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From 2011.igem.org

Contents 1 Construct 2 High concentration Mechanism 3 Low Concentration Mechanism 4 Photoreceptor

Construct

The construct combining the three previously mentioned plasmids, the green receptor activates the expression of the RecA final product, this RecA protein binds into the operators, allowing the expression of Pbad´s, in presence of arabinose, depending in the concentration of arabinose, if there is a high concentration, the low concentration plasmid is inactivated by the iTast. If there is a low concentration, the plasmid will activate de Pbad´s and the keys and anti-keys to avoid the expression of both fluorescent proteins.

High concentration Mechanism

This plasmid works almost as the same as the low concentration mechanism, also the presence of RecA unfolds the loop for PBad, in this case has a low affinity (PBadwk), then also expresses the key (Tawk) for the lock (Crxwk) but the difference in this mechanism is the production of an anti-key (itast) this anti-key prevents the binding of the Tast of the low concentration mechanism preventing the expression of GFP. Also the GFP is actually GFP-LVA tag, this tail allows the rapid degradation of the GFP proteins.

Low Concentration Mechanism

This Plasmid contains a constitutive promoter for the expression of the lambda repressor which binds into the OR1,OR2 ,OR3 building a loop, this loop avoids the expression of the pBad St, which in presence of arabinose promotes the expression of the next biobricks, the lambda repressor is removed from the operators (OR1, OR2, OR3), when this loops unfolds allows the expression of pBADst, then the expression of Tast is complementary to the Crxst, after binding, allows the expression of the RBS for the expression of GFP.

Photoreceptor

The green receptor acts in the presence of a specific wavelength; we see this specific wave as green light. After receiving the green light the plasmid product would be RecA.

Green-absorbing form (Pg λmax 535 nm)

GREEN RECEPTOR: Is going to be used to initiate the mechanism that was developed, this using green light.

The constitutive promoter is used for the gene to be expressed always, it is attached to a ribosome binding site (RBS) that is a sequence on the messenger RNA to which the ribosome can bind and initiate protein translation and produce the ho1 (heme oxigenase 1) that converts heme to biliverdin IXalpha.

• Another RBS that initiates the protein translation to produce pcy4A (phycocyanobilinferredoxin oxidoreductase) that allows the convertion of the biliverdin IXalpha to phycocyanobilin.
• Other RBS is used to produce CcaS-EnvZ which is a chimeric between ScaS and the EnvZ protein light responsive domain. CcaS-EnvZ is the green light receptor that induces the expression of phycobilisome linker protein.
• Using green light OmpR (a response regulator for osmoregulation that regulates production of outer membrane proteins) will be generated in order to activate de OmpR (+) promoter that will allow the expression of a certain protein (we don’t know what protein yet) that in some way will activate the expression of the promoters for arabinose concentration.