes: if we insert an
operator sequence at a promoter, we can inhibit (repress) transcription by adding a
sufficient amount of repressor. The presence of a repressor makes transcription less likely.
MIRNA
• MicroRNAs (miRNAs) are small single-stranded

RNA molecules that can bind to mRNA
• These can degrade mRNA or block its translation
Fig. 18-13
Hairpin miRNA Hydrogen

bond
Dicer
miRNA miRNA-
5 3 protein
(a) Primary miRNAtranscript
 complex
mRNA degraded Translation blocked

(b) Generation and function of miRNAs
• The phenomenon of inhibition of gene expression by
RNA molecules is called RNA interference (RNAi)
• RNAi is caused by small interfering RNAs
(siRNAs)
• siRNAs and miRNAs are similar but form from
different RNA precursors
IRESS(INTERNAL RIBOSOME ENTRY SITE)
• IRES: internal ribosome entry site. IRESs are a unique class of sequences that allow 5'
cap-independent initiation of translation. Typically, eukaryotic translation always starts at
the 5' cap (a modified G nucleotide), and is called "cap-dependent initiation". Some
viruses have developed sequences that when transcribed to RNA will fold in structures
resembling various initiation factors or mimic conformations required for initiation; there
are other ways IRESs function as well, e.g. by base-pairing with the RNA components of
the ribosome.
FP(FLUORESCENT PROTEINS)
• Fluorescent proteins have truly enabled a revolution in

molecular biology and biotechnology by allowing us to
visualize protein production and localization. Over the
years, fluorescent protein technology has come a long
way. One can order proteins that mature (fold and
become ready to fluoresce) fast or slow, emit in a range
of wavelengths and can even be split (the two halves of
the protein can be brought together in cells to fluoresce;
this is a useful tool to see if/where/when two proteins
interact). The most famous FP is GFP, the green
fluorescent protein. In the problems you will also see the
protein mKate, which is a red fluorescent protein.
• Yongjian Qian (Roger Y. Tsien)
• Japanese scientist Shuji Nakamura, American scientist Martin Chalfie, and
Chinese-American scientist Yongjian Qian shared the 2008 Nobel Prize in
Chemistry for their contribution to the study of green fluorescent protein.
Among the three, Shuji Nakamura first discovered the protein that emitted green
fluorescence under ultraviolet light in jellyfish on the west coast of North
America in 1962, which is known as the green fluorescent protein. Martin
Chalfie later contributed significantly to using green fluorescent protein as a
biological tracer molecule. Yongjian Qian deepened the scientific understanding
of the luminescence mechanism of green fluorescent protein and expanded the
range of fluorescent proteins beyond green, laying the foundation for tracking
multiple biological cell changes simultaneously.
WEEK 2 ARRANGEMENT
• We are going to discuss the detailed steps to design a modified bacteria.

Week 1 of IGEM

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SYNTHETIC

OLD vs. NEW

(a) Prokaryotic cell. In a cell lacking a nucleus, mRNA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Protein Trp Phe Gly Ser

Third mRNA base (3 end)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

– Elongation RNA polymerase 1 Initiation. After RNA polymerase binds to

Unwound RNA DNA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

A modified guanine nucleotide 50 to 250 adenine nucleotides

RNA PROCESSING Pre-mRNA Protein-coding segment Polyadenylation signal

5 Exon Intron Exon Intron Exon 3

mRNA Coding Introns cut out and

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• The presence of introns

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Figure 17.12 Polypeptide

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

– Is roughly L-shaped Amino acid

Figure 17.14a Anticodon

2 ATP loses two P groups

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

(a) Computer model of functioning ribosome. This is a model of a bacterial

(b) Schematic model showing binding sites. A ribosome has an mRNA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Next amino acid

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

mRNA binding site Small Translation initiation complex

2 Peptide bond formation. An

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• Or from genetic dissection

• Furthermore, many repressor proteins can be activated or inactivated by small molecules

DNA lacI lacZ

(a) Lactose absent, repressor active, operon off

DNA lacI lacZ lacY lacA

Protein -Galactosidase Permease Transacetylase

(b) Lactose present, repressor inactive, operon on

• MicroRNAs (miRNAs) are small single-stranded

Hairpin miRNA Hydrogen

mRNA degraded Translation blocked

• Fluorescent proteins have truly enabled a revolution in

• We are going to discuss the detailed steps to design a modified bacteria.

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