Biology Reference
In-Depth Information
12
CHAPTER
Tools for Genome Synthesis
Mitsuhiro Itaya
Keio University, Tsuruoka, Yamagata, Japan
INTRODUCTION
A cellular system is complex. Present terrestrial lives from unicellular to multicellular have
adapted to the present diverse environments on the Earth. The two putative definitions are
in favor by the author and sufficient for this chapter: the premise of the biological systems
should: (1) constitute biological materials such as proteins, nucleic acids, lipids, and other
metabolites; and (2) yield proliferative offspring inherited by genome DNA. Genomes, as
information molecules, govern cellular activities in accordance with information flow
known as the central dogma: DNA
metabolites. Fundamental knowledge
on biology has been obtained through various genetic, biochemical, and biophysical studies
applied on existing lives in nature and long-cultivated domesticated ones in laboratories.
The framework of further complicated biological systems will be ultimately proven if
man-made ones are available. What are man-made cells deemed to be or recognized by
most researchers? Fundamental answers should eventually come when those cells use
genetic circuits of interacting genes and proteins to implement diverse cellular
functions.
1,2
-
RNA
-
protein
-
225
A fundamental topic underlying this chapter is how to practically produce genomes that
should lead to innovation of newly engineered cells. The genome, the largest unbranched
polymer molecule among biological substances, is a warehouse of all genes in a given cell.
Hundreds or thousands of genes are embedded in the genome, as illustrated in
Figure 12.1
.
The gene, a major object in modern biology on the other hand, is included in small DNA
molecules. Thanks to the genetic engineering technologies developed in the late 1980s and
later established as essential fundamental tools, small DNA segments are readily available in
all aspects of the current molecular biology field. Genes encoded in small DNA segments
are stable in test tubes. Once cloned, they are ready to be amplified by
Escherichia coli
plasmids followed by manipulations aiming at various goals. In contrast, large DNAs
become unstable in test tubes, even if they are carefully isolated. The primary reason is
that hydrodynamic shearing in solution under regular laboratory manipulations will
result in fragmentation to small DNAs, due to intrinsic physicochemical properties
associated with the long polymer. Regular DNA implementations, agitation by vortex or
precipitation by addition of ethanol, provide fragmented DNA not exceeding 50 kbp.
Unnoticed nucleases contaminated during biochemical isolation procedures might be the
secondary reason making damage-free genome DNA preparation considerably difficult.
A DNA molecule of
50 kbp is designated as a large DNA molecule to discriminate
small DNA fragments, unless specified otherwise in this chapter (
Fig. 12.1
). Protocols
working well for small DNA engineering have not simply been expanded to those for
large DNA engineering.
.
