Biology Reference
In-Depth Information
Early examples of assembly technology were carried out in vitro, such as the PCR-based
method of Stemmer and coworkers.
67
Using a mixture of overlapping 40 nucleotide
oligomers, they were able to generate a 1.1 kb
-lactamase gene via several cycles of PCR.
Following standard PCR amplification of the gene, restriction digestion, and ligation into
a plasmid, the assembled gene was successfully cloned and expressed in
E. coli
.They
analogously demonstrated assembly of a 2.7 kb plasmid, which was maintained in
E. coli
following circularization and transformation. In 2003, a similar approach was taken to
assemble the complete 5386 bp genome of
β
X174.
68
While the synthetic DNA was able to
produce infectious virions following
E. coli
transformation, reduced infectivity was observed,
indicating a rate of approximately one lethal error per 500 bp. More recently, the Golden
Gate assembly method and the Gibson assembly method have been developed for
high-efficiency in vitro assembly of large constructs, making possible the accurate assembly
of large natural product gene clusters.
69,70
The details of these methods can be found in
Chapter 3.
ϕ
As an alternative to total in vitro assembly, techniques that make partial or complete use
of in vivo recombination have been devised. Two examples are Sequence and Ligation-
Independent Cloning (SLIC)
71
and Circular Polymerase Extension Cloning (CPEC),
72
which were covered in Chapter 3. Of particular note in natural product biosynthesis is
the method of Reisinger and coworkers, who assembled the complete 31.6 kb
6-deoxyerythronolide B gene cluster.
73
They utilized a PCR-based assembly of 40 bp
oligomers to generate 500 bp fragments in vitro, followed by in vivo ligation-independent
cloning to generate 3
6 kb intermediate plasmids. Finally, digestion with type II restriction
enzymes and ligation-based selection yielded the fully assembled gene cluster. An example
of a completely in vivo assembly technique is the DNA assembler method of Shao and
coworkers.
74
This method, which relies on the high homologous recombination proficiency
of
Saccharomyces cerevisiae
, was demonstrated to construct an 11 kb five-gene biosynthetic
pathway to the carotenoid natural product zeaxanthin. At the same time, Gibson and
coworkers demonstrated in vivo assembly of the entire
Mycoplasma genitalium
genome in
S. cerevisiae
via simultaneous transformation of 25 DNA fragments of
189
24 kb.
75
This
impressive feat clearly demonstrates the utility of in vivo assembly for the preparation
of large DNA constructs, underscoring the applicability of such techniques in the
field of natural product biosynthesis.
B
OPTIMIZATION OF ASSEMBLED PATHWAYS
In traditional engineering disciplines, the standardization of parts enables the rapid
assembly of novel devices and the reliable prediction of their functionality a priori. As the
field of synthetic biology matures, efforts are also being made to standardize biological
parts in the form of a standard parts registry of so-called BioBrick components.
76,77
Nevertheless, the ideal configuration of components to maximize flux through a
biosynthetic pathway is often difficult or impossible to predict in the context of a complex
biological system. As a result, a number of experimental tools have been developed to
facilitate the optimization of an assembled pathway.
One method by which to optimize a pathway is through combinatorial assembly of
isofunctional variants of each gene. These variants could either be derived from different
species or could be engineered versions of a parental construct. Many of the assembly tools
described in the previous section, such as Golden Gate, DNA assembler, and Gibson
assembly, are largely sequence-independent, and thus can readily be applied to the
combinatorial assembly of pathway libraries from diverse fragments with compatible ends.
Such a library generation approach was recently demonstrated by Wingler and Cornish.
78
By combining homologous recombination with type II restriction digestion in vivo,
they demonstrated the construction of a library of
10
4
biosynthetic pathways.
.
