Abstract
Our project goal is to structure viable artificial cell framed by all known genetic elements, for the purpose of unraveling the life phenomena and functions. The precise E-cell simulation[5] will be possible by getting valuable quantitative data from the viable artificial cells. In order to accomplish these tasks, following three major experiments should be completed. 1. The artificial genome construction[1,2,3,4,10,11]; developing the high-through-put method to construct artificial genome in vitro. 2. The natural genome destruction[1,2,3]; constructing the plasmid, which can replace natural genome with artificial genome. 3. The injectable genome region map; getting a clear picture of the region, which will cause incompatibility1.
Last term, a prototype protocol of E.coli genome BAC library for searching the injectable genome region has been developed[11]. In summer, these clones have been affirmed through restriction enzyme map and DNA sequencing. Tough, they have only short genome fragments. It’s seems to have been about 150kb, but indeed, they have only about 20kb fragments. Some reagent were contaminated by some kinds of inhibitor, therefore, incomplete insert check results showed not monomer (20kb) but polymer (150kb). This term, several BAC library protocols were tested, and an accurate method of searching the injectable genome region has been established. This method is accurate but, takes much time and can’t cover all regions of genomes. This report introduces the overview of the E.coli genome BAC library, and the optimization experiments for high-through-put methods of searching the injectable genome region.
1: Incompatibility: several regions in the genome that
appear to be uncloneable or unstable, this regions may effect incompatibility.
2, 3: these experiments are carried out with Komai Hiromi and
Kenji Higashi.
INTRODUCTION
Our project overview
At the present time, there is no organism whose genetic elements and restriction expression mechanism are fully known. Even the well-known and studied model organism, E.coli, still holds great amount of unknown factors. We aim to structure viable artificial cell framed by known genetic elements, for the purpose of unraveling the life phenomena and functions. This viable artificial cell will be very helpful to get valuable quantitative data for precise E-cell simulation[5], in consequence, this simulation results will promote detailed understanding of the cell’s behavior.
In our project, to achieve above
purpose, following three major experiments: 2artificial genome construction, 3natural genome destruction, and searching the
injectable genome region, are undergoing in parallel. The procedure to
structure viable artificial cell is as follows(See FIG.1). First of all, design
an artificial genome through an informatics approach, and then construct the
designed artificial genome in vitro.
Next, introduce the constructed artificial genome to an Escherichia coli host cell using a genome destruction plasmid,
which can replace natural genome with artificial genome. At this step, also
refer to the injectable genome region map obtained from the genome BAC library.
These technologies are completely new approach to biological
science, and also, have widespread applications. For instance, the valuable
quantitative analysis of the cell’s metabolome will be
possible, due to construction an organism containing a minimal genome
structured by all known genetic elements. Additionally, functional analysis
will be accomplished by the cloning of specific genes into the artificial
genome.
Last term, constructing a prototype protocol of E.coli genome BAC library for searching
the injectable genome region has been done[11]. This term, protocol
optimization has finished and also high-through-put methods are just getting
underway.
FIG.1 Outline flowchart of
constructing the viable artificial cell framed by known genetic elements.
FIG.2 Outline flowchart of
searching the injcetable genome region
Searching the injectable genome region
Our approach has the step, that 2 type of the genome, both natural genome and artificial genome, exists in the same step. This situation may cause incompatibility. Therefore, we need to know the injectable genome region and non-injectable genome region, in advance. Constructing genome library will tell us useful region information of the incompatibility effect.
FIG.2 shows the outline flowchart of this experiment. First, natural genome will be extracted from host cell, and its genome will be cut into pieces using several rare base cutters, such as NotI. As a consequence of cutting genome, approximately 200kb fragments can be picked it up by plasmid vectors. Then, these cloned plasmid vectors will be inserted into competent cell by electro-poration. Cloned colonies can check by selecting using antibiotic marker. Then, extracts plasmid and detects insert size and region. These regions have no influence of incompatibility. Thus, in the fail case, non- injectable sequence will be cut into half size and can be retried separately. After a repetition of this rechecking process, non-injectable region can be detected. Thorough these investigation, we can make the injectable region map and non-injectable region map. Non-injectable region map shows the influence of incompatibility, therefore, both map will be very helpful at the time when real artificial genome will be inserted. This protocol is very similar to constructing of genome library.
For the reasons stated above, we decided to construct Escherichia coli W31104 Genome BAC library.
4: In our project, artificial genome is designed from Escherichia coli K12 W3110 genome
sequence.
MATERIALS AND
METHODS
There are two types of insert DNA preparation method. One is complete digestion using rare base cutter enzyme like NotI. And the other one is partial digestion using common enzyme like BamHI. Each method has merit and demerit. The complete digestion can predicted DNA fragment size, and region so insert DNA can be check by restriction enzyme map, but it can’t cover all of the genome region and, can’t chose wanted fragment size. On the other hand, partial digestion can cover all of a genome region randomly, and can align wanted fragment size, but it can’t predict DNA fragment size and region, so it can’t check by restriction enzyme map. Thus, partial digestion condition is so sensitive and not constant, so insert DNA making process is difficult.
This report is divided into two sections, BAC library using complete digestion, and BAC library using partial digestion.
BAC library using complete digestion
Construction of BAC
libraries by
complete digestion is outlined in FIG.2 using pBeloBAC11 vector(see FIG.3).
First the vector is digested with NotI,
and then dephosphorylated to prevent self ligation(see FIG.7). Next, high
molecular weight DNA is completely digested with NotI, and each DNA fragment is size-selected on a CHEF gel(see FIG.6).
Finally,
the vector and genomic DNA are ligated and then electroporated into E. coli. The most widely used E. coli strain for BAC cloning
is
DH10B(Hanahan etal, 1991). Key features of this strain include mutations that
block: (1)restriction of foreign DNA by endogenous restriction
endonucleases(hsdRMS); (2) restriction of DNA containing methylated DNA (5'
methylcytosine or methyl adenine residues,and 5' hydroxymethyl cytosine)
(mcrA,mcrB, mcrC, and mrr); 3) recombination (recA1). Recombinant transformants
are selected on media containing chloramphenicol, After recombinant transformants are detected, their size is
assayed by a simple DNA mini preparation followed by digestion with NotI to free the DNA insert from the vector, and conventional
electrophoresis, if transformants have long size of insert DNA, use CHEF electrophoresis. Cloned
library is checked by Restriction enzyme map(See FIG.8) and DNA sequencing(data
is not shown).
FIG.
3 A
map of the pBeloBAC11 vector. The primary characteristic of pBeloBAC11 is the
addition of a lacZ gene into the
multiple cloning site of pBAC108L. On plates supplemented with X-gal/IPTG, an
intact lacZ gene encodes b-galactosidase which catalyses the supplemented
substrate into a blue substance. Successful ligation of insert DNA into the
vector inactivates lacZ; hence white
colonies indicate the presence of a successful vector-insert ligation. However,
pBeloBAC11 is still a low-copy number plasmid from the presence of parA and parB.
BAC
library using partial digestion
Mostly same as complete digestion
protocol, except enzyme(NotI--->BamHI), size selection step(each
size--->Average 150kb ), selection plate(Cm---> Cm/Xgal/IPTG). The
construction of BAC libraries by partial digestion is outlined in FIG.4 using
pBeloBAC11(see FIG.3). First the vector is digested with Bam HI and then dephosphorylated to prevent self ligation. Next,
high molecular weightDNA is partially digested wit Bam HI and DNA >150 kb is size-selected on a CHEF gel. Because
small DNA fragments are trapped during the first size-selection(Woo et al,
1994) found that it is essential to perform two such size-selections to
increase the average insert size of BACs. Finally, the vector and genomic DNA
are ligated and then electroporatedinto E. coli. The most widely used E. coli strain for BAC cloning
is DH10B.
Recombinant transformants are selected on media containingchloramphenicol,
X-Gal, and IPTG. After recombinant transformants are detected, their size
is assayed by a simple DNA mini preparation followed by digestion with BamHI to free the DNA insert from the
vector, and CHEF electrophoresis. Cloned library is checked by only DNA
sequencing.
Currently,(2005.Jan.28th), partial digestion method is under
way, this report introduces just partial digestion condition test result(see
FIG.9). Thus, partial digestion has possibility to apply high-through-put
method.
FIG. 4 An outline of construction
of BAC libraries by partial digestion.
RESULTS AND
DISCUSSION
BAC library using complete digestion
Inhibitor
check
Last term, a prototype protocol of E.coli genome BAC library for searching the injectable genome region has been developed[11]. In summer, these clones have been affirmed through restriction enzyme map and DNA sequencing. Tough, they have only short genome fragments. It’s seems to have been about 150kb, but indeed, they have only about 20kb fragments. Some reagent were contaminated by some kinds of inhibitor, therefore, incomplete insert check results showed not monomer (20kb) but polymer (150kb).(See FIG.5) The reason why contaminated some inhibitors into reagents is not detected. Although, about 15kb genome DNA fragments were successfully cloned.
FIG.
5 Inhibitor check result by NotI digestion for insert DNA size
check. Compare between left 3
samples with new reagents(enzyme, buffer, and DDW ), and right 3 samples with
old reagents(enzyme, buffer, and DDW). Left side samples can cut correctly to
free insert DNA(15kb) as monomer. Right side samples can’t cut because of some
kinds of inhibitor, so it can be seen
dimmer(150kb) and trimmer.(300kb).
Gel extraction
After reexamination of last-term prototype protocol, we decided to optimize this protocol for getting long size fragments(>150kb). The main optimized points are as follows. (1)To prepare genome DNA embedded in agarose, which can protect large size of DNA fragments from pyshical damage. (2)At the insert DNA fragment preparation step, add gel extract step. This step may promote to get large size of DNA fragments, by selecting DNA size before ligation.
FIG.6 shows predicted DNA
fragments on CHEF gel. Genome DNA fragments are predicted by analysis software
GENETYX[6].
FIG.
6 Insert DNA fragment
selection on CHEF gel. Genome DNA was completely digested with NotI, so that Each A~E area contains
predicted DNA fragments.
Insert check
FIG.7 shows insert DNA fragments size by NotI digestion. As a result, there are no clones, which have large size of DNA fragments. This reason may thought be as follows.
(1) Need
more gently treatment to avoid pyshical shearing for large size of DNA
fragment. (2) Need more time to dissolve the large size of DNA fragment after
ethanol precipitation. This procedure can make large DNA fragments
concentration levels up. Although,
compare with FIG.7-A and FIG. 7-B, cloned efficiency was successfully promoted.
FIG.
7 Comparison of cloned efficiency between non-optimized protocol (A) and optimized protocol (B). Both (A) and (B) are NotI digestion insert check analysis by conventional
electrophoresis; Black point indicates successfully cloned sample.
Restriction enzyme map
FIG. 8 shows a restriction enzyme map for confirming cloned sequence. Compare with predicted restriction map and real restriction map, several clones were successfully confirmed(NotI digested genome fragment: about 15kb). We also confirmed by DNA sequence(data is not shown).
FIG.
8 Restrict
enzyme map of a cloned transformant. Compare with predicted restriction enzyme
map using analysis software GENETYX [6] (data is
not shown).
BAC library using partial digestion
Partial digestion test
Currently, partial digestion method is under way, in parallel. FIG.9 shows the results of partial digestion condition test.
FIG.
9 Genome Partial digestion
condition test result. by CHEF electrophoresis. The middle lane shows a best
condition of partial digestion of E.coli genome
embedded in agarose plug. Successfully cut into around 100kb to 200kb DNA
fragments.
As a result, 3units of enzyme for 1plug and, 20min
reaction is the best condition. Thus, partial
digestion has possibility to apply high-through-put method. BamHI sites of pBeloBAC11 is on the lacZ
gene(see FIG3), so bule white selection can be used. Also, colony picking
machine with 96-well plate and, high-through-put BAC clone extraction kit
R.E.A.L [9] can speed up the construction of BAC library.
ACNOWLEDGMENTS
We are grateful to Mr.
Yoichi Nakayama, Prof. Mitsuhiro Itaya, Mr. Tomoya Baba, and, Ms, Azusa Kuroki
for scientific discussion and technical supports. We also acknowledge our
project member Ms. Hiromi Komai, Mr. Kenji Higashi, and, Prof. Masaru Tomita
for his comprehensive supports.
REFERENCES
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