Team:UANL Mty-Mexico/Project/Circuit

Once explained the mechanism through which the community processes the information, it is time to have a look at the whole genetic circuit. A brief description of each figure is provided in sake of clarity.

Most of the circuit is made up of biobricks, listed at the bottom of the page. However, some of the parts are yet to be submitted to the registry of biological parts.

Red light induced, green light repressed. Red light induces cI's expression as well as PAI auntoinducer synthetase (QS1 producer) from pTetMnt and pMnt, respectively. cI can activate rather pRM or pRM434, depending on its concentration. pRM transcribes GFP and cI434, the latter repressing pRM434. The community then enters state 1. As [cI] increases, pRM gets repressed and pRM434 is allowed to be activated by cI; YFP is then transcribed from pRM434. That would mean state 2.

In the dotted box, green light-activated HHL auntoinducer synthetase from cell two which produces HHL molecules (QS2). RhIR is constitutively produced in cell one. When QS2 molecules reach cell one, they bind to RhIR and the complex activates transcription of TetR and cI434 from pRhIR/HHL. TetR represses pTetMnt inhibiting cI's further transcription; cI434 represses pRM434 enforcing cell's one off state. Note that aisPAI does not get repressed by green light. The λ operators OL and OR, spaced ∼2.4 kb apart, allow DNA looping required for the biphasic switch .

Green light induced, red light repressed. Remember cells one and two share a very similar mechanism. Green light induces cI's expression as well as HHL auntoinducer synthetase (QS2 producer) from pCpcG2/TetR and pCpcG2, respectively. cI can activate rather pRM or pRM434, depending on its concentration. pRM transcribes RFP and cI434, the latter repressing pRM434. The community then enters state 3. As [cI] increases, pRM gets repressed and pRM434 is allowed to be activated by cI; BFP is then transcribed from pRM434. That would be state 4.

In the dotted box, red light-activated HHL auntoinducer synthetase from cell one which produces PAI molecules (QS1). LasR is constitutively produced in cell two. When QS1 molecules reach cell two, they bind to LasR and the complex activates transcription of TetR and cI434 from pLasR/PAI. TetR represses pCpcG2/tetR inhibiting cI's further transcription; cI434 represses pRM434 enforcing cell's two off state. Note that aisHHL does not get repressed by red light. The λ operators OL and OR, spaced ∼2.4 kb apart, allow DNA looping required for the biphasic switch .

Green AND Red Light induced. When both lights are on, cells one and two repress each other but keep producing QS molecules (through aisPAI and aisHHL, in the dotted boxes); cell three then gets active. LasR and RhIR are constitutively expressed in cell three. Mnt represses pMnt, inhibiting CFP's transcription. There are two Mnt genes placed under a different promoter each. PAI molecules (QS1) coming from cell one bind to LasR and the complex activates cI's transcription from pLasR/PAI. cI then represses pR, inhibiting Mnt's transcription. HHL molecules (QS2) coming from cell two bind to RhIR and the complex activates TetR's transcription from pRhIR/HHL. TetR then represses the second Mnt's transcription from pTet. Only when both Mnt genes are repressed (Mnt NOR Mnt), CFP is allowed to be expressed from pMnt; then the community enters state 5. Even if a NOR gate is directly implied in CFP's expression, we consider this globally as an AND gate as both lights need to be present to induce CFP expression.

pConst+RBS BBa_K081005 Constitutive promoter plus Strong Ribosome Binding Site RBS (strong) BBa_B0030 Strong Ribosome Binding Site mRFP BBa_J06505 Red Fluorescent Protein with LVA tag GFP BBa_K145015 Green Fluorescent Protein with LVA tag CFP BBa_E0022 Cyan Fluorescent Protein with LVA tag YFP BBa_E0032 Yellow Fluorescent Protein with LVA tag BFP BBa_K156010 Blue Fluorescent Protein with LVA tag Double Ter BBa_B0015 Double transcriptional terminator Double Ter BBa_B0014 Double transcriptional terminator Mnt BBa_B0030 Mnt Repressor pRM434 BBa_I12006 Hybrid Promoter, induced by cI and repressed by cI434 pLasR/PAl BBa_R0079 LasR & PAl regulated Promoter (Quorum sensing) pRhlR/HHL BBa_R0071 RhlR & HHL regulated Promoter (Quorum sensing) aisHHL BBa_C0070 Autoinducer synthesis protein that produces N-butyryl-HHL wich binds to RhlR RhlR BBa_C0071 Transcriptional regulator, in complex with RhlR binds to the RhlR promoter aisPAl BBa_C0078 Autoinducer synthesis protein that produces PAI-1 [N-(3-oxododecanoyl)-L-homoserine lactone] which binds to LasR LasR BBa_C0079 Transcriptional regulator, in complex with PAI binds to LasR promoter pMnt BBa_R0073 Mnt Reprressible Promoter RBS.2 (weak) BBa_B0030 Weak Ribosome Binding Site RBS.3 (medium) BBa_B0032 Medium Ribosome Binding Site RBS.4 (weaker) BBa_B0033 Weaker Ribosome Binding Site TetR BBa_C0040 Tetracycline Repressor with LVA tag cI BBa_C0051 cI protein from phage lambda cI434 BBa_C0052 cI protein from phage 434 pTetR BBa_R0040 TetR repressible Promoter pR BBa_R0051 Right Promoter from Phage lambda pTetMnt BBa_K091105 Mnt and TetR repressible Promoter