Team:HokkaidoU Japan/RBS/Methods

From 2013.igem.org

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<h2>RBS family parts</h2>
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<p>
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  We constructed new RBS family, SD2, SD4, SD6, SD8.
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  These RBSs have Enhancer sequence (GCTCTTTAACAATTTATCA) and SD sequence (SD2:GG, SD4:GAGG, SD6:AGGAGG, SD8:TAAGGAGG).
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  We constructed SD8 from synthetic oligos (forward:SD8-f, reverse:SD8-r).
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  We constructed SD2, SD4, SD6 by PCR (forward:EX-f, reverse:SD2-r, SD4-r, SD6-r, template:SD8).
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</p>
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<div class="fig800">
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  <img src="https://static.igem.org/mediawiki/2013/a/ab/HokkaidoU_RBS_methods1_800.png">
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  <div>Fig.1: oligos; RED: enhancer sequence, BLUE: SD sequence.</div>
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</div>
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<div class="fig800">
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  <img src="https://static.igem.org/mediawiki/2013/9/9e/HokkaidoU_RBS_methods2_400.png">
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  <div>Fig.2: RBS construction</div>
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</div>
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<div class="fig400">
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  <img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_RBS_methods3_400.png">
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  <div>Fig.3: our parts</div>
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</div>
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<h2>Assay</h2>
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<p>
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  We ligated TetR repressible promoter (pTET), each of the new RBSs', LacZ&alpha; and double terminator.
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  Using this construct we performed β-Galactosidase assay.
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</p>

Revision as of 03:28, 27 September 2013

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.

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