C++Guns – RoboBlog


Biologie++ - Silver Assembly

Filed under: Allgemein — Tags: — Thomas @ 14:03

Im Glas rekombinationsmthoden haben eine Einschritt Erstellung von großen DNA Sequenezen
von mehreren einzelnen ermöglicht. Obwohl synthetische biologische Schaltungen im Prinzip
auf der selben Art erstellt werden können, enthalten sie normalerweise sich wiederholende Sequenzteile,
wie promoters und terminator, die bei der Recombination stören.
Wir benutzen ein Computer gestützten Ansatz um biologische, synthetische inaktive unique nucleotide
sequences (UNSes) zu designen, die das genaue Erstellen erleichtern.
Wichtig: unsere designten UNSes machen es möglich Teile zu assemblieren die wiederholende
terminator und insulator Sequenzen enthalten. Und damit isolierte, funktionale genetische Schaltungen
in Bakterien und Säugetierzellen zu erstellen.

In vitro recombination methods have enabled one-
step construction of large DNA sequences from
multiple parts. Although synthetic biological
circuits can in principle be assembled in the same
fashion, they typically contain repeated sequence
elements such as standard promoters and termin-
ators that interfere with homologous recombination.
Here we use a computational approach to design
synthetic, biologically inactive unique nucleotide
sequences (UNSes) that facilitate accurate ordered
assembly. Importantly, our designed UNSes make it
possible to assemble parts with repeated terminator
and insulator sequences, and thereby create
insulated functional genetic circuits in bacteria and
mammalian cells. Using UNS-guided assembly to construct
repeating promoter-gene-terminator parts, we systematically varied gene expression to
optimize production of a deoxychromoviridans bio-
synthetic pathway in Escherichia coli. We then used
this system to construct complex eukaryotic AND-
logic gates for genomic integration into embryonic
stem cells. Construction was performed by using
a standardized series of UNS-bearing BioBrick-
compatible vectors, which enable modular assembly
and facilitate reuse of individual parts. UNS-guided
isothermal assembly is broadly applicable to the
construction and optimization of genetic circuits
and particularly those requiring tight insulation,
such as complex biosynthetic pathways, sensors,
counters and logic gates.

[1] Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Silver et al.

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