BTN Protocol Guide-08

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nsion of combinatorial cloning
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ast date of revision

eptember 2007
V

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IBA Headquarters
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IBA GmbH
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n Center| Cloning
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StarGate

®
– Combinatorial Cloning in One Tube
Introduction The Technology
Efﬁ cient procedures to generate functional recombinant proteins StarGate
®
is a technology that allows the systematic combination of
or protein complexes are of key importance in state-of-the-art life promoters (i.e., hosts), puriﬁ cation tag sequences or other genetic
sciences. Many tools like various expression hosts (bacteria, yeast, elements with any gene of interest (GOI) in a convenient cloning
insect and mammalian cells), promoters, afﬁ nity or ﬂ uorescent system. The core element of this new technology is the site-speciﬁ c
tags are currently available to express, purify, detect or immobilize combinatorial enzyme formulation StarCombinase
TM
, that makes
recombinant proteins. Due to the diverse nature of proteins, cloning versatile, fast, easy and safe.
however, it is impossible to predict which combination of these tools
will perform best. Therefore, many have to be tried to identify the
optimal solution.
To systemize and accelerate this initial search which is crucial for
successful subsequent proteomic research, we have developed the
Step 1:
StarGate
®
Cloning System. StarGate
®
offers rapid and highly efﬁ cient
Donor Vector generation
subcloning of an arbitrary gene – initially cloned into a Donor Vector
In a first step, the gene of interest (GOI) is
– to simultaneously fuse it with many different genetic surroundings
equipped at both ends with combinatorial
via transfer into Acceptor Vectors to generate Destination Vectors. sites (4 bases) by PCR and is inserted into an
The latter enable the efﬁ cient expression of your protein with various
Entry Vector by a simple one-tube reaction.
features (e.g., different tags and different promoters) in different
hosts.
The StarGate
®
Top Beneﬁ ts
• One-tube subcloning procedure (1 hour)
Step 2:
Destination Vector generation
• Cutting bands out of gels becomes superﬂ uous
After sequence confirmation, the resulting
• Extremely short combinatorial sites (4 bases only) have minimal
Donor Vector is the origin for the highly
effect on the gene of interest
parallel subcloning of GOI into a multitude
• Improved screening of different host/tag combinations of Acceptor Vectors, each providing a
• Acceptor Vectors for different hosts with correlating features different genetic surrounding like host
always yield exactly the same protein sequence
specific promoters and different purification
• Highest level cloning efﬁ ciency due to a directed reaction (no
tags, by a second simple one-tube reaction.
equilibrium)
The resulting Destination Vectors are then
• Once Donor Vector is sequenced no further veriﬁ cation is
transformed into the corresponding host cells
for further experiments.
required
Further Useful Features
• StarGate
®
Fusion Cloning System, e.g., for generation of fusion
proteins or of synthetic operons for expression of protein
complexes
• Versatile StarGate
®
Mutagenesis System “StarChange”
• Free StarPrimer D’Signer Software 2.0
• Royalty-free License Policy “OpenGate
®
”
• Custom StarGate
®
Services
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 17
StarGate_short_Protocol 17 10/25/07 6:52:18 PM
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