Notebook:Federico Castro M/Projects/Repressilator

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Current revision (17:38, 18 November 2008) (view source)
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<small>Last updated January/17/2008</small>
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<small>Last updated November/18/2008</small>
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{{p|Repressilator|
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<!-- {{p|Repressilator|
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}} -->
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Project status: <font color="green">'''Active'''</font>
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Project status: <font color="skyblue">'''Frozen'''</font>
== The Design ==
== The Design ==
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In order to improve lab skills and to see with my own eyes the repressilator I intended to assemble it with the biobricks available at 2007 part kits.  
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I intend to assemble the repressilator from [http://partsregistry.org/wiki/index.php?title=Part:BBa_I5612 BBa_I5612] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I6032 BBa_I6032]. I would like to do so in order to improve my lab skills. Besides the parts obtained will be extremely usefull for the [[User:Federico_Castro_M/Notebook/Spatial_Patterns | Spatial Patterns project ]] as BBa_I6032 might be used to observe  [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0030 EYFP] and BBa_I5612 will be usefull just in case we would like to change the reporter gene used for the repressillator.
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At first I tough I could build the repressilator with 3 inverters, one protein generator and one single part... then I realized that almost all the devise was already assembled into one huge biobrick DUH! While searching within the registry I found that only EYFP had the correct degradation tag; other fluorescent proteins had a tag that was to strong for this devise or had no tag at all.
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[[Image:Fed2.JPG|center|Repressilator No2]]
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It seems that I will have to use EYFP (I kind of dislike EYFP) so the final construction will be exactly like <bbpart>BBa_I5611</bbpart> O_o Even when the final construction is already available the biobricks obtained and the practice should be worth the time and hard work. Besides, the whole process should be very simple and It shouldn't take more than a week of work.
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== Issues ==
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Now I only have to transform <bbpart>BBa_S03151</bbpart> and <bbpart>BBa_E0430</bbpart> and assemble them.  
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I'm a little worried about using EYFP as it doesn't have a [http://partsregistry.org/wiki/index.php?title=Part:BBa_M0050 ssrA tag]. Yet the whole repressillator available in the iGEM kit plates as [http://partsregistry.org/wiki/index.php?title=Part:BBa_I5611 BBa_I5611] seems to use EYFP without the tag. I could use [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0022 ECFP] instead of EYFP since it has the ssrA tag that Ellowitz used yet [[User:Jason_R._Kelly | Jason R. Kelly]] [[User_talk:Federico_Castro_M | advised me]] against using that part.
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[[Image:Fed1.JPG|center|Repressilator No1]]
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Alternatively I could use the parts <bbpart>BBa_I5612</bbpart> and <bbpart>BBa_I13602</bbpart>.
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For now I'd recomend just to assemble the repressillator with BBa_I5612 and BBa_I6032 and it it fails we can modify it. Also I'd recomend to recover BBa_I5611 just to check how the final devise should work.
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[[Image:FEDXI.JPG|center|Repressilator No2]]
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== Work to do ==
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I don't know why there are so few GFPs with LVA tags at the registry. Elowitz used a GFP instead of a EYFP. I'm not such a purist so I will make my own devise devise with EYFP in the plasmid pSB1A2 and I wont use IPTG (anyway Elowitz was unable to synchronize the cells with IPTG).
 
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== Issues ==
 
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Have you seen the zize of I5612? It's 3337 bp and thats a lot for a plasmid and yet I want to add some 886 bp... I wonder if that would burden the plasmid so much that it would be unable to replicate along with the bacteria yet the whole construction with 4223bp (<bbpart>BBa_I5611</bbpart>) is available.
 
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There is only one way to know if a plasmid can hold 4000+ bp hahahaha.
 
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=Project Entries=
 
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[[Image:OWWLabNotebook100.png‎|right|Notebook]]
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Current revision


Last updated November/18/2008

Contents


Project status: Frozen

The Design

I intend to assemble the repressilator from BBa_I5612 and BBa_I6032. I would like to do so in order to improve my lab skills. Besides the parts obtained will be extremely usefull for the Spatial Patterns project as BBa_I6032 might be used to observe EYFP and BBa_I5612 will be usefull just in case we would like to change the reporter gene used for the repressillator.

Repressilator No2

Issues

I'm a little worried about using EYFP as it doesn't have a ssrA tag. Yet the whole repressillator available in the iGEM kit plates as BBa_I5611 seems to use EYFP without the tag. I could use ECFP instead of EYFP since it has the ssrA tag that Ellowitz used yet Jason R. Kelly advised me against using that part.

For now I'd recomend just to assemble the repressillator with BBa_I5612 and BBa_I6032 and it it fails we can modify it. Also I'd recomend to recover BBa_I5611 just to check how the final devise should work.

Work to do

Notebook

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