Booperator test: Inverter NOR biasing in monocistronic design
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
This is the third test chassis for the Booperator and is particular to the monocistronic design. It tests the biasing of the Inverter NOR pair.
RhlR and LasR constitutive generation (using TetR)
Produces RhlR and LasR constitutively under control of a TetR self-repressor.
Sara Neves, Alvin Carter Powers (Fighting Darwins)
lasR receiver test: -to test basal activity, give no input of chemical signal, and reporter indicates TIPs leakage -to test binding, put in solution of chemical signal made by lasI (AI-1), the inducer binds to the lasR protein to activate the promoter R0079, to make TIPs that will express reporter
Booperator aiia test 6: Effect of tetR on production of LuxR
intermediate for lac relaxation oscillator
Intermediate for lac relaxation oscillator (final stage)
There is a single base insertion (of an A) at position 286, this is in the mixed site between R0011 and B0034 (first subpart of E0034) so suspect it should have little effect on device performance. But no experimental confirmation.
Test: AspA attractant sender (I3100.I3200.B0015)
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Tests Lac trigger ability to produce AspA. After trigger induction, the amount of aspartate secreted into the medium can be measured directly.
Sara Neves, Chris Walsh (Fighting Darwins)
lasI test: with input of TIPs from I0500, determines how much chemical signal is ultimately made by the lasI protein amount of signal can be determined chemically (?)
434 cI/lambda cI857 (Lambda II) RS flip-flop
RS flip-flop with lambda cI857 (Lambda II) as the set and 434 cI as the reset. It will also take UV exposure as the set and heat (40??C) as the reset.
Test: Quorum threshold level (I3510.E0033.B0015)
Tests threshold level of HSL concentration needed to induce the switch quorum response.
This testbed is designed to respond to a manual input of Lux HSL. The output is through the LacZ reporter.
with an input of TIPs from I0500, tests the amount of chemical signal produced by the rhlI protein
QPI Test Construct Intermediate (Q01400.E0430)
Lambda cI switch output device (R0050.B0034.C0051)
One output from the Hk022cI-lambda cI wired-nor bistable switch.
Hk022cI switch input device (B0034.C0050.B0015)
One input into the Hk022cI-lambda cI wired-nor bistable switch.
Tests input levels needed to trigger switch, bistability, noise resistance, and switch time. Switch has three independently controllable trigger inputs and two reporter outputs, ON and OFF.
The layout of the switch mimics the layout of the proposed Polkadorks E. colibrator (I3000). Two switch outputs are insulated by appearing first. Hk022cI is attached to the CFP reporter (I3410.E0422). Lambda cI is attached to the YFP reporter (I3411.E0432). The three individually controlled trigger inputs follow the outputs in the layout. Lac trigger controls one hk022cI input (I3100.I3400), and TetR trigger controls the other (I3101.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, AraC trigger controls the lambda cI input (I3102.I3401).
E. colibrator design 1 has the OR gate embedded within the switch via a double instance of I3400 Hk022cI switch input device. Design 1 also has no explicit activity during the switch OFF state, as illustrated by the terminator immediately following I3411 Lambda cI switch output device. This causes the OFF state to rely completely on cellular diffusion to reset the system.
The layout of E. colibrator (I3000) is designed to insulate the most sensitive devices, namely those in the switch that store state. The hk022cI switch output (ON) is insulated by appearing first. This output is then attached to the AspA attractant sender and the CFP reporter (I3410.I3200.E0422). The quorum receiver and its control of the lambda cI switch input (OFF) occur next (I3510.I3401) because leaky TIPS through this module will encourage the OFF state dicated by the downstream lambda cI switch output (I3411.B0015). Note that the OFF switch output simply ends at termination of the output TIPS (B0015) in this design. The rest of the switch inputs occur downstream because they do not store state and therefore do not need to be as insulated from leaky TIPS. Lac trigger controls one hk022cI input (I3100.I3400), and Tar-EnvZ receptor (I3300 upstream ompC version) controls the other (I3300.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, the quorum sender device (I3500) is last because it is meant to be constitutively on at some basal level, so insulation is not required. Note: Depending on the efficacy of the OmpR promoter, this design can be implemented with any of I3300, I3301, I3302 (variations of Tar-EnvZ devices with different OmpR promoters). The default implementation uses I3300.
Expression of lambda cI controlled by IPTG (requires LacIq strain). Expression of 434 cI controlled by arabinose. If I12006 promoter works, YFP is activated by lambda cI and repressed by 434 cI.
This is a potential synchronized relaxation oscillator simmilar to the synchronization element of I13905
Prakash *: Syncronator Cell-Signalling Module
434 cI/lambda cI857 (E07700) RS flip-flop
RS flip-flop with lambda cI (E07700) as the set and 434 cI as the reset. It will also take UV exposure as the set and heat (40??C) as the reset.
Cancelled due to failure to successfully mutate BBa_J06502 and BBa_J06802.
Constitutive Promoter I Test (R0074.E0422)
tests the strength of R0074, indicated by strength of expression of reporter
Exogenous LuxI Relaxation Oscillator Test Construct Intermediate (I0500.C0261.E0434)
Intermediate construct for Chris' relaxation oscillator test construct.
Constitutive Promoter II Test (R1075.E0422)
Alvin Carter Powers, Chris Walsh (Fighting Darwins)
tests the strength of R1075, indicated by reporter expression
Tests ability of switch to respond to aspartate input by connecting the Tar-EnvZ receptor (I3300 ompC upstream version) to one of the switch inputs. The remaining testbed is analogous to the switch functionality testbed (I3420).
The layout of the switch mimics the layout of the proposed Polkadorks E. colibrator (I3000). Two switch outputs are insulated by appearing first. Hk022cI is attached to the CFP reporter (I3410.E0422). Lambda cI is attached to the YFP reporter (I3411.E0432). The three individually controlled trigger inputs follow the outputs in the layout. Lac trigger controls one hk022cI input (I3100.I3400), and Tar-EnvZ receptor (I3300 upstream ompC version) controls the other (I3300.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, AraC trigger controls the lambda cI input (I3102.I3401).
Booperator aiia test: degradation of HSL "A" by aiia
Exogenous LuxI Relaxation Oscillator Test Construct Intermediate (R0062.C0260.E0424)
Intermediate construct for Chris' relaxation oscillator test constructs.
Lambda cI/LacI ts (mut 265) RS flip-flop with EYFP
RS flip-flop with LacI ts (mut 265) as the set and lambda cI as the reset. It will also take UV exposure as the set and heat (40??C) as the reset.
Sequenced. However, shows a 23 bp deletion in the LacI+pL regulated promoter (R0011).
3OC6HSL + LuxR reporter and aiia (LVA+) tuning device
This device uses the S01656 module to encode IPTG (or lactose) inducible aiia (LVA+), allowing the tuning of 3OC6HSL degradation activity in lactose tune-able E.coli strains. The presence of the 3OC6HSL and the LuxR protein is read out by the I6042 module.
Tests ability of switch to respond to aspartate input by connecting the Tar-EnvZ receptor (I3301 truncated ompC upstream version) to one of the switch inputs. The remaining testbed is analogous to the switch functionality testbed (I3420).
The layout of the switch mimics the layout of the proposed Polkadorks E. colibrator (I3000). Two switch outputs are insulated by appearing first. Hk022cI is attached to the CFP reporter (I3410.E0422). Lambda cI is attached to the YFP reporter (I3411.E0432). The three individually controlled trigger inputs follow the outputs in the layout. Lac trigger controls one hk022cI input (I3100.I3400), and Tar-EnvZ receptor (I3301 truncated upstream ompC version) controls the other (I3301.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, AraC trigger controls the lambda cI input (I3102.I3401).
LacI regulated cI/LuxI with constitutive LuxR
Polycistronic lambda cI and LuxI proteins regulated by Lac Repressor (LacI). Downstream is consitutive LuxR expression cassette. System flanked by dual transcription terminators.
BL oscillator, no cooperativity, reporter protein, kickstart
This part is a Barkai-Leibler oscillator with no cooperativity for the repressor (one 434 cI OR site). A reporter protein (EYFP) and a kickstart is included.
There is no cooperativity for 434 cI in this oscillator. A protein EYFP (with degradation tag) coding region is placed directly after the lambda cI coding region in order to keep track of the lambda cI levels. This part also includes a "kickstart" that will yield lambda cI if ATC is added.
Quorum sender has luxI device under control of the lux pR promoter to create HSL.
Part for screening plasmid test (to be combined with an upstream inverter library).
intermediate part for the lac version of the synchronized relaxation oscillator
Sequence verified, however there is an N at position 1021, however upon looking at the actual sequence trace it looks like it is most likely the correct base.
RS flip-flop with LacI as the set and lambda cI as the reset.
constitutive promoter, RBS, rhlR coding region, terminator rhlR protein binds AI-2 chemical signal to induce operator
Ring Oscillator Synchronization Device (lac)
This device is meant to be coupled to the elowitz repressilator plasmid to synchronization oscillations accross a population of cells. Sequence
Test: Tar-EnvZ attractant receiver (I3301.E0422)
Tests Tar-EnvZ attractant receiver device (truncated ompC upstream version) by attaching TIPS output to CFP reporter.
Tar-EnvZ attractant receiver device (R1075.B0034.C0082.B0015.R0084)
Constitutive promoter makes Tar-EnvZ fusion protein, which binds aspartate to create TIPS.
Intermediate part in lac synchronization device
This part is the result of a build step which was done backwards by accident. The correctly orientated version was remade as I13978
test (lambda-cI nor_input) I0500.E0422.I0500.I9009
E. colibrator design 2 has an explicit OR gate before the switch via an araC OR gate device (I3701, I3700). Design 2 has no explicit activity during the switch OFF state, as illustrated by the terminator immediately following I3411 Lambda cI switch output device. This causes the OFF state to rely completely on cellular diffusion to reset the system.
The layout of E. colibrator (I3001) is designed to insulate the most sensitive devices, namely those in the switch that store state. The hk022cI switch output (ON) is insulated by appearing first. This output is then attached to the AspA attractant sender and the CFP reporter (I3410.I3200.E0422). The quorum receiver and its control of the lambda cI switch input (OFF) occur next (I3510.I3401) because leaky TIPS through this module will encourage the OFF state dicated by the downstream lambda cI switch output (I3411.B0015). Note that the OFF switch output simply ends at termination of the output TIPS (B0015) in this design. The rest of the switch inputs occur downstream because they do not store state and therefore do not need to be as insulated from leaky TIPS. Lac trigger controls the araC input device for the OR gate (I3100.I3701), and Tar-EnvZ receptor (I3300 upstream ompC version) controls the second OR input, embedded in the araC buffer device (I3300.I3700). The araC OR gate in turn outputs TIPS into the hk022cI switch input (I3400). Finally, the quorum sender device (I3500) is last because it is meant to be constitutively on at some basal level, so insulation is not required. Note: Depending on the efficacy of the OmpR promoter, this design can be implemented with any of I3300, I3301, I3302 (variations of Tar-EnvZ devices with different OmpR promoters). The default implementation uses I3300.
Booperator test: Inverter NOR biasing in polycistronic design
This is the fourth test chassis for the Booperator and is particular to the polycistronic design. It tests the biasing of the Inverter NOR pair.
QPI Test Construct Intermediate (Q04400.E0430)
Sub-part of the synchro oscillator assemblies
Alvin Carter Powers, Yong-Su Jin (Fighting Darwins)
test of cI-857: input TIPs with I0500, E0022 reporter indicates amount of TIPs, cI-857 is expressed, represses R0051, reporter E0032 indicates TIPs leakage
Sara Neves, Chris Walsh, Alvin Carter Powers (Fighting Darwins)
lasI/lasR signal test: test lasR receiver when receiving signal from lasI sender (ie. test how they work together)
Test: Quorum threshold level and chemotaxis response, receiver cells (I3601.I3520.I3500)
Tests cellular density of asparate-secreting cells needed to induce quorum threshold response. Sender cells are nonmotile and have a trigger controlling the AspA attractant sender and CFP reporter indicating transcription of aspA. Receiver cells have a constitutive promoter controlling YFP reporter. Chemotaxis can be observed by the movement of the YFP-marked receiver cells. Quorum threshold is detected when the LacZ reporter is observed.
Sender cells are the nonchemotactic E. coli strain RP5232. Receiver cells are wildtype E. coli. This test combines the chemotaxis response test with the quorum threshold test.
LuxR generator. POPS generate cI(LVA) and LuxI(LVA)
Accepts POPS to produce lambda cI (LVA) and LuxI (LVA). Constitive LuxR generator in the absence of TetR.
This circuit is regulated by two proteins, TetR and CI. In the presence of TetR, LasR production will be repressed. In the presence of CI, LasI production will be repressed. Without repressors present the circuit may be leaky enough to trigger the positive feedback LasI/LasR/PAI response.
Test: Chemotaxis response to secreted aspartate, sender cells (I3100.I3200.E0422)
Tests the chemotaxis cell-cell signalling machinery based on aspartate secretions. Sender cells are nonmotile and have a trigger controlling the AspA attractant sender and CFP reporter indicating transcription of aspA. Receiver cells have a constitutive promoter controlling YFP reporter. Chemotaxis can be observed by the movement of the YFP-marked receiver cells.
Sender cells are the nonchemotactic E. coli strain RP5232. Receiver cells are wildtype E. coli.
Ring Oscillator Synchronization Device (tet)
This device is meant to be coupled to the elowitz repressilator plasmid to synchronization oscillations accross a population of cells.
RS flip-flop with lambda cI as the set and 434 cI as the reset.
Tester module for BBa_I15001. Flanked by dual terminators. LacI regulated polycistronic cI/LuxI/LacZalpha
RhlR, LuxR, and LasR constitutive generation (using TetR)
AraC buffer device for OR gate (I3701.R0080)
Positive regulation of araC promoter by AraC CDS. This buffer takes TIPS in as one of two OR gate inputs and outputs TIPS. The second input to the OR gate is I3701.
BL oscillator (I12036 based) with IPTG kickstart
Modified BL oscillator based on I12036. It can be "reset" by adding IPTG which stimulates production of the A protein (lambda cI) if used in LacIq strain. The B protein is 434 cI. The YFP-AAV reporter tracks the dynamics of the A protein.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
This part combines multiple Biobrick elements into a single module for testing and debugging elements of the XOR genetic gate. It tests systems that use cI protein as output. The promoter within the testing module is cI repressible. The advantage of constructing the test module de novo is the ease of transferring the entire unit to elements we would like to test within the XOR gate. YFP expression levels are the output of the test module and serve as a marker for system function.
synthetic element, using a combination of Biobrick parts--cI promoter, RBS, YFP, transcriptional terminators. used for testing systems that use cI protein as outputs.
Olmecs: Oscillator 1 of Synchro-Dualator
Kickstart for the Barkai-Leibler oscillator
This part is designed for "kickstarting" the Barkai-Leibler oscillator by producing lambda cI.
Addition of ATC will repress the tetR and this will result in production of lambda cI.
Tar-EnvZ attractant receiver device (R1075.B0034.C0082.B0015.R0083)
QPI Test Construct Intermediate (Q04121.E0430)
Exogenous AiiA Relaxation Oscillator Test Construct Intermediate (R0062.B0034.C0061.E0434)
BL oscillator (I12040 based) with IPTG kickstart
Modified BL oscillator based on I12040. It can be "reset" by adding IPTG which stimulates production of the A protein (lambda cI) if used in LacIq strain. The B protein is 434 cI. The YFP-AAV reporter tracks the dynamics of the A protein.
Relaxation Oscillator Test Construct (I13202.B0015.I0460)
QPI Test Construct Intermediate (Q01121.E0430)
Tests ability of switch to respond to aspartate input by connecting the Tar-EnvZ receptor (I3302 ompF upstream version) to one of the switch inputs. The remaining testbed is analogous to the switch functionality testbed (I3420).
The layout of the switch mimics the layout of the proposed Polkadorks E. colibrator (I3000). Two switch outputs are insulated by appearing first. Hk022cI is attached to the CFP reporter (I3410.E0422). Lambda cI is attached to the YFP reporter (I3411.E0432). The three individually controlled trigger inputs follow the outputs in the layout. Lac trigger controls one hk022cI input (I3100.I3400), and Tar-EnvZ receptor (I3302 upstream ompF version) controls the other (I3302.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, AraC trigger controls the lambda cI input (I3102.I3401).
When used in LacIq strain, addition of IPTG should increase YFP fluorescence if I12007 is working.
434 cI/lambda cI857 (Lambda II) RS flip-flop with EYFP
Synchronized Brickilator Test Construct (I13201.Q04400.I0461)
Booperator test: LasR biasing, LasR/LuxR/RhlR crosstalk
3OC6HSL + LuxR reporter and aiia tuning device
This part is analogous to J13202, but encodes a more stable aiia protein, with presumably more anti-3OC6HSL activity.
Test: Chemotaxis response to secreted aspartate, receiver cells (R1075.E0432)
Tests ability of switch to respond to quorum sensing input by connecting the quorum receiver to one of the switch inputs. The remaining testbed is analogous to the switch functionality testbed (I3420).
The layout of the switch mimics the layout of the proposed Polkadorks E. colibrator (I3000). Two switch outputs are insulated by appearing first. Hk022cI is attached to the CFP reporter (I3410.E0422). Lambda cI is attached to the YFP reporter (I3411.E0432). The three individually controlled trigger inputs follow the outputs in the layout. Lac trigger controls one hk022cI input (I3100.I3400), and TetR trigger controls the other (I3101.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, the quorum receiver device controls the lambda cI input (I3510.I3401).
Lambda cI/LacI ts (mut 265) RS flip-flop
Tar-EnvZ attractant receiver device (R1075.B0034.C0082.B0015.R0082)
Booperator test: RhlR biasing, RhlR/LuxR/LasR crosstalk
Booperator: LuxR/RhlR/aiia, monocistronic
Test: Quorum threshold level and chemotaxis response, sender cells (I3600.I3520.I3500)
Booperator test: LuxR biasing, LuxR/RhlR/LasR crosstalk
434 cI/lambda cI857 (E07700) RS flip-flop with EYFP
QPI Test Construct Intermediate (Q04510.E0430)
Barkai-Leibler design experiment Part A (Lambda cI)
This forms the activator of a Barkai-Leibler oscillator.
This part to work in conjunction with BBa_I12002. This part creates one of the proteins involved in the Modified Barkai-Leibler relaxation oscillator design (activator protein: lambda cI). In the presence of Lambda cI, more Lambda cI will be produced (positively autoregulated). However, if there is 434 cI repressor present in the system, the production of Lambda cI should be repressed. This was implemented in the subpart BBa_I12006, which represents the Operating Regions for the promoter. The fact that the OR-3 site was replaced with the OR-1 site of 434 cI repressor theoretically should mean that when 434 cI binds in, the production of the Lambda cI protein should be blocked.
AspA attractant sender device (B0034.C0083)
System flanked on both sides by dual transcription terminators. Bears a joint Lux promoter-lambda operator, strong RBS, with LacZ alpha peptide.
p(pen).medium RBS. LacI. medium RBS. araC. strong RBS. YFP. double terminator
RBS, LacZ coding region, terminator reporter device that makes a relatively stable blue product
BL oscillator (I12006-based) with IPTG kickstart
Modified BL oscillator based on I12006. It can be "reset" by adding IPTG which stimulates production of the A protein (lambda cI) if used in LacIq strain. The B protein is 434 cI. The YFP-AAV reporter tracks the dynamics of the A protein.
(Chassis E. coli, carrier plasmid pSB1C3) Length: 1047 bp Background: This Epsilon/MazF part is one of the two components of a double toxin-antitoxin system. The part was designed to be carried on a plasmid while its counterpart MazE/Zeta was carried on another. Unfortunately, only Epsilon/MazF part was successfully cloned in E. coli. This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
Epsilon and MazF are under the control of the promoter Pveg (constitutive promotor for B. subtilis) followed by a strong RBS. A theophylline-dependent riboswitch was used to prevent the MazE toxin expression during the cloning steps in presence of theophylline (Topp and Gallivan, 2008). Indeed, with an expressed toxin, it would be impossible to clone the Biobrick in E. coli.SacII and SalI are the restriction sites placed respectively before and after the riboswitch so that this fragment can easily be replaced depending on the control we want to apply to the bacteria.
The gene Epsilon codes for a toxin countered by the anti-toxin Zeta (Zielenkiewicz and Cegłowski, 2005 ; Mutschler et al., 2011). The gene MazF encodes the anti-toxin for the MazE toxin (Bravo et al., 1987, Zhang et al., 2005, Wang et al., 2013).
Tests I3101 TetR trigger device by attaching TIPS output to CFP reporter.
RhlR and LuxR constitutive generation (using TetR)
Produces RhlR and LuxR constitutively under control of a TetR self-repressor.
This device is a strong amplifier of 3OC6HSL. R0065 is a leaky promoter that produces enough LuxI to generate ample HSL to switch this device to a stable high expression state, the magnitude of which is dependent upon LuxR (or possibly lambda cI repressor) concentration.
Tetracycline-induced trigger using tetR.
Tests I3102 AraC trigger device by attaching TIPS output to CFP reporter.
Booperator test: RhlR biasing, RhlR/LuxR crosstalk
This is the second test chassis for the Booperator and it tests for crosstalk across the HSL signals and biasing of the RhlR receiver and inverter.
Booperator test: LuxR biasing, LuxR/RhlR crosstalk
This is the first test chassis for the Booperator and it tests for crosstalk across the HSL signals and the biasing of the LuxR receiver and inverter.
Arabinose-inducible trigger using araC. The trigger consists of a constitutive promoter controlling an araC buffer.
Lac stochastic trigger device (R1075.B0034.C0012.B0015.R0010)
IPTG-inducible stochastic trigger using lacI.
Temperature sensor that is tunable with aTc. Assumes cell line provide source of TetR.
Exogenous LuxI Relaxation Oscillator Test Construct (I13940.I13941)
R1075 was replaced with R0053, a medium-strength promoter. Trigger component added.
intermediate for lac ring oscillator synchronization device
Lambda cI switch input device (B0034.C0051.B0015)
Olmecs: Repressilator version 1 (HK022 cI)
Unnatural Selector Design (Fighting Darwins)
Sara Neves, Alvin Carter Powers, Yong-Su Jin, Chris Walsh, Jacky Mallett
Tunable Temperature Sensor made with parts availble in Registry 7.05 DNA-1 and cI857ts.
Tests ability of switch to output aspartate given particular inputs. The remaining testbed is analogous to the switch functionality testbed (I3420).
The layout of the switch mimics the layout of the proposed Polkadorks E. colibrator (I3000). Two switch outputs are insulated by appearing first. Hk022cI is attached to the AspA attractant sender (I3410.I3200). Lambda cI is attached to the YFP reporter (I3411.E0432). The three individually controlled trigger inputs follow the outputs in the layout. Lac trigger controls one hk022cI input (I3100.I3400), and TetR trigger controls the other (I3101.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, AraC trigger controls the lambda cI input (I3102.I3401).
QPI Test Construct Intermediate (Q04530.E0430)
Hk022cI switch output device (R0051.B0034.C0050)
3OC6HSL + LuxR reporter and cI tuning device
This device uses the S03182 module to produce lambda cI in an IPTG (or lactose) dependent fashion. Lambda cI concentration should be tune-able in certain E.coli strains. The I6042 module is used to read out 3OC6HSL and LuxR concentrations.
Synchronator 2 w/ CFP under control of Lac (I4202.I13601)
This is the Synchronator Design 2 with an additional CFP generator under control of the Lac promoter for debug purposes.
Booperator aiia test 3: effect of tetR on production of RhlR
Expression of lambda cI controlled by IPTG (requires LacIq strain). Expression of 434 cI controlled by arabinose. If BBa_I12036 promoter works, YFP is activated by lambda cI and repressed by 434 cI.
Designed in the exact same way as BBa_I12028 (Test construct for I12006) except with BBa_I12036 swapped in for BBa_I12006
Booperator aiia test 5: effect of tetR on production of LuxR
Exogenous AiiA Relaxation Oscillator Test Construct (I13942.I13943)
Synchronized Brickilator Test Construct (I13201.Q04510.I0461)
AraC input device for OR gate (B0034.C0080.B0015)
This input device takes TIPS in as one of two OR gate inputs and produces AraC. AraC then acts on the araC promoter in I3700 to complete the OR gate.
Booperator aiia test: degradation of HSL "B" by aiia
Lambda cI/LacI ts (mut 241) RS flip-flop with EYFP
RS flip-flop with LacI ts (mut 241) as the set and lambda cI as the reset. It will also take UV exposure as the set and heat (40??C) as the reset.
Contains a XmaI site and an AvrII site. A separate (non-BB) part contains a complete BB site within XmaI/AvrII sites.
rhlI/rhlR signal test: test rhlR receiver when receiving signal from rhlI sender (ie. test how they work together)
Test: Tar-EnvZ attractant receiver (I3300.E0422)
Tests Tar-EnvZ attractant receiver device (ompC upstream version) by attaching TIPS output to CFP reporter.
Full Heat Trigger Test (R1075.Q04811.E0432)
heat trigger test: tests the trigger as an entire device; constitutive promoter should prevent TIPs leakage (indicated by the reporter), and when heat shocked, reporter should produce YFP
c1 Delay Oscillator Intermediate Construct (I13202.C0051.B0015)
Natural Lux Cassette + c1 + Term. This construct will be put together with the synthetic c1 promoter being synthesized by Caltech and the various AiiA Generation Constructs to create a variety of c1 Delay Oscillators.
temperature sensor: cold =green, hot =red.
rhlR receiver test: -to test basal activity, give no input of chemical signal, and reporter indicates TIPs leakage -to test binding, put in solution of chemical signal made by rhlI (AI-2), the inducer binds to the lasR protein to activate the promoter R0071, to make TIPs that will express reporter
E. colibrator design 3 has the OR gate embedded within the switch via a double instance of I3400 Hk022cI switch input device. Design 3 has an aiiA mechanism turn on during the switch OFF state, as illustrated by I0460 AiiA device immediately following I3411 Lambda cI switch output device. This allows the OFF state to speed up reset of the system by degrading residual HSL once quorum threshold is reached.
The layout of E. colibrator (I3002) is designed to insulate the most sensitive devices, namely those in the switch that store state. The hk022cI switch output (ON) is insulated by appearing first. This output is then attached to the AspA attractant sender and the CFP reporter (I3410.I3200.E0422). The quorum receiver and its control of the lambda cI switch input (OFF) occur next (I3510.I3401) because leaky TIPS through this module will encourage the OFF state dicated by the downstream lambda cI switch output (I3411.I0460). Note that the OFF switch output leads to aiiA synthesis to promote HSL degradation and system reset in this design. The rest of the switch inputs occur downstream because they do not store state and therefore do not need to be as insulated from leaky TIPS. Lac trigger controls one hk022cI input (I3100.I3400), and Tar-EnvZ receptor (I3300 upstream ompC version) controls the other (I3300.I3400). The dual control forms the built-in OR gate in one of the switch inputs. Finally, the quorum sender device (I3500) is last because it is meant to be constitutively on at some basal level, so insulation is not required. Note: Depending on the efficacy of the OmpR promoter, this design can be implemented with any of I3300, I3301, I3302 (variations of Tar-EnvZ devices with different OmpR promoters). The default implementation uses I3300.
constitutive promoter, RBS, lasR coding region, terminator lasR protein binds with AI-1 chemical signal to induce operator
Tests I3100 Lac trigger device by attaching TIPS output to CFP reporter.
Lambda cI/LacI ts (mut 241) RS flip-flop
RS flip-flop with LacI ts (mut 241) as the set and lambda cI as the reset. It will also take UV exposure as the set and heat (40°C) as the reset.
Tunable Temperature Sensor v3 (Registry 7.05 DNA-1 + Harvard Parts)
Tuneable Temperature Sensor made with parts available in Registry 7.05 DNA-1. At higher temperatures, the lacI dimer degrades into lacI monomers. The LacI promoter is no longer inhibited, hence more RFP is produced. Also, cI 434 is produced resulting in Prm+ being repressed, thus lowering the levels of GFP. At lower temperatures, the lacI dimer is allowed to form, hence the LacI promoter is repressed. RFP levels decrease, and cI levels drop. This results in Prm+ no longer being repressed, hence GFP levels increase. Behaviour is tuneable by adjusting the levels of lacI ts via L-arabinose activating pBad. The net result is that the fluorescence (green to red) can be correlated to a temperature (at a given level of L-arabinose).
LacI repressed lambda cI(LVA) + LuxI(LVA) + LacZalpha
LacI repressed polycistronic cI/LuxI/LacZalpha. Requires LacZomega complementation. See notes on E0033.
pOmpR dependent dual repressor device for I15030 and J13030
In the presence of phosphorylated ompR the proteins CI and TetR will be coexpressed from a single mRNA. These two proteins repress the HSL and PAI amplifiers I15030 and J13030. When using Cph1/EnvZ (I15010) as the OmpR regulator, these repressors will be expressed in the dark.
Test: Tar-EnvZ attractant receiver (I3302.E0422)
Tests Tar-EnvZ attractant receiver device (ompF upstream version) by attaching TIPS output to CFP reporter.
Exogenous AiiA Relaxation Oscillator Test Construct Intermediate (I0500.C0260.E0424)
Booperator aiia test 4: effect of tetR on production of RhlR
Tunable Temperature Sensor v2 (using LacI ts + other parts from Registry 7.05)
Tuneable Temperature Sensor made with parts available in Registry 7.05 DNA-1. At higher temperatures, the lacI dimer degrades into lacI monomers. The LacI promoter is no longer inhibited, hence more RFP is produced. Also, cI 434 is produced resulting in Prm+ being repressed, thus lowering the levels of GFP. At lower temperatures, the lacI dimer is allowed to form, hence the LacI promoter is repressed. RFP levels decrease, and cI levels drop. This results in Prm+ no longer being repressed, hence GFP levels increase. Behaviour is tuneable by adjusting the levels of lacI ts via L-arabinose activating pBad. The net result is that the fluorescence (green to red) can be correlated to a temperature (at a given level of L-arabinose). Note IPTG induces R0010, offering a second degree of tuning.
Booperator: aiia/LuxR/RhlR, polycistronic
Quorum receiver device (R0063.I0462.R0062)
Quorum receiver has luxR device under the control of lux pL promoter to produce luxR, which binds HSL and activates lux pR promoter.