Biology Reference
In-Depth Information
essential genes can be obtained. A date-driven concept of minimal genome
49
and minimal
set of genes
50
appeared for the first time. It should be emphasized here that a minimal set
of genes (MSG) is primarily determined by growth environments. Suffice it to mention that
genes for amino acid biosynthesis are regarded as dispensable if abundant amino acids are
present in the culture medium. Therefore, in terminology, MSG is not equivalent to a
minimal genome that is a real DNA molecule including all information at the sequence
level described above. In an extreme and ideal environment where all nutrients are supplied
in growth medium at proper concentrations and at constant pH, and temperature, the core
set of genes for hypothetical minimal bacterial cell are highlighted. The most
straightforward manner for construction of a minimal genome seems to be starting from
any existing small genome. Eradication of all the nonessential genes may be the starting
point to obtain reduced
Mycoplasma
species genomes.
51
Alternatively,
B. subtilis
is shown to
require approximately 300 genes under fixed growth conditions.
52
COSTS TO SYNTHESIZE GENOMES
Genome synthesis is offering versatile and broad applications for cell engineering never
discussed previously. Meanwhile, the state-of-the-art technology still remains at a nascent
stage. Genome synthesis is surprisingly expensive in both time and cost. If KEIO and/or
JCVI would repeat the guest genome synthesis already published by the same highly skillful
staff and materials, costs are roughly 0.5 million US$ per 1000 kbp at a synthesis rate of
1000 kbp/staff/year on average. Synthesis of the familiar
E. coli
K-12 genome
(4650 kbp
4) might require 3 years and 3 million US$ for completion.
Higher-performance oligomer synthesis and reduced cost should allow applications to
various guest genomes.
1000 kbp
.
3
237
RELEVANT METHODS TO SUPPORT GENOME SYNTHESIS
Production of Domino DNA by Connection of Small DNA in B. subtilis
The bottom-up assembly approach requires a rapid, precise, and efficient method to connect
small DNA segments. Connection of many small DNAs in fewer reaction steps ensures a
rapid and prerequisite method for bottom-up assembly. A special method to produce
through
B. subtilis
was innovated and developed by our group.
17,18
Details of the method,
called OGAB, standing for Ordered Gene Assembly in
B. subtilis
, can be separately referred
to in these reviews.
53,54
Rapid preparation of a domino fragment is one of the key factors to reduce the labor cost,
as well as assembly time. If domino is made in a BAC vector, the total time to transform
B.
subtilis
ought to decrease as indicated earlier. Use of dominos prepared in the BAC of
E. coli
have already been reported.
45
However, inefficiency lies in the step of preparation of high-
quality BAC DNA suited for the domino method. The need for a careful purification
procedure may be a bottleneck for its general use.
9
Rapid and Simple Preparation of Dominos in E. coli Lysate
Recently, we invented a protocol for rapid preparation of plasmid DNA carried by special
E. coli
by which plasmids are purified at high yield and have no damage at all.
10,11
An
E. coli
strain lysogenic with lambda prophage carrying cI857ts mutation can grow normally at
30
C, but is unable to grow at restricted temperature above 37
C. The strain starts inducing
lambda phage and lyses if exposed at a high temperature, for example, at 42
C. In the
resulting clear
E. coli
lysate, what are expected include cell debris and degraded DNA, but
not the genome and plasmids. To our great surprise, plasmid DNA was found undamaged
and stayed stable for certain periods.
10
The amount and quality of plasmids in
E. coli
lysate
remained high enough to be used for transformation of
B. subtilis
. This finding led to the
