Team:USP-UNESP-Brazil/Associative Memory/Experiments

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(Difference between revisions)
(Experiments of Multi Regulated Promoter)
(Test of Repression by cl434)
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===Test of Repression by cl434===  
===Test of Repression by cl434===  
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated.  By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence.  
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated.  By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence.  
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There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it.   
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it.   
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Assembly diagrams
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===Assembly diagrams===
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Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.
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To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.
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We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram.  Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram.  Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.

Revision as of 21:32, 22 September 2012