Team:HokkaidoU Japan/RBS/Methods

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Maestro E.coli

RBS

RBS family parts

We constructed new RBS family, SD2, SD4, SD6, SD8. These RBSs have Enhancer sequence (GCTCTTTAACAATTTATCA) and SD sequence (SD2:GG, SD4:GAGG, SD6:AGGAGG, SD8:TAAGGAGG). We constructed SD8 from synthetic oligos (forward:SD8-f, reverse:SD8-r). We constructed SD2, SD4, SD6 by PCR (forward:EX-f, reverse:SD2-r, SD4-r, SD6-r, template:SD8).

fig.1: oligos; RED: enhancer sequence, BLUE: SD sequence.
fig.2: RBS construction
fig.3: our parts

Assay

We ligated TetR repressible promoter (pTET), each of the new RBSs', LacZα and double terminator. Using this construct we performed β-Galactosidase assay.