"
Team:HokkaidoU Japan/RBS/Methods
From 2013.igem.org
(Difference between revisions)
| (4 intermediate revisions not shown) | |||
| Line 3: | Line 3: | ||
<div id="common-header-bottom-background"> | <div id="common-header-bottom-background"> | ||
<div class="wrapper"> | <div class="wrapper"> | ||
| - | <h1 id="common-header-title">Maestro E.coli</h1> | + | <h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1> |
<h2 id="common-header-subtitle">RBS</h2> | <h2 id="common-header-subtitle">RBS</h2> | ||
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"> | <img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"> | ||
| Line 17: | Line 17: | ||
<p> | <p> | ||
We constructed new RBS family, SD2, SD4, SD6, SD8. | We constructed new RBS family, SD2, SD4, SD6, SD8. | ||
| - | These RBSs have | + | 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 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). | We constructed SD2, SD4, SD6 by PCR (forward:EX-f, reverse:SD2-r, SD4-r, SD6-r, template:SD8). | ||
| Line 24: | Line 24: | ||
<div class="fig fig800"> | <div class="fig fig800"> | ||
<img src="https://static.igem.org/mediawiki/2013/a/ab/HokkaidoU_RBS_methods1_800.png"> | <img src="https://static.igem.org/mediawiki/2013/a/ab/HokkaidoU_RBS_methods1_800.png"> | ||
| - | <div>fig.1: oligos | + | <div><span class="bold">fig.1: oligos.</span> RED: enhancer sequence, BLUE: SD sequence.</div> |
</div> | </div> | ||
<div class="fig fig400 para"> | <div class="fig fig400 para"> | ||
<img src="https://static.igem.org/mediawiki/2013/9/9e/HokkaidoU_RBS_methods2_400.png"> | <img src="https://static.igem.org/mediawiki/2013/9/9e/HokkaidoU_RBS_methods2_400.png"> | ||
| - | <div>fig.2: RBS construction</div> | + | <div><span class="bold">fig.2: RBS construction.</span></div> |
</div> | </div> | ||
| Line 35: | Line 35: | ||
<div class="fig fig400 para"> | <div class="fig fig400 para"> | ||
<img src="https://static.igem.org/mediawiki/2013/1/1a/HokkaidoU2013_RBS_methods3revision_400.png"> | <img src="https://static.igem.org/mediawiki/2013/1/1a/HokkaidoU2013_RBS_methods3revision_400.png"> | ||
| - | <div>fig.3: our parts</div> | + | <div><span class="bold">fig.3: our parts.</span></div> |
</div> | </div> | ||
<div class="clearfix"></div> | <div class="clearfix"></div> | ||
<h2>Assay</h2> | <h2>Assay</h2> | ||
<p> | <p> | ||
| - | We ligated TetR repressible promoter ( | + | We ligated TetR repressible promoter (pTet), each of the new RBSs', LacZα and double terminator. |
Using this construct we performed β-Galactosidase assay. | Using this construct we performed β-Galactosidase assay. | ||
</p> | </p> | ||
Latest revision as of 02:52, 29 October 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.
