IGEM:IMPERIAL/2006/project/Oscillator/Parts

Part Status

See the Imperial iGEM Lab Status page for the current status of required parts, plus information on colour coding

Table of parts info (WordDoc)

aiiA

This is the progress of the aiiA gene

Parts for Testing Phase Part T9002: Prey Transfer Function Characterisation/ AHL Assay Cell BBa_T9002 (Parts Registry) - (controlling GFP) well 19B, plate 2 (available and working) T9002 Information Page This construct is used to measure the ability of AHL to activate gene expression from pLuxR. LuxR expression can also be modulated from pTet to work out how LuxR levels effect pLuxR activation. We can then use this data to plot a calibration curve of Activation vs AHL concentration. Color added Part T9002 testing using Fuji-5400 machine Part J37015: Prey Cell Positve Feeback Test <bbpart>J37015</bbpart> (Parts Registry) J37015 Information Page This construct is used to characterise the positive feedback loop, with AHL levels being determined by the assay construct above. Part F2620: Quorum sensing receiver BBa_F2620 - Well 6B, plate 1 (available and working) Part C0261: C0261 - 24A Plate 1 Part I13504: I13504 - 12D Plate 1 Part J37016: Predator Transfer Function Characterisation (Single Promoter) BBa_J37016(Parts Registry) J37016 Information Page This construct is used to characterise the pLuxR transfer function, and noteably its effect on YFP expression, which would be equivalent to AiiA expression in the full system with one promoter. Part R0062: Promoter (luxR & HSL regulated - lux pR) R0062 - 9G Plate 1 Part B0034: RBS B0034 - 3O Plate 1 Part C0062: Repressor/Activator, luxR C0062 - 7A Plate 1 Part I13504: RBS & GFP I13504 - 12D Plate 1 Part J37020: Predator Transfer Function Characterisation (Double Promoter) BBa_J37020(Parts Registry) J37020 Information Page This construct is used to characterise the pLuxR transfer function, and noteably its effect on YFP expression, which would be equivalent to AiiA expression in the full system with two promoters. Part J37018 & J37019: For predator double promoter test construct BBa_J37018 (Parts Registry)- to be built ! Part I13504: For predator double promoter test construct BBa_I13504 - 12D Plate 1 Part J37022: AiiA Activity Assay BBa_J37022 - AiiA test construct (production) - with LVA and FLAG tags added Protocol and Modelling Page for J37022 same as: ] AiiA with FLAG immunotag and LVA tag controlled by LacI under IPTG promotion Part I13207: HSL/AiiA test construct (- not to be used anymore) BBa_I13207 (Parts Registry) - Well 2P, plate 1 (available) Protocol and Modelling Page for I13207

Parts for Final Assembly Measuring the cell population + Cre-Lox system Cre-Lox Overview Tagging LuxR LuxR Tag Overview Cre Lox Part File:Redoing Cre.doc Part F2620: Quorum sensing receiver BBa_F2620 - Well 6B, plate 1 (available and working) Part F1610: Quorum sensing sender LuxI BBa_F1610 - Well 1B, plate 2 (available and working) Part J37019: For predator single promoter final assembly BBa_J37019 Part R0062: pLuxR Promoter BBa_R0062 - Well 9G, plate 1 (available and working) Part B0034: RBS BBa_B0034 - Well 3O plate 1 (available and working) Part C0062: LuxR coding region BBa_C0062 - Well 7A, plate 1 (available) Part J37018 & J37019: For predator double promoter final assembly BBa_J37018 BBa_J37019 Part J37019: Terminators and pLuxR promoter BBa_I13033 - Well 2H, plate 1 (available) Part J37025: AiiA enzyme coding region BBa_J37025 Needs creating

Parts for Final Assembly

Measuring the cell population + Cre-Lox system

[Overview]

Tagging LuxR

[overview]

Cre Lox Part

File:Redoing Cre.doc

BBa_F2620 - Quorum sensing receiver

• BBa_F2620 - Well 6B, plate 1 (available and working)

BBa_F1610 - Quorum sensing sender LuxI

• BBa_F1610 - Well 1B, plate 2 (available and working)

BBa_J37019 - For predator single promoter final assembly

• BBa_R0062 - pLuxR Promoter: Well 9G, plate 1 (available and working)
• BBa_B0034 - RBS: Well 3O plate 1 (available and working)
• BBa_C0062 - LuxR coding region: Well 7A, plate 1 (available)

BBa_J37018 - For predator double promoter final assembly

• BBa_J37019 - See above
• BBa_I13033 - Terminators and pLuxR promoter Well 2H, plate 1 (available)

BBa_J37025 - AiiA enzyme coding region

• Needs creating

Parts for Testing Phase

• BBa_T9002 (controlling GFP) well 19B, plate 2 (available and working)
• BBa_I13207 (HSL/AiiA test construct) well 2P, plate 1 (available) Protocol and Modelling Page for I13207 - not to be used anymore
• BBa_J37022 - AiiA test construct (production) - with LVA and FLAG tags added Protocol and Modelling Page for J37022

Prey Transfer Function Characterisation/AHL Assay Cell

• T9002 Information Page - (Parts Registry)

This construct is used to measure the ability of AHL to activate gene expression from pLuxR. LuxR expression can also be modulated from pTet to work out how LuxR levels effect pLuxR activation. We can then use this data to plot a calibration curve of Activation vs AHL concentration.

Color added Part T9002 testing using Fuji-5400 machine

Prey Cell Positve Feeback Test

• J37015 Information Page - (Parts Registry)

This construct is used to characterise the positive feedback loop, with AHL levels being determined by the assay construct above.

• F2620 - 6B Plate 1

• C0261 - 24A Plate 1

• I13504 - 12D Plate 1

Predator Transfer Function Characterisation (Single Promoter)

• J37016 Information Page - (Parts Registry)

This construct is used to characterise the pLuxR transfer function, and noteably its effect on YFP expression, which would be equivalent to AiiA expression in the full system with one promoter.

• R0062 - 9G Plate 1
• B0034 - 3O Plate 1
• C0062 - 7A Plate 1
• I13504 - 12D Plate 1

Predator Transfer Function Characterisation (Double Promoter)

• J37020 Information Page - (Parts Registry)

This construct is used to characterise the pLuxR transfer function, and noteably its effect on YFP expression, which would be equivalent to AiiA expression in the full system with two promoters.

• J37018 - To be built!
• I13504 - 12D Plate 1

AiiA Activity Assay

• J37022- (Parts Registry)

SAME AS:
]
AiiA with FLAG immunotag and LVA tag controlled by LacI under IPTG promotion

E-mail from Mr. Barry Canton of MIT regarding parts

16 July 2006: Parts for Final Project

Hi John, I was forwarded your email below as I have done some work with the quorum sensing systems you are asking about.

Specifically, we tested F1610 as part of I13015 and showed that it could induce receiver cells. We tested F2621 a little bit and showed that it could be induced by the sender cells. I believe the transfer curve looked very similar to that of F2620 but I would have to try and find the data for that.

Most of our testing was with F2620. The best place to read about our characterization of F2620 is on openwetware by going to this page - http://openwetware.org/wiki/Endy:F2620 All the experiments we have done are described there.

You are asking about time to send and receive signals. We haven't done much work on transport times for the molecules through liquid media or over solid media but if you can specify what the experimental set-up is I might have some qualitative comments. Our experience has been that the receivers activate transcription within minutes after addition of HSL to liquid media.

Ron Weiss at Princeton has done some tests where he looked at receiver cells placed at different distances from sender cells. He made some movies showing the closer receivers being induced first. I think that some of that data should be available in his papers.

Anyway, let me know if you have some specific questions and I can try and give you more specific info.

Good luck with the oscillators, I'll be watching the progress on OWW!

Barry

16 July 2006: AiiA Testing Construct

Hi John, we did very little testing with I13207 and the results were not especially conclusive. Had we continued the testing somewhat more rigorously, we may have had better results.

If you download the presentation linked below, which we gave at the Jamboree in 2004 you can have a look at slide 41 which has some of the data we got for I13207. Basically we induced receiver cells in the presence or absence of arabinose which should induce aiia production. The drawback of that experiment was that we were using MC4100 for which arabinose is toxic. You might have better results in another strain. I just had time to transform into MG1655 and do one experiment before I switched over to working full time on my thesis project. The results in that experiment suggested that there was low levels of induction of the receiver when aiiA was expressed. I was inducing with 0.02% arabinose at the time.

2004 Jamboree Presentation

All our experiments with these receiver devices were basically the same. Grow an ovenight in supplemented M9, dilute back 1/500 in the morning, wait until an OD of ~0.1 and then induce with HSL and arabinose if appropriate. We use a 96 well plate reader to measure GFP accumulation over time. You can read more about the experimental set-up on the F2620 page of OWW, its basically the same.

Hope this is of some use and let me know if you have further questions,

Barry