Difference between revisions of "Part:BBa K079045"

Revision as of 09:30, 29 October 2008

Lac operator library

Parts K079045 through K079048 are libraries intended to introduce standardization and modularity for operator sequences. Each of the 4 collections has a different repressor specificity (Lac, Tet, Lambda and LexA) and consists of 3 different members with distinct binding affinities. Since operators flanked by Biobrick ends are unpractical and costly to obtain, both by dna sinthesys and phosphorylated oligos, we designed a "Standard Collection Vector".

This layout allows to get multiple small sequences with a single synthesis process and specifically enable the extraction of one or a combination of operators using exclusively Biobrick prefix and suffix restriction enzymes. Digestion and religation of the collection vector with Xba and Pst yields a single Operators 1 and Operator 3 vectors respectively (Es: Lac2 and Lac Symmetric) avoiding the troubles and optimization related to the run and purification of very small bands on agarose gel.

A similar approach using EcoRI give rise to a multiple Operator Vector comprising Operator 1 and Operator 2 sequences, usable per se as a composite regulatory site or further restrictable with PstI to extract the single Operator 2 (Es: Lac1)

The Libraries have been syntetized by Geneart in their standard pGA18 and pMA synthesis vector, with both ColE1 high copy number origin of replication and Ampicillin resistance.

Right now we're ultimating the design and testing of an equally simple protocol to transfer operator sequences in Biobrick Standard Assembly Vectors using standard cloning reagents and avoiding as much as possible experimentally fragile or optimization dependent steps.

Sequence and Features