IGEM:Imperial/2010/Lab Work

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(LAB TEAMS: help dry lab)
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(LAB TEAMS)
 
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{{Imperial2010/Header}}
==LAB TEAMS==
==LAB TEAMS==
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Our group has divided into three lab pairs in order to cope with the load of wet lab work. Each lab team will be responsible for the pathway indicated by the team's name while Wolfgang would act as a central co-ordinator. The co-ordinator would have the role ensuring all three groups are on schedule but also have a deep understanding of all components of the project. This will ensure that Wolfgang would be able to present the project with confidence in Boston (alongside with Ben).  
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Our group has divided into three lab pairs in order to cope with the load of wet lab work. Each lab team will be responsible for the pathway indicated by the team's name while Wolfgang would act as a central co-ordinator. The co-ordinator would have the role ensuring all three groups are on schedule but also have a deep understanding of all components of the project. This will ensure that Wolfgang would be able to present the project with confidence in Boston (alongside with Ben). For summerized information on the overall progress click [[IGEM:Imperial/2010/Strategy | here]].
   
   
*[[IGEM:Imperial/2010/XylE_team | XylE team (Maddie and Nicolas)]]
*[[IGEM:Imperial/2010/XylE_team | XylE team (Maddie and Nicolas)]]
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==DRY LAB WISH LIST==
==DRY LAB WISH LIST==
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'''Experiments we wish that could be carried out for us'''
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* Determine maximum concentration of sD that cells can produce – crucial! (''in vivo''):
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Compare activities with the next one
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* Determine how much XylE we need to produce prior adding catechol (the threshold value to be determined). (''in vitro then maybe in vivo'')
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In vivo IPTG? -> compare the cultures that have less of it
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* Determine XylE and TEV production. (''in vivo'')
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XylE -> maybe, but very noisy. Use robot to induce production and measure activities
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TEV -> FRET pairs with TEV link
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* Degradation of XylE (''in vivo'' or ''in vitro'' if the cell division is not taken into account)
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Yes -> Monitoring activity and then approximate the concentration. Remove the IPTG. Use robot
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* Can we determine TEV kinetics (kcat, Km?) on XylE-GFP? (''in vitro'')
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Yes
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<!-- Old wishes - please don't remove - just keeping them in case wanted to check something
===Parameters to be obtained===
===Parameters to be obtained===
* rate of production of Dioxygenase and TEV in B.sub
* rate of production of Dioxygenase and TEV in B.sub
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* threshold in XylE concentration for detectable signal!
===Research===
===Research===
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* Mechanism of ComE, ComD singalling:
* Mechanism of ComE, ComD singalling:
Piotr: I have trouble making sense out of information given in papers. Help with this one very much appreciated.
Piotr: I have trouble making sense out of information given in papers. Help with this one very much appreciated.
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Understanding so far:
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ComD + CSP <-> ComD-P  (Autophosphorylation)
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ComD-P + ComE -> ComE-P + ComD
Objective: '''Describe how ComD works. Does it phospohrylate continously after AIP binding or is it rather 1 binding → 1 phosphorylation. What is the slowest reaction step?'''
Objective: '''Describe how ComD works. Does it phospohrylate continously after AIP binding or is it rather 1 binding → 1 phosphorylation. What is the slowest reaction step?'''
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Please update what you have read (even if useless) so we don't keep reading the same papers all over again.
Please update what you have read (even if useless) so we don't keep reading the same papers all over again.
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-->

Current revision

LAB TEAMS

Our group has divided into three lab pairs in order to cope with the load of wet lab work. Each lab team will be responsible for the pathway indicated by the team's name while Wolfgang would act as a central co-ordinator. The co-ordinator would have the role ensuring all three groups are on schedule but also have a deep understanding of all components of the project. This will ensure that Wolfgang would be able to present the project with confidence in Boston (alongside with Ben). For summerized information on the overall progress click here.

Lab supervisor: Wolfgang

DRY LAB WISH LIST

Experiments we wish that could be carried out for us

  • Determine maximum concentration of sD that cells can produce – crucial! (in vivo):

Compare activities with the next one

  • Determine how much XylE we need to produce prior adding catechol (the threshold value to be determined). (in vitro then maybe in vivo)

In vivo IPTG? -> compare the cultures that have less of it

  • Determine XylE and TEV production. (in vivo)

XylE -> maybe, but very noisy. Use robot to induce production and measure activities TEV -> FRET pairs with TEV link

  • Degradation of XylE (in vivo or in vitro if the cell division is not taken into account)

Yes -> Monitoring activity and then approximate the concentration. Remove the IPTG. Use robot

  • Can we determine TEV kinetics (kcat, Km?) on XylE-GFP? (in vitro)

Yes


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