Difference between revisions of "Part:BBa K177038:Experience"

 
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==Experimental Results==
  
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'''UPO-Sevilla Team''' ([http://2011.igem.org/Team:UPO-Sevilla Wiki])
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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how you used this part and how it worked out.
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===Applications of BBa_K177038===
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Two are the most important features of a flip flop: Time of the change of state and stability when the inducer is retired.Then these are the condition that we have observed to characterize this genetic device. I set overnight inoculums inducing a condition (adding 0,1 mM IPTG or growing at 42ºC). The morning after I make a 1/100 dilution, and let it grow until de optic density reaches 0,2. In that moment I change the conditions, to test the change between states or just retire the inducers, to observe the stability of one state in a long time.
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It’s important not to use LB media, because it contains cyclic aminoacids which make it fluorescent. For all this experiments we used Minimal Media supplemented with 0,1% casminoacids. Another problem we faced is that RFP is also excited in the wavelength we use for GFP, making our measure a little bit inaccurate. A solution to this may be insert the bistable in the bacterial chromosome, using the Mini-Tn7 based system my team-mate has developed for biobricks. The less copies of the construction, the better the regulation is, because there is more repressors for each copy of the promoters.
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In the following charts, the Y-axis represents the amount of fluorescence and the other axis the time in minutes.
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https://static.igem.org/mediawiki/2011/2/27/UPOSevilla42-IPTG.png
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The data shows how the GFP increases and the RFP lowers, just as expected. But when you test longer times, this effect no longer exists. The behaviour is the opposite as the one in the beginning of the experiments.
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https://static.igem.org/mediawiki/2011/c/c0/UPOSevillaIPTG-42.png
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The results obtained for the reverse switch fit perfectly to the expectations. Two repetitions of each switch were made, giving unequal results as it is shown in the graphs. We expect this great variation to be reduced in the improved models we are developing.
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We’ve only tried the stability of only the RFP state, finding out it last 16 h at least. This effect is very noticeable when the growth takes place in a plaque, where you can see the change of colour without UV-light.
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==Mathematical Modeling==
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Modeling/Basic_Bistable Basic Bistable]
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Modeling/Toggle_Switch Toggle Switch]
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Modeling/Other_Models Other Models]
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==Multiagent Modeling==
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/Overview Overview]
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/How_does_it_work How does it work?]
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/How_to_use_it How to use it?]
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* [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/Results Results]
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==Applications of BBa_K177038==
  
 
===User Reviews===
 
===User Reviews===
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<partinfo>BBa_K177038 AddReview 1</partinfo>
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<I>Tokyo-Tech iGEM 2013</I>
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We constructed primers every 500 bases and tried to analyze the whole base sequence, but we could only confirm the existence of LacI and RFP.

Latest revision as of 18:41, 26 September 2013

Experimental Results

UPO-Sevilla Team ([http://2011.igem.org/Team:UPO-Sevilla Wiki])

Two are the most important features of a flip flop: Time of the change of state and stability when the inducer is retired.Then these are the condition that we have observed to characterize this genetic device. I set overnight inoculums inducing a condition (adding 0,1 mM IPTG or growing at 42ºC). The morning after I make a 1/100 dilution, and let it grow until de optic density reaches 0,2. In that moment I change the conditions, to test the change between states or just retire the inducers, to observe the stability of one state in a long time.

It’s important not to use LB media, because it contains cyclic aminoacids which make it fluorescent. For all this experiments we used Minimal Media supplemented with 0,1% casminoacids. Another problem we faced is that RFP is also excited in the wavelength we use for GFP, making our measure a little bit inaccurate. A solution to this may be insert the bistable in the bacterial chromosome, using the Mini-Tn7 based system my team-mate has developed for biobricks. The less copies of the construction, the better the regulation is, because there is more repressors for each copy of the promoters. In the following charts, the Y-axis represents the amount of fluorescence and the other axis the time in minutes.

UPOSevilla42-IPTG.png

The data shows how the GFP increases and the RFP lowers, just as expected. But when you test longer times, this effect no longer exists. The behaviour is the opposite as the one in the beginning of the experiments.

UPOSevillaIPTG-42.png

The results obtained for the reverse switch fit perfectly to the expectations. Two repetitions of each switch were made, giving unequal results as it is shown in the graphs. We expect this great variation to be reduced in the improved models we are developing.

We’ve only tried the stability of only the RFP state, finding out it last 16 h at least. This effect is very noticeable when the growth takes place in a plaque, where you can see the change of colour without UV-light.

Mathematical Modeling

  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Modeling/Basic_Bistable Basic Bistable]
  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Modeling/Toggle_Switch Toggle Switch]
  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Modeling/Other_Models Other Models]

Multiagent Modeling

  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/Overview Overview]
  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/How_does_it_work How does it work?]
  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/How_to_use_it How to use it?]
  • [http://2011.igem.org/Team:UPO-Sevilla/Project/Basic_Flip_Flop/Multiagent_System/Results Results]

Applications of BBa_K177038

User Reviews

UNIQffdf3cd09b04d8cb-partinfo-00000000-QINU UNIQffdf3cd09b04d8cb-partinfo-00000001-QINU


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Tokyo-Tech iGEM 2013


We constructed primers every 500 bases and tried to analyze the whole base sequence, but we could only confirm the existence of LacI and RFP.