Biology Reference
In-Depth Information
TABLE 14.1
Composition of the PURE System, adapted from
20
No.
Factor
No.
Factor
1
Ala RS
20
Val RS
2
Arg RS
21
MTF
3
Asn RS
22
IF1
4
Asp RS
23
IF2
5
Cys RS
24
IF3
6
Gln RS
25
EF-G
7
Gly RS
26
EF-Tu
8
His RS
27
ET-Ts
9
Ile RS
28
RF1
10
Leu RS
29
RF2
11
Lys RS
30
RF3
12
Met RS
31
RRF
13
Phe RS
32
Myokinase
14
Pro RS
33
Creatine kinase
15
Ser RS
34
NDK
16
Ser RS
35
PPiase
17
Thr RS
36
T7 RNA polymerase
18
Trp RS
37
Ribosome
19
Tyr RS
38
tRNAs mix
RS: tRNA synthetase; MTF: methionyl-tRNA transformylase; IF: initiation factor; EF: elongation factor;
RF: release factor; NDK: nucleoside-diphosphate kinase; PPiase: pyrophosphatase.
a first gene (under SP6 promoter), was transcribed by SP6 RNA polymerase. The gene
encoded for T7 RNA polymerase. Therefore, after translation, functional T7 RNA polymerase
was produced inside liposomes. In turn, T7 RNA polymerase started the transcription of the
second gene (under T7 promoter). The second gene encoded for a reporter protein, the GFP,
which was revealed fluorimetrically.
The second
22
deals with the problem of low permeability of the lipid vesicle membrane,
and consists of the expression of a pore-forming protein (the
266
-hemolysin) inside
liposomes. Thanks to the formation of the pore, the synthetic cell could be fed by low-
molecular-weight compounds added externally, so that the synthesis of a reporter protein
could be prolonged up to 100 hours, whereas in the absence of the pore, it generally stops
after a couple of hours. The size of the pore (molecular weight cut-off: 3 kDa), although
permitting the diffusive flow of small molecules inward and outward, did not allow the
escape of large internal macromolecules from the synthetic cell.
The third
23
deals with the replication of genetic information, and consists in the replication
of genetic material operated by a replicase produced in situ by a cell-free system. In
particular, RNA was taken as a genetic polymer. It encoded the sequence for Q
α
β
-replicase.
RNA was translated so that Q
β
-replicase was effectively produced inside liposomes. Once
β
formed, Q
produced the RNA complementary strand. The
latter was so designed that it could be translated into an enzyme (
-replicase
acting on RNA
-galactosidase) that was
revealed fluorimetrically. The RNA complementary strand could also act as a template for
Q
β
-replicase for producing the first RNA strand. In other words, the RNA and its
complementary sequences were both produced by Q
β
-replicase, which is an RNA-dependent
RNA-polymerase. This report demonstrates that a genetic polymer can produce an enzyme
that is able to replicate the genetic polymer.
β
The fourth example
24
deals with the synthesis of phospholipids inside lipid vesicles,
aimed at obtaining an autopoietic lipid production. In this work, the target was the
synthesis of phosphatidic acid, which occurs in two steps from simple precursors like
