IGEM:IMPERIAL/2006/project/Oscillator/Parts

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Contents

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



image:T9002.JPG
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
Color added Part T9002 testing using Fuji-5400 machine



Part J37015:
Prey Cell Positve Feeback Test



image:J37015.JPG
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)
image:F2620.JPG


Part C0261:
Part I13504:


Part J37016:
Predator Transfer Function Characterisation (Single Promoter)



image:J37016.JPG
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)
Part B0034:
RBS
Part C0062:
Repressor/Activator, luxR
Part I13504:
RBS & GFP


Part J37020:
Predator Transfer Function Characterisation (Double Promoter)



image:J37020.JPG
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
image:J37018.JPG



Part I13504:
For predator double promoter test construct


Part J37022:
AiiA Activity Assay



Image:J37022.png


same as:
Image:J37022a.png]


AiiA with FLAG immunotag and LVA tag controlled by LacI under IPTG promotion


Part I13207:
HSL/AiiA test construct
(- not to be used anymore)



Parts for Final Assembly

Measuring the cell population + Cre-Lox system


Tagging LuxR


Cre Lox Part
Image:Redoing Cre.doc


Part F2620:
Quorum sensing receiver
  • BBa_F2620 - Well 6B, plate 1 (available and working)
image:F2620.JPG




Part F1610:
Quorum sensing sender LuxI
  • BBa_F1610 - Well 1B, plate 2 (available and working)
image:F1610.JPG




Part J37019:
For predator single promoter final assembly
image:J37019.JPG



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



Part J37018 & J37019:
For predator double promoter final assembly
image:J37018.JPG
Image:J37019.PNG



Part J37019:
Terminators and pLuxR promoter



Part J37025:
AiiA enzyme coding region




Parts for Final Assembly

Measuring the cell population + Cre-Lox system

[Overview]

Tagging LuxR

[overview]

Cre Lox Part

Image:Redoing Cre.doc

BBa_F2620 - Quorum sensing receiver

image:F2620.JPG

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


BBa_F1610 - Quorum sensing sender LuxI

image:F1610.JPG

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


BBa_J37019 - For predator single promoter final assembly

image:J37019.JPG

  • 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

image:J37018.JPG


BBa_J37025 - AiiA enzyme coding region

  • Needs creating




Parts for Testing Phase


Prey Transfer Function Characterisation/AHL Assay Cell



image:T9002.JPG

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
Color added Part T9002 testing using Fuji-5400 machine


Prey Cell Positve Feeback Test



image:J37015.JPG

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

Predator Transfer Function Characterisation (Single Promoter)



image:J37016.JPG

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.

Predator Transfer Function Characterisation (Double Promoter)



image:J37020.JPG

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.



AiiA Activity Assay



Image:J37022.png
SAME AS:
Image:J37022a.png]
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

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