Team:NYMU-Taipei/ymis3.html

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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!!


Enzyme check of our plasmid shows that gene construction 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 construction



Biobrick of Cys I sulfite reductase, BBa_K896001

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, BBa_K896002

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