Team:Paris/Addressing testing

From 2009.igem.org

(Difference between revisions)
(→WetLab - Addressing the message)
(→Functional Testing:)
 
(76 intermediate revisions not shown)
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-
'''global constructions :'''
+
 
 +
<font style="color:black;font-weight:bold; font-size:21px">'''Global constructions :'''</font>
 +
 
 +
 
 +
 
 +
<center><font style="color:green;font-weight:bold; font-size:16px">The green color means the experiment was a success</font></center>
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Time required : A lOT !!!!!
 
 +
 +
<font style="color:black;font-weight:bold; font-size:18px"><I>'''Experiments ran :'''</I></font>
-
'''Experiments ran :'''
 
{|
{|
-
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold; "
+
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold;font-size:14px "
-
|width="300px" ; bgcolor="#F8F8F8"| column 1
+
|width="300px" ; bgcolor="#F8F8F8"| Column 1
-
|width="300px" ; bgcolor="#E0E3FE"| column 2
+
|width="300px" ; bgcolor="#E0E3FE"| Column 2
-
|width="300px" ; bgcolor="#CBD1FD"| column 3
+
|width="300px" ; bgcolor="#CBD1FD"| Column 3
-
|width="300px" ; bgcolor="#BCCDFD"| column 4
+
|width="300px" ; bgcolor="#BCCDFD"| Column 4
|- style="background: #bebebe; text-align: center;"
|- style="background: #bebebe; text-align: center;"
-
|'''PCR :'''  
+
|bgcolor="#F8F8F8"|'''PCR :'''  
-
ClyA Cterm  matrix : [https://2009.igem.org/Team:Paris/Fridge F4] Oligo :[https://2009.igem.org/Team:Paris/Freezer_Primers#O10 O10] and [https://2009.igem.org/Team:Paris/Freezer_Primers#O31 O31]  TM :55°C
+
pBAD: plate 2008
-
ClyA Nterm  matrix : [https://2009.igem.org/Team:Paris/Fridge F4] Oligo : [https://2009.igem.org/Team:Paris/Freezer_Primers#O32 O32] and [https://2009.igem.org/Team:Paris/Freezer_Primers#O9 O9] TM :55°C
 
-
mRFP Cterm  matrix : ? Oligo :O59 and O60  TM :
+
ClyA Cterm  matrix : [https://2009.igem.org/Team:Paris/Fridge F4]
-
mRFP Nterm  plasmid : ? Oligo :O57 and O58 TM :
+
Oligo :[https://2009.igem.org/Team:Paris/Freezer_Primers#O10 O10] and [https://2009.igem.org/Team:Paris/Freezer_Primers#O31 O31]  TM :55°C
-
|'''PCR :'''
+
-
-
 
-
|'''PCR :'''
 
-
-
+
mRFP Nterm  plasmid : pSB1A3
-
|'''PCR :'''
+
 
 +
Oligo :O57 and O58 TM :
 +
 
 +
 
 +
ClyA Nterm  matrix : [https://2009.igem.org/Team:Paris/Fridge F4]
 +
 
 +
Oligo : [https://2009.igem.org/Team:Paris/Freezer_Primers#O32 O32] and [https://2009.igem.org/Team:Paris/Freezer_Primers#O9 O9] TM :55°C
 +
 
 +
 
 +
mRFP Cterm  matrix : pSB1A3
 +
 
 +
Oligo :O59 and O60  TM :
 +
 
 +
|bgcolor="#E0E3FE"|
 +
-
 +
|bgcolor="#CBD1FD"|
 +
-
 +
|bgcolor="#BCCDFD"|
-
-
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''Verification on gel :'''
+
|bgcolor="#F8F8F8"|'''Verification on gel :'''
ok
ok
-
|'''Verification on gel :'''
+
|bgcolor="#E0E3FE"|
-
 
+
-
-
-
|'''Verification on gel :'''
+
|bgcolor="#CBD1FD"|
-
-
-
|'''Verification on gel :'''
+
|bgcolor="#BCCDFD"|
-
 
+
-
-
 +
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''Purification on gel :'''
+
|bgcolor="#F8F8F8"| '''Purification on gel :'''
ok
ok
-
|'''Purification on gel :'''
+
|bgcolor="#E0E3FE"|
-
 
+
-
-
-
|'''Purification on gel :'''
 
 +
|bgcolor="#CBD1FD"|
-
-
-
|'''Purification on gel :'''
 
 +
|bgcolor="#BCCDFD"|
-
-
 +
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''digestion:'''  
+
|bgcolor="#F8F8F8"|'''Digestion:'''  
ClyA Cterm S/P
ClyA Cterm S/P
 +
RFP  Nterm X/P
RFP  Nterm X/P
-
PBAD  vector : ? E/S
 
 +
pBAD  vector : ? E/S
-
|'''digestion:'''
 
-
ClyA Cter-RFP Nter vector PSB1A3 E/X
+
|bgcolor="#E0E3FE"|'''Digestion:'''
-
|'''digestion:'''
+
ClyA Cter-RFP Nter vector PSB1A3 E/X
 +
|bgcolor="#CBD1FD"|
-
-
-
|'''digestion:'''
+
|bgcolor="#BCCDFD"|
-
 
+
-
-
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''verification digestion:'''
+
|bgcolor="#F8F8F8"| '''Verification digestion:'''
ok
ok
-
|'''verification digestion:'''
+
|bgcolor="#E0E3FE"|'''Verification digestion:'''
ok
ok
-
|'''verification digestion:'''
+
|bgcolor="#CBD1FD"|-
-
 
+
|bgcolor="#BCCDFD"|
-
-
-
|'''verification digestion:'''
 
-
 
-
-
 
-
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''ligation:'''  
+
|bgcolor="#F8F8F8"|'''Ligation:'''  
ClyA Cter-RFP Nter  vector PSB1A3
ClyA Cter-RFP Nter  vector PSB1A3
-
|'''ligation:'''  
+
|bgcolor="#E0E3FE"|'''Ligation:'''  
PBAD  E/S  ClyA Cter-RFP Nter PSB1A3 E/X (x2)
PBAD  E/S  ClyA Cter-RFP Nter PSB1A3 E/X (x2)
-
PBAD  S/P  ClyA Cter-RFP Nter PSB?  X/P (x2)
 
-
|'''ligation:'''
+
 
 +
PBAD  S/P  ClyA Cter-RFP Nter PSB2K3  X/P (x2)
 +
 
 +
|bgcolor="#CBD1FD"|'''Ligation:'''
-
-
-
|'''ligation:'''
+
|bgcolor="#BCCDFD"|
-
+
 
-
-
+
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''PCR colo :'''
+
|bgcolor="#F8F8F8"| '''Colony PCR :'''
ok
ok
-
|'''PCR colo :'''
+
|bgcolor="#E0E3FE"|'''Colony PCR :'''
ok
ok
-
|'''PCR colo :'''
+
|bgcolor="#CBD1FD"|'''Colony PCR :'''
-
-
+
ok
-
|'''PCR colo :'''
+
|bgcolor="#BCCDFD"|
-
-
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''miniprep:'''
+
|bgcolor="#F8F8F8"|'''Miniprep:'''
clone :
clone :
-
|'''miniprep:'''
+
|bgcolor="#E0E3FE"|'''Miniprep:'''
clone :
clone :
-
|'''miniprep:'''
+
|bgcolor="#CBD1FD"|'''Miniprep:'''
-
-
+
clone 3
-
|'''miniprep:'''
+
 
 +
clone 5
 +
 
 +
|bgcolor="#BCCDFD"|
-
-
 
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''sequencing :'''
+
|bgcolor="#F8F8F8"|'''Sequencing :'''
ok
ok
-
|'''sequencing :'''
+
|bgcolor="#E0E3FE"|'''Sequencing :'''
ok
ok
-
|'''sequencing :'''
+
|bgcolor="#CBD1FD"|'''Sequencing :'''
-
-
+
ok
-
|'''sequencing :'''
+
 +
|bgcolor="#BCCDFD"|
-
-
 +
|- style="background: #d8d8d8; text-align: center;"   
|- style="background: #d8d8d8; text-align: center;"   
-
|'''stock glycerol:'''
+
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
ClyA CTer: S47(clone 3) and S48(clone 7)
ClyA CTer: S47(clone 3) and S48(clone 7)
 +
ClyA Nter: S72
ClyA Nter: S72
 +
RFP Cter: S55 (Clone 3)
RFP Cter: S55 (Clone 3)
 +
RFP Nter: S56 (Clone 3)
RFP Nter: S56 (Clone 3)
-
|'''stock glycerol'''
+
|bgcolor="#E0E3FE"|'''Stock glycerol'''
Cly A(Cter)-(Nter)RFP: S72(clone 8)
Cly A(Cter)-(Nter)RFP: S72(clone 8)
-
|'''stock glycerol'''
+
|bgcolor="#CBD1FD"|'''Stock glycerol'''
-
-
+
pBAD ClyA RFP : S89 (clone 3)
-
|'''stock glycerol'''
+
-
-
+
pBAD ClyA RFP: S90 (clone 5)
 +
 
 +
|bgcolor="#BCCDFD"|
Line 280: Line 300:
-
PBAD ClyA RFP on PSB3T5 was transformed into Delta Tol bacteria , in this case we are supposed to see fluorescent vesicles went the medium contains 1 % arabinose , and to have no fluorescent on 1% glucose.
+
PBAD ClyA RFP on PSB3T5 was transformed into Delta Tol bacteria , in this case we are supposed to see fluorescent vesicles when the medium contains 1 % arabinose , and to have no fluorescent on 1% glucose.
 +
 
 +
 
 +
To learn more, see the [[Team:Paris/Conclusion | Conclusion page]]
Line 295: Line 318:
-
Time required : A week.
 
 +
<font style="color:black;font-weight:bold; font-size:18px"><I>'''Experiments ran :'''</I></font>
-
Experiments ran :
 
{|
{|
-
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold; "
+
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold;font-size:14px "
-
|width="300px" ; bgcolor="#F8F8F8"| column 1
+
|width="300px" ; bgcolor="#F8F8F8"| Column 1
-
|width="300px" ; bgcolor="#E0E3FE"| column 2
+
|width="300px" ; bgcolor="#E0E3FE"| Column 2
-
|width="300px" ; bgcolor="#CBD1FD"| column 3
+
|width="300px" ; bgcolor="#CBD1FD"| Column 3
-
|width="300px" ; bgcolor="#BCCDFD"| column 4
+
|width="300px" ; bgcolor="#BCCDFD"| Column 4
-
 
+
|- style="background: #bebebe; text-align: center;"
|- style="background: #bebebe; text-align: center;"
-
|'''PCR :'''  
+
|bgcolor="#F8F8F8"|'''PCR :'''  
TatABCE matrix :
TatABCE matrix :
-
|'''PCR :'''
+
|bgcolor="#E0E3FE"|-
-
|'''PCR :'''
+
|bgcolor="#CBD1FD"|-
-
|'''PCR :'''
+
|bgcolor="#BCCDFD"|-
 +
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''Verification on gel :'''
+
|bgcolor="#F8F8F8"|'''Verification on gel :'''
ok
ok
-
|'''Verification on gel :'''
+
|bgcolor="#E0E3FE"|-
-
-
+
|bgcolor="#CBD1FD"|
-
 
+
-
|'''Verification on gel :'''
+
-
-
-
|'''Verification on gel :'''
+
|bgcolor="#BCCDFD"|
-
-
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''Purification on gel :'''
+
|bgcolor="#F8F8F8"|'''Purification on gel :'''
ok
ok
-
|'''Purification on gel :'''
+
|bgcolor="#E0E3FE"|
-
-
-
|'''Purification on gel :'''
+
|bgcolor="#CBD1FD"|
-
-
-
|'''Purification on gel :'''
+
|bgcolor="#BCCDFD"|
-
-
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|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''digestion:'''  
+
|bgcolor="#F8F8F8"|'''Digestion:'''  
-
TatABCE
+
none
-
|'''digestion:'''  
+
|bgcolor="#E0E3FE"|'''Digestion:'''  
-
|'''digestion:'''  
+
none
-
|'''digestion:'''  
+
|bgcolor="#CBD1FD"|'''Digestion:'''  
 +
none
 +
|bgcolor="#BCCDFD"|'''Digestion:'''  
 +
none
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''verification digestion:'''
+
|bgcolor="#F8F8F8"|'''Digestion checking:'''
 +
none
-
ok
+
|bgcolor="#E0E3FE"|'''Digestion checking:'''
 +
none
-
|'''verification digestion:'''
 
-
-
+
|bgcolor="#CBD1FD"|'''Digestion checking:'''
-
|'''verification digestion:'''
+
none
 +
|bgcolor="#BCCDFD"|'''Digestion checking:'''
-
-
+
none
-
|'''verification digestion:'''
+
-
 
+
-
-
+
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''ligation:'''  
+
|bgcolor="#F8F8F8"|'''Ligation:'''  
-
TatABCE
+
none
-
 
+
|bgcolor="#E0E3FE"|'''Ligation:'''  
-
|'''ligation:'''  
+
none
-
|'''ligation:'''  
+
|bgcolor="#CBD1FD"|'''Ligation:'''  
-
|'''ligation:'''  
+
none
 +
|bgcolor="#BCCDFD"|'''Ligation:'''  
 +
none
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''PCR colo :'''
+
|bgcolor="#F8F8F8"|'''Colony PCR :'''
 +
none
-
ok
+
|bgcolor="#E0E3FE"|'''Colony PCR :'''
 +
none
-
|'''PCR colo :'''
+
|bgcolor="#CBD1FD"|'''Colony PCR :'''
 +
none
 +
|bgcolor="#BCCDFD"|'''Colony PCR :'''
 +
none
-
-
+
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Miniprep:'''
-
|'''PCR colo :'''
+
none
-
-
+
|bgcolor="#E0E3FE"|'''Miniprep:'''
-
|'''PCR colo :'''
+
-
-
+
none
-
|- style="background: #d8d8d8; text-align: center;"
+
|bgcolor="#CBD1FD"|'''Miniprep:'''
-
|'''miniprep:'''
+
-
STOPPED
+
none
-
|'''miniprep:'''
+
|bgcolor="#BCCDFD"|'''Miniprep:'''
-
-
+
none
-
|'''miniprep:'''
+
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Sequencing :'''
-
-
+
none
-
|'''miniprep:'''
+
|bgcolor="#E0E3FE"|'''Sequencing :'''
-
-
+
none
-
|- style="background: #bebebe; text-align: center;"
+
|bgcolor="#CBD1FD"|'''Sequencing :'''
-
|'''sequencing :'''
+
-
-
+
none
 +
|bgcolor="#BCCDFD"|'''Sequencing :'''
-
|'''sequencing :'''
+
none
-
-
+
|- style="background: #d8d8d8; text-align: center;" 
 +
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
-
|'''sequencing :'''
+
none
 +
|bgcolor="#E0E3FE"|'''Stock glycerol'''
-
-
+
none
-
|'''sequencing :'''
+
|bgcolor="#CBD1FD"|'''Stock glycerol'''
-
-
+
none
 +
|bgcolor="#BCCDFD"|'''Stock glycerol'''
 +
 
 +
none
|}
|}

Latest revision as of 03:53, 22 October 2009

iGEM > Paris > WetLab > Addressing


WetLab - Addressing the message



Global constructions :


The green color means the experiment was a success


plasmid = PSB3T5




Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

pBAD: plate 2008


ClyA Cterm matrix : F4

Oligo :O10 and O31 TM :55°C


mRFP Nterm plasmid : pSB1A3

Oligo :O57 and O58 TM :


ClyA Nterm matrix : F4

Oligo : O32 and O9 TM :55°C


mRFP Cterm matrix : pSB1A3

Oligo :O59 and O60 TM :


-

-

-

Verification on gel :

ok

-

-

-


Purification on gel :

ok

-


-

-


Digestion:

ClyA Cterm S/P


RFP Nterm X/P


pBAD vector : ? E/S


Digestion:

ClyA Cter-RFP Nter vector PSB1A3 E/X

-

-

Verification digestion:

ok

Verification digestion:

ok

-

-

Ligation:

ClyA Cter-RFP Nter vector PSB1A3

Ligation:

PBAD E/S ClyA Cter-RFP Nter PSB1A3 E/X (x2)


PBAD S/P ClyA Cter-RFP Nter PSB2K3 X/P (x2)

Ligation:

-


Colony PCR :

ok

Colony PCR :

ok

Colony PCR :

ok


Miniprep:

clone :

Miniprep:

clone :

Miniprep:

clone 3

clone 5


Sequencing :

ok

Sequencing :

ok

Sequencing :

ok

-


Stock glycerol:

ClyA CTer: S47(clone 3) and S48(clone 7)

ClyA Nter: S72

RFP Cter: S55 (Clone 3)

RFP Nter: S56 (Clone 3)


Stock glycerol

Cly A(Cter)-(Nter)RFP: S72(clone 8)

Stock glycerol

pBAD ClyA RFP : S89 (clone 3)

pBAD ClyA RFP: S90 (clone 5)



Functional Testing:

PBAD ClyA RFP was transformed into Top10 bacteria in order to localize the fluorescence, we are supposed to have a superior fluorescence in the membrane.


PBAD ClyA RFP on PSB3T5 was transformed into Delta Tol bacteria , in this case we are supposed to see fluorescent vesicles when the medium contains 1 % arabinose , and to have no fluorescent on 1% glucose.


To learn more, see the Conclusion page


Export system

We finally thought that it won't be neccesary to overexpress the Tat system, nevertheless we have run a few experiments before starting to focus on others parts of the project.



Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

TatABCE matrix :

- - -


Verification on gel :

ok

-

-

-

Purification on gel :

ok

-

-

-


Digestion:

none

Digestion:

none

Digestion:

none

Digestion:

none

Digestion checking:

none

Digestion checking:

none


Digestion checking:

none

Digestion checking:

none


Ligation:

none

Ligation:

none

Ligation:

none

Ligation:

none

Colony PCR :

none

Colony PCR :

none

Colony PCR :

none

Colony PCR :

none

Miniprep:

none

Miniprep:

none

Miniprep:

none

Miniprep:

none

Sequencing :

none

Sequencing :

none

Sequencing :

none

Sequencing :

none

Stock glycerol:

none

Stock glycerol

none

Stock glycerol

none

Stock glycerol

none


Retrieved from "http://2009.igem.org/Team:Paris/Addressing_testing"