Team:ETHZ Basel/Biology/Status

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{{ETHZ_Basel10}}
{{ETHZ_Basel10}}
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{{ETHZ_Basel10_Biology}}
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= generation of subparts =
+
= generation of subparts, linkers and vectors =
{| border="1"
{| border="1"
|+ subparts
|+ subparts
-
! !! Protein !! primers !! PCR product !! library of transformants !! single colonies !! storage vector !! insert
+
! !! Protein !! primers !! PCR product !! storage vector !! digest !! sequencing !! correct construct
|-
|-
! che proteins
! che proteins
-
| CheY-A || recieved || purified on stock || on agar plate || not available || purified on stock || on stock
+
| CheY-A || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| CheY-C || recieved || purified in stock || on agar plate || not available || purified on stock || on stock
+
| CheY-C || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| CheB-A || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock || on stock
+
| CheB-A || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| CheB-C || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock || on stock
+
| CheB-C || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| CheR-A || recieved || purified in stock || on agar plate || 3 on agar plate || digest has to be redone ||
+
| CheR-A || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| CheR-C || recieved || purified in stock || on agar plate || 3 on agar plate || digest has to be redone ||
+
| CheR-C || yes || purified || yes || worked || in progress
|-
|-
 +
! localizers
! localizers
-
| TrigF-A || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock ||
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| TrigF-A || yes || purified || yes || in progress ||
|-
|-
!  
!  
-
| MreB-C || recieved || purified in stock || in process || ||  ||
+
| MreB-C || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| tetR-A || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock || on stock
+
| tetR-A || yes || purified || yes || worked ||
|-
|-
-
! couplers
+
!  
-
| PIF3-A || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock ||
+
| tetO7 || yes || purified || yes || worked || verified || tetO7_2
 +
|-
 +
 
 +
! linkers
 +
| PIF3-A || yes || purified || yes || in progress ||
|-
|-
!  
!  
-
| PIF3-C || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock ||
+
| PIF3-C || yes || purified || yes || in progress ||
|-
|-
!  
!  
-
| PhyB-C || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock ||
+
| PhyB-C || yes || purified || yes || in progress ||
|-
|-
! reporters
! reporters
-
| cyFP-A || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock || on stock
+
| cyFP-A || yes || purified || yes || worked || verified
|-
|-
!  
!  
-
| cyFP-C || recieved || purified in stock || on agar plate || 3 on agar plate || purified on stock ||
+
| cyFP-C || yes || purified || yes || in progress ||
|-  
|-  
|}
|}
 +
<br>Linker oligos are recieved and don't need to be treated further. They can be used directly for the generation of the BioBricks.
 +
<br>storage and acceptor vectors are available.
 +
<br>PIF3 and PhyB were additionally ordered for synthesis (codon optimized). Not yet recieved.
= generation of biobricks =
= generation of biobricks =
 +
We'll talk to Fabian. Apparently he has a nice protocol to generate acceptor vectors and transform ''E. coli'' sequentially in a 96 well plate.
-
== testing of che-fusions ==
+
= testing of biobricks =
 +
=== testing of che-fusions ===
We're currently trying to establish an assay to test for the chemotactic behavour of ''E. coli''. A first try with a "syringe test" failed. We'll redo the test and are looking for other possibilites. Maybe a swarm test?
We're currently trying to establish an assay to test for the chemotactic behavour of ''E. coli''. A first try with a "syringe test" failed. We'll redo the test and are looking for other possibilites. Maybe a swarm test?
 +
*tested biobricks: che-fusions
 +
*negative control: ''E. coli'' mutated for the accoding che-protein
 +
*positive control: wild type ''E. coli''
-
 
+
=== testing of localicer-fusions ===
-
 
+
-
== testing of localicer-fusions ==
+
We are planning to use a fluorescence microscope in order to visualize the distribution of localizer-cyFP fusions within a single ''E. coli''.
We are planning to use a fluorescence microscope in order to visualize the distribution of localizer-cyFP fusions within a single ''E. coli''.
-
== testing of couplers ==
+
=== testing of couplers ===
-
We're planning to use the setup as for the testing of the localizers. In this case we're going to use the following biobricks:
+
We're planning to use the setup as for the testing of the localizers.
-
*localizer-PhyB and PIF3-cyFP or
+
*tested biobricks: localizer-PhyB and PIF3-cyFP, localizer-PIF3 and PhyB-cyFP
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*localizer-PIF3 and PhyB-cyFP
+
Images should look as in testing of localizer-fusions. Preliminary clarificaiton of the suitability of the approach are to be done.
-
Images should look as in testing of localizers
+
-
<br>Preliminary clarificaiton of the suitability of the approach are to be done.
+
-
 
+
-
= testing of biobricks =
+
= bringing ''E. lemming'' alive =
= bringing ''E. lemming'' alive =

Latest revision as of 11:49, 12 October 2010

generation of subparts, linkers and vectors

subparts
Protein primers PCR product storage vector digest sequencing correct construct
che proteins CheY-A yes purified yes worked verified
CheY-C yes purified yes worked verified
CheB-A yes purified yes worked verified
CheB-C yes purified yes worked verified
CheR-A yes purified yes worked verified
CheR-C yes purified yes worked in progress
localizers TrigF-A yes purified yes in progress
MreB-C yes purified yes worked verified
tetR-A yes purified yes worked
tetO7 yes purified yes worked verified tetO7_2
linkers PIF3-A yes purified yes in progress
PIF3-C yes purified yes in progress
PhyB-C yes purified yes in progress
reporters cyFP-A yes purified yes worked verified
cyFP-C yes purified yes in progress


Linker oligos are recieved and don't need to be treated further. They can be used directly for the generation of the BioBricks.
storage and acceptor vectors are available.
PIF3 and PhyB were additionally ordered for synthesis (codon optimized). Not yet recieved.

generation of biobricks

We'll talk to Fabian. Apparently he has a nice protocol to generate acceptor vectors and transform E. coli sequentially in a 96 well plate.

testing of biobricks

testing of che-fusions

We're currently trying to establish an assay to test for the chemotactic behavour of E. coli. A first try with a "syringe test" failed. We'll redo the test and are looking for other possibilites. Maybe a swarm test?

  • tested biobricks: che-fusions
  • negative control: E. coli mutated for the accoding che-protein
  • positive control: wild type E. coli

testing of localicer-fusions

We are planning to use a fluorescence microscope in order to visualize the distribution of localizer-cyFP fusions within a single E. coli.

testing of couplers

We're planning to use the setup as for the testing of the localizers.

  • tested biobricks: localizer-PhyB and PIF3-cyFP, localizer-PIF3 and PhyB-cyFP

Images should look as in testing of localizer-fusions. Preliminary clarificaiton of the suitability of the approach are to be done.

bringing E. lemming alive

timetable

experimental plan
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