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Team:Evry/Interlab Study/Results
From 2014.igem.org
Interlab study - Aim & Results
The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from the iGEM teams all around the world. This will permit to observe the result repeatability across technics, strains and teams.
The study is also extended to the entire Anderson library of constitutive prokaryotic promoters (J23100 to J23119). This library corresponds to 19 constitutive promoters containing some nucleotide mutations on the -35 and the -10, as shown figure x. The idea is to compare the expression strength of promoters according to mutations, from the maximum amount of fluorescence data in order to have significant results.
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. Our team decided to test the fluorescence with a TECAN infiniteM200 in 96 well plates.
The goal of the interlab study is to obtain GFP fluorescence data from the iGEM teams all around the world for about twenty constructions. This will permit to observe the result repeatability across technics, strains and teams.
There were two sub-parts in this study:
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. Our team decided to test the fluorescence with a TECAN infiniteM200 in 96 well plates.
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 .
There were two sub-parts in this study:
- The mandatory one: obtain fluorescence data for three specific genetic devices expressing GFP and compare them. (donner les constructions) (J23100 to J23119).
- The Extra Credit assignments: we choose to study the entire Anderson library of constitutive prokaryotic promoters. This library corresponds to 19 constitutive promoters containing some nucleotide mutations on the -35 and the -10, as shown figure x. The idea is to compare the expression strength of promoters according to mutations, from the maximum amount of fluorescence data in order to have significant results.
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. Our team decided to test the fluorescence with a TECAN infiniteM200 in 96 well plates.
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.
Required Devices
 The three required constructions shown various fluorescence intensity at OD 600 nm = 0.5, as shown on bar chart on the right. The same trend was observed between OD 600 nm = 0.1 to 0.8(Data shown on Interlab study notebook). Fluorescence intensity curve profile according to OD 600 nm corresponded to the expected profile for a constitutive promoter (Data shown on Interlab study notebook). |
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Entire Anderson library of constitutive promoters (J23100-J23119)
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