DNAassembly techniques- BioBrick, Golden gate, OE-PCR, CPEC, Gibson assembly, DNA assembler, LCR

1. DNA assembly
techniques
Nikunj Aggarwal
MSc. Biotechnology (3rd sem)
Department Of Biotechnology
Central University of Haryana

2. INTRODUCTION
Design efficient metabolic routes
Construct and optimize biochemical pathways
Garcia-Ruiz, E., HamediRad, M., & Zhao, H. (2016). Pathway design, engineering, and optimization. In Synthetic Biology–Metabolic Engineering (pp. 77-116).
Springer, Cham.Chicago
PATHWAY CONSTRUCTION
Pathways of interest are
selected and designed
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To produce chemicals of high
interest

3. They have to be assembled and constructed
After the pathways of interest are selected and designed
DNA constructs as large as the size of entire genomes and with as many as 25 parts
have been assembled
restriction enzyme based
in vivo
in vitro
bridging oligo based
DNA
assembly
methods
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Sequence
Homology
Based Methods

4. DNA assembly methods
Garcia-Ruiz, E., HamediRad, M., & Zhao, H. (2016). Pathway design, engineering, and optimization. In Synthetic Biology–Metabolic Engineering (pp. 77-116).
Springer, Cham.Chicago 4

5. Restriction Enzymes Based Methods
• Uses restriction enzymes and DNA ligase
BioBrick
https://international.neb.com/applications/cloning-and-synthetic-
biology/dna-assembly-and-cloning/biobrick-assembly
Isocaudomers
 Used for building up larger DNAs for
standardized intermediates (parts)
https://www.igem.tudarmstadt.de/igem_2/projekt_2/biobricks_1/biobricks.
en.jsp
Enzymes that recognizes a slightly
different sequence, but produces the same ends
generate two compatible sticky ends
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6. Golden Gate method
https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly
Type ll Restriction
• Cleave DNA outside of their recognition site and
produce an overhang of 4 arbitrary nucleotides
• Two digested fragments can be ligated to
generate a product lacking the original restriction
sites
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7. Sequence Homology Based Methods: in vitro
• Utilize longer arbitrary overlapping regions between parts
 DNA parts are first amplified in separate PCRs
with homologous ends between them
 With the corresponding homologous regions,
these DNA parts can anneal to each other and
extended by DNA polymerase in the second round
of PCR to yield spliced DNA molecules
 The resulting larger DNA fragments can then be
inserted into plasmids using the restriction
digestion and ligation method
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Overlap extension polymerase chain reaction (OE-PCR)
(Splicing by Overlap Extension or SOEing)
https://openwetware.org/wiki/PCR_Overlap_Extension

8. Circular polymerase extension cloning (CPEC)
• Allows assembly of multiple inserts with any vector in a one-step OE-PCR reaction
• With extended homologous regions, the fused DNA molecules circularize with a nick in each
strand
• After transformation, host Escherichia coli fixes the nicks to form intact vectors
• Non-linearized vectors can be used as templates for extension followed by DpnI digestion
to eliminate the original plasmids
Bryksin, A. V., & Matsumura, I. (2010). Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques, 48(6), 463-465.
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9. Gibson assembly
https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-
and-cloning/gibson-assembly
 Single-pot assembly of multiple parts at the same time
The parts being assembled usually have around 25-bp homology which guides
the assembly
Three enzymatic activities
1. 5’ exonuclease (T5 exonuclease) generates long overhangs
2. Phusion Polymerase fills in the gaps of the annealed single strand regions
3. DNA ligase seals the nicks of the annealed and filled-in gaps
9https://international.neb.com/tools-and-resources/feature-articles/gibson-
assembly-building-a-synthetic-biology-toolset

10. Sequence Homology Based Methods: in vivo
DNA Assembler- Yeast
• Homologous recombination occurs naturally in Saccharomyces cerevisiae which was exploited
to construct the genome by assembling multiple fragments to construct a large pathway by
assembling multiple fragments nearly at the same time
•All DNA parts to be assembled can be obtained either from PCR amplification or restriction
digestion with homologous arms between neighboring parts in the pathway
•All the linear DNA parts directly transformed into S. cerevisiae.
•Circular plasmids are then constructed by its endogenous homologous recombination
machinery
•DNA assembly was also performed in other organisms such as Bacillus subtilis
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11. 11
DNA cloning- E. Coli
• RecE and RecT facilitate highly efficient homologous recombination between two linear DNA
molecules in E. Coli
• Digested or sheared DNA was transformed together with PCR-amplified vector containing
terminal homology arms into the host which over-expressed full-length RecET promoting
formation of an intact vector
Wang, X., Sa, N., Tian, P. F., & Tan, T. W. (2011). Classifying DNA assembly protocols for
devising cellular architectures. Biotechnology advances, 29(1), 156-163.

12. Bridging Oligo Based Methods
• Single stranded bridging oligos are designed to be complementary to two ends of neighboring
DNA parts
https://international.neb.com/tools-and-resources/video-library/nebuilder-hifi-dna-assembly-bridging-dsdna-with-a-ssdna-oligo
Ligase cycling reaction (LCR)
• DNA parts to be assembled via LCR were amplified using 5’-phosphorylated primers
• During the reaction, the dsDNA parts are denatured, and then the upper (or lower) strands
from the two parts to be adjoined anneal with the bridging oligo at a lower temperature and are
subsequently joined scarlessly by thermostable ligase via a phosphodiester bond
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13. • Ligated strand serves as the template to assemble the complementary strands. By running
multiple thermal cycles, linear DNA parts can be assembled into circular DNA constructs and
then transformed into E. coli competent cells for amplification
Chandran, S. (2017). Rapid assembly of DNA via ligase cycling reaction (LCR). In Synthetic DNA (pp. 105-110). Humana Press, New York, NY.13

14. Restriction
Enzymes Based
Methods
Sequence
Homology Based
Methods: in vivo
Sequence
Homology Based
Methods: in vivo
Bridging Oligo
Based Methods
BioBrick
• Powerful and flexible
technique
• Consists scar
sequences
Golden Gate
method
• Scarless
• Reliable assembly of
multiple parts
• Fast
OE-PCR
•Scarless
•Consists scar
sequences
Gibson assembly
• Scarless
• Reliable assembly of
multiple parts
• Fast
DNA Assembler
• Flexible
• Reliable
• Recommended for
large constructs
LCR
• Scarless
•Modular method
useful for combinatorial
assemblies
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15. • Garcia-Ruiz, E., HamediRad, M., & Zhao, H. (2016). Pathway design, engineering, and optimization.
In Synthetic Biology–Metabolic Engineering (pp. 77-116). Springer, Cham.Chicago
• Chao, R., Yuan, Y., & Zhao, H. (2015). Recent advances in DNA assembly technologies. FEMS yeast
research, 15(1), 1-9.
• https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-
cloning/biobrick-assembly
• https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-
cloning/golden-gate-assembly
• https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-
cloning/gibson-assembly
• https://international.neb.com/tools-and-resources/video-library/nebuilder-hifi-dna-assembly-
bridging-dsdna-with-a-ssdna-oligo
• Chandran, S. (2017). Rapid assembly of DNA via ligase cycling reaction (LCR). In Synthetic DNA (pp.
105-110). Humana Press, New York, NY.
REFERENCES
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16. THANK YOU!
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