Team:Nanjing China Bio/results

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All parts that we make consist of an anaerobic promoter and a luciferase gene. The luciferase gene was cloned from the iGEM part K387009, which has a RBS region, luciferase gene and two terminals.
There are 17 anaerobic promoters cloned in two different ways. Firstly, we ask 8 anaerobic promoter from iGEM. Secondly, we cloned 9 anaerobic promoters from BL21 genomic DNA , one E.coli strain. The information of these promoters is from the paper Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant Escherichia coli, and thanks to their great work.
The anaerobic promoters from iGEM parts are listed below

Parts Name Length Plasmid Resistance
BBa_K387003 PfdhF: fdhF promoter, a hypoxia inducible promoter 99bp pSB1C3 C
BBa_K404106 phTERT promoter 459bp pSB1C3 C/C
BBa_K227007 puc promoter 651 bp pSB1K3 K
BBa_K376003 J6 Oxygen Sensitive Promoter 83 bp pSB1C3 C
BBa_K239005 NarK promoter, Fnr activated under anaerobic conditions 139 bp pUC57 A
BBa_K239006 Modified NarK promoter 89 bp pUC57 A
BBa_K258005 Oxygen promoter-Vitreoscilla hemoglobin(VHb) promoter in E. coli. 137 bp pSB1A2 A
BBa_J61043 pBca1020-r0040 pSB1A2 A

The promoters that are cloned from BL21 are linked with luciferase. The parts that we made are listed below.
parts name plasmid resistance
BBa_K905001 Padhe pSB1C3 C
BBa_K905000 PnirB pSB1C3 C
BBa_K905002 PgadA pSB1C3 C
BBa_K905003 PgadA pSB1C3 C
BBa_K905004 PhdeA pSB1C3 C
BBa_K905005 PhdeD pSB1C3 C
BBa_K905006 PldhA pSB1C3 C
BBa_K905007 PpflA pSB1C3 C
BBa_K905008 PftnA pSB1C3 C
BBa_K905009 ParcA pSB1C3 C

Then, we link hypoxia-induced nirB promoter to the plasmid pSB1C3, because we want more iGEMers can use the promoter to do their research.
Then we put plasmids that each has an anaerobic promoter and a luciferase gene into VNP20009 by electroporation. Then we tested the strength and anaerobic sensitivity of our promoters by measuring the fluorescence strength of each VNP strain that has a different promoter after the anaerobic culture.