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Team:Evry/Interlab Study/Used Devices
From 2014.igem.org
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<left> Anderson collection promoter sequences and mutations. </left> | <left> Anderson collection promoter sequences and mutations. </left> | ||
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<right>Promoter name, plate and well location into the iGEM distribution kit 2014, vector, resistance cassette, part size in bp, used primers and PCR product profile.</right> | <right>Promoter name, plate and well location into the iGEM distribution kit 2014, vector, resistance cassette, part size in bp, used primers and PCR product profile.</right> | ||
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<h2>Construction way</h2> | <h2>Construction way</h2> | ||
Revision as of 19:49, 17 October 2014
Interlab study - Used devices
Used Biobricks and plasmids
Construction way
Constructions were built using classical Biobrick restriction sites.
- Required device 1: originally this device was carried by PSB1K3 and was sud-cloned onto PSB1C3.
- Required device 2, 3 and the 19 constructions for the entire Anderson library: promoters were inserted into PSB1C3 carrying E0240. Process is described on following figure.
To test the expression of promoters, they are cloned into the PSB1C3 vector followed by the
BBa_E0240, a GFP generator. Constructions are inserted in E. coli DH5 alpha that grown on LB medium added with the selection agent, chloramphenicol.
Analysis protocol
Our team decided to test the fluorescence with a
TECAN infiniteM200 in 96 well plates. Each assay were performed after a 16 - 18 h preculture on 5 ml of LB medium at 37°C with 220 rpm agitation. Cultures were distributed 200 µl per well. Three strains per constructions were tested in five replicates. Two controls were added on plates: LB with chloramphenicol and the strain carrying the promoterless vector on LB with chloramphenicol.
Fluorescence data were analyzed via following formula that come from 2010 De Jong et al Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria .
Fluorescence data were analyzed via following formula that come from 2010 De Jong et al Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria .
Equation A: A(t) is the corrected absorbance of DH5 alpha with the functional reporter system, Au(t)the uncorrected absorbance of DH5 alpha with the functional reporter system and Ab(t)the background absorbance corresponding to LB with chloramphenicol medium absorbance.
Equation B: B(t) is the absorbance of the strain carrying the promoterless vector, Iu(t) the uncorrected fluorescence intensity of DH5 alpha with the functional reporter system and Ib(t) the background fluorescence intensity of LB chloramphenicol medium with the DH5 alpha carrying the promoterless vector.
Equation B: B(t) is the absorbance of the strain carrying the promoterless vector, Iu(t) the uncorrected fluorescence intensity of DH5 alpha with the functional reporter system and Ib(t) the background fluorescence intensity of LB chloramphenicol medium with the DH5 alpha carrying the promoterless vector.
