Difference between revisions of "Team:Grenoble-Alpes/construction"

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<p>The BBa_J04450 biobrick is composed of a RFP gene which is controlled by a LacI promoter inducible by IPTG. Moreover, it has a resistance gene for chloramphenicol to be able to select only bacteria that have been transformed.</p>
  
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<figure><center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Grenoble-Alpes--select_figure4.png"><figcaption>Figure 2: Organisation of the backbone (vector) BBa_J04450 for the cloning.    </figcaption></center></figure>
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<p>Several teams showed that BBa_J04450 LacI promoter leaks. <a href="https://2017.igem.org/Team:Grenoble-Alpes/LabBook">iGEM Grenoble 2017 </a> (https://2017.igem.org/Team:Grenoble-Alpes/LabBook) highlights the role of IPTG, at least 180 minutes after induction. IPTG appeared to be important in the detection because it triggers a shorter rise of the fluorescence. As we know, it triggers the transcription of lac operon inducing the expression of the reporter gene.</p><p>
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So BBa_J04450 was chosen because this iGEM part enables users to produce mRFP1, a fluorophore which is an engineered mutant of red fluorescent protein from Discosoma striata. This reporter gene is LacI sensitive and can be induced with IPTG. This biobrick was chosen to be able to see fluorescence even if no IPTG was added or adding it to have a more rapid kinetic production.</p>
  
  

Revision as of 20:43, 8 October 2018

Template loop detected: Template:Grenoble-Alpes

PROBE CONSTRUCTION

PSEUDOMONAS AERUGINOSA DETECTION

LYSIS

1. Plasmid backbone selection

Among all the backbones proposed by iGEM, we decided to choose one for our probe insertion. In fact, the 2017 IGEM Grenoble team showed by their experiments, that the pSB1C3-BBa_J04450 backbone was the most suitable for the detection of a red fluorescence.


Figure 1: Backbone tested for hybridization of the target to the probe detecting bacterial lysis

The BBa_J04450 biobrick is composed of a RFP gene which is controlled by a LacI promoter inducible by IPTG. Moreover, it has a resistance gene for chloramphenicol to be able to select only bacteria that have been transformed.


Figure 2: Organisation of the backbone (vector) BBa_J04450 for the cloning.

Several teams showed that BBa_J04450 LacI promoter leaks. iGEM Grenoble 2017 (https://2017.igem.org/Team:Grenoble-Alpes/LabBook) highlights the role of IPTG, at least 180 minutes after induction. IPTG appeared to be important in the detection because it triggers a shorter rise of the fluorescence. As we know, it triggers the transcription of lac operon inducing the expression of the reporter gene.

So BBa_J04450 was chosen because this iGEM part enables users to produce mRFP1, a fluorophore which is an engineered mutant of red fluorescent protein from Discosoma striata. This reporter gene is LacI sensitive and can be induced with IPTG. This biobrick was chosen to be able to see fluorescence even if no IPTG was added or adding it to have a more rapid kinetic production.