ECONEY Project

There is a unique phenomenon of E.coli activity called “Catabolite Repression”. Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes, even though the inducer of the pathway was present in the environment.

Glucose is known to repress a large number of inducible enzymes in many different bacteria. Glucose represses the induction of inducible operons by inhibiting the synthesis of cyclic AMP (cAMP), a nucleotide that is required for the initiation of transcription of a large number of inducible enzyme systems.

The role of cyclic a cAMP is complicated. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNAp polymerase to the promoter for the initiation of transcription. In the presence of glucose, cAMP levels are low, and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose)

CONSTRUCTING ECONEY

We found plasmid BBa_J04450 in iGEM Parts Registry that works according to catabolite repression phenomenon. It works in the presence of lactose by emitting red color from mRFP gene expression. mRFP gene expression will decrease in the presence of glucose. We use this plasmid to measure the glucose concentration.

Unfortunately, we faced a problem that in high glucose concentration, the red color from mRFP gene expression goes dimer, while we want the device to express brighter red color in high glucose concentration. Thus, we modified the system by changing sensitivity of catabolite repression in order to make the system has ability to express red color in high glucose concentration.

We add 2 new circuits behind plasmid BBa_J04450. The first circuit consists of constitutive promoter, RBS, Adenylate Cyclase (AC) gene, and a terminator. The second circuit consists of constitutive promoter, RBS, IIAglc gene, and a terminator.

ECONEY CIRCUITS

Our ECONEY consists of 2 circuits.

Circuit #1 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, AC gene, and a terminator. Adenylate Cyclase is an enzyme that can catalyse the shynthezise of cAMP and contain 848 amino acids. Adenylate cyclase will synthesize cAMP that can help lac promoter still active in high glucose concentration. Constitutive promoter is used to guarantee that AC gene will be always expressed. This circuit will allow the system to emit red color in high glucose concentration.

Circuit #2 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, IIAglc gene, and a terminator. IIAglc is one of sub unit of cytoplasmic enzyme that involve in Phosphotransferase system (PTS). Thus with even low glucose concentration outside the cell, more glucose may enter the cells.

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