Team:NYMU-Taipei/ymis3.html

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Revision as of 14:48, 17 October 2012

NYMU iGEM

Experiment Design

Gene Cloning of Cys I Sulfite Reductase

Firstly, we get the whole gene sequence of Cys I from NCBI web (www.ncbi.nlm.nih.gov/gene/878581). Cys I contain endogenous EcoRI and PstI site, so we can’t clone into PSB1C3 directly.As a result, We create a noval cassette- new pSB1C3( Mfe I-Xba I -pSB1C3-Sbf I-Spe I) for easily cloning. Taking advantage of MfeI and EcoRI are compatible, also SbfI and XbaI, we can easily clone Cys I gene into pSB1C3 standard biobrick. Enzyme check by XbaI and SbfI, we can get ~1700bp band, which means our gene constraction is correct!!



In order to establish stable expression cyanobacteria system, we need to subclone our Cys I into stable expressing plasmid, pTrc-kan plasmid (constructed by Prof. Chang). pSB1C3-Cys I cut with MfeI and SPeI. pTrc-kan cut with EcoRI and XbaI. Finally we can get ~7100bp plasmid, pTrc-kan-Cys I gene. Enzyme check by SpeI and get ~2100bp and ~3000bp bands.


pTrc-kan-Cys I plasmid



Biobrick of Cys I sulfite reductase

Gene Cloning of Dsr Sulfite Reductase

As same as Cys I gene, we get the whole gene sequence of Dsr I and Dsr II gene
from NCBI web (http://www.ncbi.nlm.nih.gov/protein/CAC09931.1). DsrI and DsrII also contain endogenous EcoRI and PstI site, so we clone them into noval cassette- new pSB1C3( Mfe I-Xba I -pSB1C3-Sbf I-Spe I). We create a BamH I site inside of DsrI and DsrII, in order to combine them into a whole part, “Dsr”. Enzyme check by XbaI and Spe I , we can get ~5800 bp, which means our gene constraction is correct!! Subclone our Dsr into stable expressing plasmid, pTrc-kan plasmid (constructed by Prof. Chang). pSB1C3-Dsr cut with MfeI and SPeI, pTrc-kan cut with EcoRI and XbaI. Finally we can get ~9800bp plasmid, pTrc-kan-Dsr gene. Enzyme check by BamHI, we can ~1200bp.




Biobrick of Dsr Sulfite Reductase