Regulatory

Part:BBa_K079048

Designed by: Francesca Ceroni   Group: iGEM08_Bologna   (2008-10-25)

LexA 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". 320x240

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.


LacPlasmid.jpg
LAC LIBRARY
Lac1
Lac2
Lac SymL
Tet.jpg
TET LIBRARY
TeT O
TetO-4C
TetO-wt/4C5G
CI.jpg
LAMBDA LIBRARY
Lambda OR1
Lambda OR2
Lambda OR3
LexLib.jpg
LEXA LIBRARY
LexA 1
LexA 2






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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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