In silico cloning - Bits.vib.be

Tutorial 4: Gateway cloning
The Workbench allows to perform in silico Gateway cloning, including Multisite Gateway cloning.
In the Gateway cloning system your gene of interest is inserted into an entry vector. The entry vector
contains attP recombination sites. To insert your gene of interest into the entry vector, you need
to create a PCR product with terminal attB sites, using primers containing a 25-base pair attB sequence,
plus four terminal G's. This PCR product can then be inserted in the entry vector using the
BP reaction, resulting in the formation of an entry clone (purple vector at the top) carrying the gene
of interst flanked by attL sites. Once the entry clone has been formed it is available to express your
gene in a variety of other destination vectors (red vector on the top) which contain attR sites using
an LR reaction. The result is an expression clone (red vector at the bottom) with your gene of interest
inserted in the destination vector.
The attB’s, attP’s, attL’s and attR’s are the key component of the Gateway system: they are the
restriction sites. All entry clones have attL's on both sides of their gene of interest. These attL's are
cut to form sticky ends by the Gateway recombination proteins. These sticky ends will then match
up with the sticky ends on a destination vector which contains attR restriction sites. This process is
called a LR reaction. The BP reaction is done similarly using other restriction sites. Note that the
LR reaction creates attB sites again in the expression clone: this makes the Gateway system totally
reversible. You can make more entry vectors from the expression clone using a BP reaction, in
which the attB sites recombine with attP's to form attL's and attR's.
The system has a high transformation efficiency since it uses both antibiotic resistance and the
ccdB gene, which yields a product that is toxic to E. coli, to ensure that only correct clones survive.
The three tools for Gateway cloning mimic the procedure followed in the lab:
1. attB sites are added to a sequence fragment
2. attB-flanked fragment is recombined in an entry vector (BP reaction) to construct an entry clone
3. target fragment from the entry clone is recombined into a destination vector (LR reaction) to
construct an expression clone.
For Multisite Gateway cloning, multiple entry clones can be created that can recombine in the LR
reaction. To perform these analyses in the CLC Main Workbench, you need to import donor and
expression vectors. These can be downloaded from Invitrogen’s web site and directly imported into
the Workbench: http://igene.invitrogen.com/products/selector/vectors.