Difference between revisions of "Part:BBa K079045"
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Revision as of 20:22, 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.
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TET LIBRARY |
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TET LIBRARY |
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LAMBDA LIBRARY |
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LEXA LIBRARY |
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Right now we're ultimating the design and testing of an equally [http://2008.igem.org/Team:Bologna/Wetlab#Operator_site_cloning_in_standard_plasmids 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]