DNA assembly methods
Gibson et al, 2010, Science
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BioBricks and genetic circuits
Standard assembly with restriction enzymes and ligase
In-Fusion assembly
SLIC assembly
Gibson assembly
DNA synthesis
Registry of Standard Biological Parts (BioBricks)‫‏‬
partsregistry.org
T9002 circuit
RBS
(Ribosome Binding Site)
Promoter
BioBrick part
Transcriptional
Terminator
Coding sequence
Scar sequence
Canton et al, 2008, Nature Biotech.
Standard Assembly
Sleight et al, 2010, Nucleic Acids Research
Restriction Enzymes
http://www.accessexcellence.org/RC/VL/GG/restriction.php
Why Standard Assembly is not ideal...
1. Does not allow for the creation of fusion proteins due to scar sequence
2. Requires restriction sites be removed before assembly
3. Requires an initial amplification of the BioBrick through transformation,
overnight growth, and plasmid extraction
4. Requires tedious extraction of restricted DNA fragments from a gel, more
intermediate enzymatic reactions, and extra time to quantitate and
optimize these reactions
In-Fusion PCR Cloning Kit
http://www.clontech.com
In-Fusion BioBrick Assembly
Sleight et al, 2010, Nucleic Acids Research
PCR (Polymerase Chain Reaction)
95C
55C
68C
Taq Polymerase
• Primers are single-stranded DNA sequences
that specify where to start
extension/polymerization
• The primer sequence will be built into the
PCR product
PCR Primers
Reverse
primer
Forward
primer
PCR Reaction
Combine:
Supermix
• Phusion polymerase
• dNTPs
• Buffer
Water
Forward primer
Reverse primer
Template DNA
(0.5 ng plasmid)
Thermocycler
In-Fusion BioBrick Assembly
Sleight et al, 2010, Nucleic Acids Research
PCR Products
E0240 = E
B0032
R0011 +
Vector
= R+V
R0011
p(lacI)
E0040
GFP
B0010
B0032
1kb 100bp R
V
R+V
E
B0012
Purified PCR Products
1kb 100bp
R+V
E
In-Fusion reaction
R+V PCR Product (~2000 bp) 33.79 ng/ul
E PCR Product (~1000 bp) 64.12 ng/ul
Want a 2:1 insert:vector molar ratio and 100 ng vector.
ng E = 2 X (1000/2000) X 100 = 100 ng
R+V = 100 ng / 33.79 ng/ul = 3 ul
E = 100 ng / 64.12 ng/ul = 1.5 ul
In-Fusion Assembly reaction:
R+V = 3 ul
E = 1.5 ul
Water = 5.5 ul
Total = 10 ul  Add to In-Fusion tube and mix thoroughly
In-Fusion reaction (continued)
10 ul
reaction tube
15 min 37C, 15 min 50C
Dilute in TE Buffer
Transformation
Transformation via electroporation
Transformants
In-Fusion reaction (continued)
Colony PCR
Overnight culture of successful transformants
Extract plasmid
Submit for DNA sequencing
DNA sequencing
• Normally a good miniprep gives about 300 ng/ul
• Add 4 ul miniprep, 1 ul primer, and 7 ul water for 12 total ul into eppy tube
• Submit labeled tube to sequencing facility with filled out form
~100 bp
~100 bp
VF2
R0011
p(lacI)
B0032
E0040
GFP
B0010
B0012
VR
Analyzing sequence data
VF2
VR
A Plasmid Editor (APE): http://www.biology.utah.edu/jorgensen/wayned/ape/
Circuit characterization
Population level
Single cell level
SLIC (Sequence and Ligation Independent Cloning)
Li and Elledge, 2007, Nature Methods
Gibson assembly
Gibson et al, 2009, Nature Methods
DNA synthesis
Czar et al, 2009, Trends in Biotech.
DNA synthesis of an artificial chromosome
Gibson et al, 2010, Science