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.

BioBrick	Part Name	Description
pConst+RBS	BBa_K081005	Constitutive promoter plus Strong Ribosome Binding Site
RBS (strong)	BBa_B0030	Strong Ribosome Binding Site
RBS (elowitz)	BBa_B0034	Standard 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_C0072	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 Repressible 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