Gibson Assembly or Gibson Cloning is a method for seamless ligation of multiple sequences in a single reaction, without the need for restriction sites. The Gibson Assembly operation allows you to simulate cloning reactions that use an exonuclease to generate overlapping fragments for ligation, including Gibson Assembly, GeneArt® Seamless Cloning (5’ exonuclease), SLIC and In-Fusion® Cloning (3’ exonuclease). It can also be used to simulate cloning operations that do not use exonuclease such as CPEC and SLiCE. For an overview of the Gibson Assembly interface, see section 14.4 .
To run a Gibson Assembly operation, optionally select a Backbone sequence, along with one or more Insert sequences, and then specify the type of exonuclease you wish to use for the reaction. You can adjust the order in which the sequences will be ligated in the Construct Layout Panel. Currently the Gibson Assembly operation can only be run using linear sequences.
Batch cloning with alternate sequences can be performed using sequence lists. Each of the individual nucleotide sequences in a list will be inserted into a separate product at the same insert position.
Exonuclease: The exonuclease activity deﬁnes which strand (if any) gets digested to expose complementary overhangs:
If there are existing, compatible, overhangs annotated on the selected sequences (e.g. those derived from restriction digest), they will be used for the ligation reaction. If existing overhang annotations are not compatible, or do not meet the speciﬁed requirements, the overhangs will be removed or ﬁlled in (depending on the overhang and the exonuclease chosen) prior to primer design.
Primer Options: Here you can adjust parameters that inﬂuence creation of primers and overhangs:
For sequences without existing complementary overlaps on both ends, a pair of primers will be created to generate complementary overlaps via Primer Extension PCR, as required. If both ends are complementary no primers will be generated. Primer design uses non-stringent conditions which may result in poor quality primers - we recommend that you check the generated primers before ordering them.
Extensions will be added to the primer corresponding to the neighboring sequence, to generate complementary overhangs. Primers are generated only for insert sequences, as it is assumed that the vector should stay unmodiﬁed. For this reason the extension length of primers extending to the vector will be the full speciﬁed minimal overlap length, whereas extensions on primers between two inserts, will each be half of the total overlap length. If you wish to manually add modiﬁcations to primer extensions, these must be annotated onto the insert sequence as type ‘Gibson Primer Extension’, otherwise they will be included within the binding region.
The melting temperature (Tm) for the annealing region between the neighbouring sequences is calculated using the Tm characteristics setting in Primer Options. In many cases the Phusion DNA polymerase is used, for which it is recommended to use the Tm formula of Breslauer et al. 1986 (See section 13.1.3 ).
For very short or long extensions, Primer3 might be unable to calculate a Tm. If Primer3 fails, Geneious will calculate the Tm using formula 5.1 (for short sequences) or 5.2 .
The primers generated in your Gibson Assembly will be listed in the Report Document, along with the calculated characteristics and any errors that occurred during the primer generation process. Furthermore any modiﬁcations (recession or maintaining overhangs, adding extensions to primers) are shown at the beginning of the document.