IGEM:IMPERIAL/2006/project/Oscillator/Parts: Difference between revisions
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==Part Status== | ==Part Status== | ||
<font color= | See the Imperial iGEM [[IGEM:IMPERIAL/2006/project/parts/lab_status|Lab Status]] page for the current status of required parts, plus information on colour coding | ||
[http://openwetware.org/images/5/5c/PartInfo.doc Table of parts info (WordDoc)] | |||
==aiiA== | |||
[[IGEM:IMPERIAL/2006/project/Oscillator/aiiA|This is the progress of the aiiA gene]] | |||
<br> | |||
{| class="wikitable" border="3" cellpadding="5" cellspacing="1" style="text-align:center" | |||
!width="400" align="left" valign="top" style="background:#E6E8FA; color:black"| | |||
==Parts for Testing Phase== | |||
<big><center><u>Part T9002:<br>Prey Transfer Function Characterisation/<br>AHL Assay Cell</u></center></big> | |||
<br> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 <font color=purple>'''BBa_T9002 '''</font>](Parts Registry) - (controlling GFP) well 19B, plate 2 (''available and working'') | |||
*<big>[[IGEM:IMPERIAL/2006/project/parts/BBa_T9002|<font color=blue>'''T9002'''</font> Information Page]]</big> | |||
<br> | |||
:[[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. | |||
<br> | |||
:[[Image:T9002test.png|thumb|left|300px|Color added Part T9002 testing using Fuji-5400 machine]] | |||
<br style="clear:both;"/> | |||
<big><center><u>Part J37015:<br>Prey Cell Positve Feeback Test</u></center></big> | |||
<br> | |||
*<bbpart>J37015</bbpart> (Parts Registry) | |||
*<big>[[IGEM:IMPERIAL/2006/project/parts/BBa_J37015|<font color=blue>'''J37015'''</font> Information Page]]</big> | |||
<br> | |||
:[[image:J37015.JPG]] | |||
:This construct is used to characterise the positive feedback loop, with AHL levels being determined by the assay construct above. | |||
<br> | |||
<big><center><u>Part F2620:<br>Quorum sensing receiver</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 <font color=green>'''BBa_F2620'''</font>] - Well 6B, plate 1 (''available and working'') | |||
:[[image:F2620.JPG]] | |||
<br> | |||
<big><center><u>Part C0261:</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0261 <font color=blue>'''C0261'''</font>] - 24A Plate 1 | |||
<big><center><u>Part I13504:</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 <font color=blue>'''I13504'''</font>] - 12D Plate 1 | |||
<big><center><u>Part J37016:<br>Predator Transfer Function Characterisation (Single Promoter)</u></center></big> | |||
<br> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37016 BBa_J37016](Parts Registry) | |||
*<big>[[IGEM:IMPERIAL/2006/project/parts/BBa_J37016|J37016 Information Page]]</big> | |||
<br> | |||
:[[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. | |||
<br> | |||
<big><center><u>Part R0062:<br>Promoter (luxR & HSL regulated - lux pR)</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 <font color=blue>'''R0062'''</font>] - 9G Plate 1 | |||
<big><center><u>Part B0034:<br>RBS</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0034 <font color=blue>'''B0034'''</font>] - 3O Plate 1 | |||
<big><center><u>Part C0062:<br>Repressor/Activator, luxR</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0062 <font color=blue>'''C0062'''</font>] - 7A Plate 1 | |||
<big><center><u>Part I13504:<br>RBS & GFP</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 <font color=green>'''I13504'''</font>] - 12D Plate 1 | |||
<big><center><u>Part J37020:<br>Predator Transfer Function Characterisation (Double Promoter)</u></center></big> | |||
<br> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37020 BBa_J37020](Parts Registry) | |||
*<big>[[IGEM:IMPERIAL/2006/project/parts/BBa_J37016|J37020 Information Page]]</big> | |||
<br> | |||
:[[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. | |||
<big><center><u>Part J37018 & J37019:<br>For predator '''double''' promoter test construct</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37018 BBa_J37018] (Parts Registry)- to be built ! | |||
:[[image:J37018.JPG]] | |||
<br> | |||
<big><center><u>Part I13504:<br>For predator '''double''' promoter test construct</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:I13504 <font color=green>'''BBa_I13504'''</font>] - 12D Plate 1 | |||
<big><center><u>Part J37022:<br>AiiA Activity Assay</u></center></big> | |||
<br> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37022 BBa_J37022] - AiiA test construct (production) - with LVA and FLAG tags added [[IGEM:IMPERIAL/2006/project/parts/BBa_J37022|Protocol and Modelling Page for J37022]] | |||
<br> | |||
:[[Image:J37022.png]] | |||
<br> | |||
:same as: | |||
:[[Image:J37022a.png]]] | |||
<br> | |||
:AiiA with FLAG immunotag and LVA tag controlled by LacI under IPTG promotion | |||
<big><center><u>Part I13207:<br>HSL/AiiA test construct</u> (- not to be used anymore)</center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13207 <font color=purple>'''BBa_I13207'''</font>] (Parts Registry) - Well 2P, plate 1 (''available'') | |||
*[[IGEM:IMPERIAL/2006/project/parts/BBa_I13207|Protocol and Modelling Page for I13207]] | |||
!width="400" align="left" valign="top" style="background:#E6E8FA; color:black"| | |||
==Parts for Final Assembly== | |||
<big><center><u>Measuring the cell population + Cre-Lox system</u></center></big> | |||
*[http://openwetware.org/index.php?title=IGEM:IMPERIAL/2006/project/Oscillator/cellcre Cre-Lox Overview] | |||
<big><center><u>Tagging LuxR</u></center></big> | |||
*[http://openwetware.org/index.php?title=IGEM:IMPERIAL/2006/project/Oscillator/luxRtag LuxR Tag Overview] | |||
<big><center><u>Cre Lox Part</u></center></big> | |||
:[[Image:Redoing Cre.doc]] | |||
<big><center><u>Part F2620:<br>Quorum sensing receiver</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 <font color=green>'''BBa_F2620'''</font>] - Well 6B, plate 1 (''available and working'') | |||
:[[image:F2620.JPG]] | |||
<br> | |||
<big><center><u>Part F1610:<br>Quorum sensing sender LuxI</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_F1610 <font color=purple>'''BBa_F1610'''</font>] - Well 1B, plate 2 (''available and working'') | |||
:[[image:F1610.JPG]] | |||
<br> | |||
<big><center><u>Part J37019:<br>For predator '''single''' promoter final assembly</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37019 BBa_J37019] | |||
:[[image:J37019.JPG]] | |||
<br> | |||
<big><center><u>Part R0062:<br>pLuxR Promoter</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 <font color=blue>'''BBa_R0062'''</font>] - Well 9G, plate 1 (''available and working'') | |||
<big><center><u>Part B0034:<br>RBS</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0034 <font color=blue>'''BBa_B0034'''</font>] - Well 3O plate 1 (''available and working'') | |||
<big><center><u>Part C0062:<br>LuxR coding region</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0062 <font color=blue>'''BBa_C0062'''</font>] - Well 7A, plate 1 (''available'') | |||
<br> | |||
<big><center><u>Part J37018 & J37019:<br>For predator '''double''' promoter final assembly</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37018 BBa_J37018] | |||
:[[image:J37018.JPG]] | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37019 BBa_J37019] | |||
:[[Image:J37019.PNG]] | |||
<br> | |||
<big><center><u>Part J37019:<br>Terminators and pLuxR promoter</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13033 <font color=green>'''BBa_I13033'''</font>] - Well 2H, plate 1 (''available'') | |||
<br> | |||
<big><center><u>Part J37025:<br>AiiA enzyme coding region</u></center></big> | |||
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37025 BBa_J37025] | |||
*Needs creating | |||
:[[Image:J37025.png|150px]] | |||
<br> | |||
|} | |||
==Parts for Final Assembly== | ==Parts for Final Assembly== | ||
===Measuring the cell population + Cre-Lox system=== | |||
[[http://openwetware.org/index.php?title=IGEM:IMPERIAL/2006/project/Oscillator/cellcre Overview]] | |||
===Tagging LuxR=== | ===Tagging LuxR=== | ||
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[[Image:J37025.png|150px]] | [[Image:J37025.png|150px]] | ||
<br> | <br> | ||
==Parts for Testing Phase== | ==Parts for Testing Phase== | ||
Line 115: | Line 334: | ||
AiiA with FLAG immunotag and LVA tag controlled by LacI under IPTG promotion | AiiA with FLAG immunotag and LVA tag controlled by LacI under IPTG promotion | ||
==E-mail from Mr. Barry Canton of MIT regarding parts== | ==E-mail from Mr. Barry Canton of MIT regarding parts== |
Latest revision as of 08:31, 8 October 2006
Part Status
See the Imperial iGEM Lab Status page for the current status of required parts, plus information on colour coding
aiiA
This is the progress of the aiiA gene
Parts for Testing PhasePrey Transfer Function Characterisation/ AHL Assay Cell
Prey Cell Positve Feeback Test
Quorum sensing receiver
Predator Transfer Function Characterisation (Single Promoter)
Promoter (luxR & HSL regulated - lux pR)
RBS
Repressor/Activator, luxR
RBS & GFP
Predator Transfer Function Characterisation (Double Promoter)
For predator double promoter test construct
For predator double promoter test construct
AiiA Activity Assay
HSL/AiiA test construct (- not to be used anymore)
|
Parts for Final Assembly
Quorum sensing receiver
Quorum sensing sender LuxI
For predator single promoter final assembly
pLuxR Promoter
RBS
LuxR coding region
For predator double promoter final assembly
Terminators and pLuxR promoter
AiiA enzyme coding region
|
---|
Parts for Final Assembly
Measuring the cell population + Cre-Lox system
[Overview]
Tagging LuxR
[overview]
Cre Lox Part
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
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.
Prey Cell Positve Feeback Test
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)
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)
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
- 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.
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