Team:Evry/Interlab Study/Used Devices

From 2014.igem.org

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<li> Required device 1: originally this device was carried by PSB1K3 and was sud-cloned onto PSB1C3.
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<li> Required device 1: originally this device was carried by PSB1K3 and was sud-cloned onto PSB1C3.</li>
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<li> 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.
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<li> 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 1.</li>
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    To test the expression of promoters, they are cloned into the PSB1C3 vector followed by the  
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<i> Figure 1: Building way and analyse schema of the required device 2, 3 and the 19 constructions.
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    <a id="igem-link" target="_blank" href="http://parts.igem.org/Part:BBa_E0240" title="Go to iGEM site">BBa_E0240</a>, a GFP generator. Constructions are inserted in E. coli DH5 alpha that grown on LB medium added with the selection agent, chloramphenicol.
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<br>There were 2 constructions steps:
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<li>Digestion of PSB1C3 carrying E0240 via EcoRI and XbaI<li>
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<li>Digestion of  carrying E0240 via EcoRI and XbaI<li>
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Revision as of 20:16, 17 October 2014

IGEM Evry 2014

Interlab study - Used devices

Used Biobricks and plasmids



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Plasmid map 1: PSB1C3 map with restriction sites (NotI, BsaI, EcoRI, PstI, SpeI and XbaI)

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Plasmid map 2: BBa_J61002 plasmid map with restriction sites and J23101 promoter ( EcoRI, PstI, SpeI and XbaI)

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Anderson collection promoter sequences and mutations.
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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.

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 1.

IMAGE

Figure 1: Building way and analyse schema of the required device 2, 3 and the 19 constructions.
There were 2 constructions steps:
  • Digestion of PSB1C3 carrying E0240 via EcoRI and XbaI
  • Digestion of carrying E0240 via EcoRI and XbaI

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 .

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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.

Contacts

Retrieved from "http://2014.igem.org/Team:Evry/Interlab_Study/Used_Devices"