Team:Paris/Transduction testing

From 2009.igem.org

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(→Fusion)
(→Fusion)
 
(75 intermediate revisions not shown)
Line 89: Line 89:
-
[[Image:Paris_construc_7_aida.jpg |700px|center|plasmid = ]]
 
 +
<center><font style="color:green;font-weight:bold; font-size:16px">The green color means the experiment was a success</font></center>
-
Time required : A lOT !!!!!
 
 +
[[Image:Paris_construc_7_aida.jpg |800px|center|plasmid = ]]
-
Experiments ran :
+
 
 +
 
 +
 
 +
 
 +
 
 +
<font style="color:black;font-weight:bold; font-size:16px"><I>'''Experiments ran :'''</I></font>
{|
{|
-
|- 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;"
|bgcolor="#F8F8F8"|'''PCR :'''  
|bgcolor="#F8F8F8"|'''PCR :'''  
-
TatABCE matrix :
+
AidA and Linker : Design, synthesize and order in two parts due to the seize.
 +
 
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
-
|bgcolor="#E0E3FE"|'''PCR :'''
 
-
|bgcolor="#CBD1FD"|'''PCR :'''
 
-
|bgcolor="#BCCDFD"|'''PCR :'''
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
Line 121: Line 126:
ok
ok
-
|bgcolor="#E0E3FE"|'''Verification on gel :'''
+
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
-
-
+
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Purification on gel :'''
-
|bgcolor="#CBD1FD"|'''Verification on gel :'''
+
ok
-
-
+
|bgcolor="#E0E3FE"|-
-
|bgcolor="#BCCDFD"|'''Verification on gel :'''
+
-
-
+
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
-
|- style="background: #bebebe; text-align: center;" 
 
-
|bgcolor="#F8F8F8"|'''Purification on gel :'''
 
-
ok
 
-
|bgcolor="#E0E3FE"|'''Purification on gel :'''
+
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Digestion:'''  
-
-
+
AidA CTerm Kpn1/HindIII
-
|bgcolor="#CBD1FD"|'''Purification on gel :'''
+
AidA NTerm Kpn1/HindIII
-
-
+
|bgcolor="#E0E3FE"|'''Digestion:'''  
-
|bgcolor="#BCCDFD"|'''Purification on gel :'''
+
-
-
+
AidA complete E/P
 +
PSB1A3 E/P
 +
We did it to obtain a biobrick first
-
|- style="background: #d8d8d8; text-align: center;"
+
|bgcolor="#CBD1FD"|'''Digestion:'''  
-
|bgcolor="#F8F8F8"|'''digestion:'''  
+
none
-
 
+
|bgcolor="#BCCDFD"|-
-
TatABCE
+
-
|bgcolor="#E0E3FE"|'''digestion:'''
 
-
|bgcolor="#CBD1FD"|'''digestion:'''
 
-
|bgcolor="#BCCDFD"|'''digestion:'''
 
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|bgcolor="#F8F8F8"|'''verification digestion:'''
+
|bgcolor="#F8F8F8"|'''Digestion checking:'''
ok
ok
-
|bgcolor="#E0E3FE"|'''verification digestion:'''
+
|bgcolor="#E0E3FE"|'''Digestion checking:'''
 +
ok
-
-
 
-
|bgcolor="#CBD1FD"|'''verification digestion:'''
+
|bgcolor="#CBD1FD"|'''Digestion checking:'''
 +
none
-
-
+
 
-
|bgcolor="#BCCDFD"|'''verification digestion:'''
+
|bgcolor="#BCCDFD"|
-
-
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|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|bgcolor="#F8F8F8"|'''ligation:'''  
+
|bgcolor="#F8F8F8"|'''Ligation:'''  
-
TatABCE
+
AidA CTerm (Kpn1/HindIII):: aidA NTerm (Kpn1/HindIII)
-
|bgcolor="#E0E3FE"|'''ligation:'''  
+
 
-
|bgcolor="#CBD1FD"|'''ligation:'''  
+
|bgcolor="#E0E3FE"|'''Ligation:'''  
-
|bgcolor="#BCCDFD"|'''ligation:'''
+
 
 +
AidA complete E/P :: PSB1A3 E/P
 +
 
 +
|bgcolor="#CBD1FD"|'''Ligation:'''  
 +
none
 +
|bgcolor="#BCCDFD"|-
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|bgcolor="#F8F8F8"|'''PCR colo :'''
+
|bgcolor="#F8F8F8"|'''Colony pCR :'''
ok
ok
-
|bgcolor="#E0E3FE"|'''PCR colo :'''
+
|bgcolor="#E0E3FE"|'''Colony PCR:'''
-
-
+
ok
-
|bgcolor="#CBD1FD"|'''PCR colo :'''
+
|bgcolor="#CBD1FD"|'''Colony PCR :'''
 +
none
-
-
+
|bgcolor="#BCCDFD"|-
-
|bgcolor="#BCCDFD"|'''PCR colo :'''
+
-
 
+
-
-
+
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|bgcolor="#F8F8F8"|'''miniprep:'''
+
|bgcolor="#F8F8F8"|'''Miniprep:'''
-
STOPPED
+
done
-
|bgcolor="#E0E3FE"|'''miniprep:'''
+
|bgcolor="#E0E3FE"|'''Miniprep:'''
-
-
+
done
-
|bgcolor="#CBD1FD"|'''miniprep:'''
+
|bgcolor="#CBD1FD"|'''Miniprep:'''
 +
none
-
-
+
|bgcolor="#BCCDFD"|-
-
|bgcolor="#BCCDFD"|'''miniprep:'''
+
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Sequencing :'''
 +
unecessary
-
-
+
|bgcolor="#E0E3FE"|'''Sequencing :'''
 +
none
-
|- style="background: #bebebe; text-align: center;" 
+
|bgcolor="#CBD1FD"|'''Sequencing :'''
-
|bgcolor="#F8F8F8"|'''sequencing :'''
+
none
-
-
 
-
|bgcolor="#E0E3FE"|'''sequencing :'''
+
|bgcolor="#BCCDFD"|-
 +
 
 +
|- style="background: #d8d8d8; text-align: center;" 
 +
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
-
-
 +
|bgcolor="#E0E3FE"|'''Stock glycerol'''
-
|bgcolor="#CBD1FD"|'''sequencing :'''
+
S75
-
-
+
|bgcolor="#CBD1FD"|'''Stock glycerol'''
-
|bgcolor="#BCCDFD"|'''sequencing :'''
+
-
-
+
none
 +
|bgcolor="#BCCDFD"|-
|}
|}
Line 238: Line 252:
-
[[Image:Paris_construc_8_junfos.jpg  |700px|center|plasmid = ]]
 
-
Time required : A lOT !!!!!
+
<center><font style="color:green;font-weight:bold; font-size:16px">The green color means the experiment was a success</font></center>
-
Experiments ran :
 
 +
[[Image:Paris_construc_8_junfos.jpg  |800px|center|plasmid = ]]
-
{|
 
-
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold; "
 
-
|width="300px" ; bgcolor="#F8F8F8"| column 1
 
-
|width="300px" ; bgcolor="#E0E3FE"| column 2
 
-
|width="300px" ; bgcolor="#CBD1FD"| column 3
 
-
|width="300px" ; bgcolor="#BCCDFD"| column 4
 
 +
 +
 +
 +
 +
<font style="color:black;font-weight:bold; font-size:16px"><I>'''Experiments ran :'''</I></font>
 +
 +
 +
{|
 +
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold;font-size:14px "
 +
|width="300px" ; bgcolor="#F8F8F8"| Column 1
 +
|width="300px" ; bgcolor="#E0E3FE"| Column 2
 +
|width="300px" ; bgcolor="#CBD1FD"| Column 3
 +
|width="300px" ; bgcolor="#BCCDFD"| Column 4
|- style="background: #bebebe; text-align: center;"
|- style="background: #bebebe; text-align: center;"
|bgcolor="#F8F8F8"|'''PCR :'''  
|bgcolor="#F8F8F8"|'''PCR :'''  
-
TatABCE matrix :
+
Jun *: design and order to be a a mutant incapable to form homodimere.
 +
 
 +
pLac : previously done
 +
 
 +
SSOmpA : previously done
 +
 
 +
Fos : previously done
 +
 
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 
-
|bgcolor="#E0E3FE"|'''PCR :'''
 
-
|bgcolor="#CBD1FD"|'''PCR :'''
 
-
|bgcolor="#BCCDFD"|'''PCR :'''
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
Line 270: Line 298:
ok
ok
-
|bgcolor="#E0E3FE"|'''Verification on gel :'''
+
|bgcolor="#E0E3FE"|
-
-
-
|bgcolor="#CBD1FD"|'''Verification on gel :'''
+
|bgcolor="#CBD1FD"|
-
 
+
-
-
+
-
|bgcolor="#BCCDFD"|'''Verification on gel :'''
+
-
-
 +
|bgcolor="#BCCDFD"|-
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
Line 286: Line 312:
ok
ok
-
|bgcolor="#E0E3FE"|'''Purification on gel :'''
+
|bgcolor="#E0E3FE"|
-
-
-
|bgcolor="#CBD1FD"|'''Purification on gel :'''
+
|bgcolor="#CBD1FD"|
-
-
-
|bgcolor="#BCCDFD"|'''Purification on gel :'''
+
|bgcolor="#BCCDFD"|
 +
 
-
-
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|bgcolor="#F8F8F8"|'''digestion:'''  
+
|bgcolor="#F8F8F8"|'''Digestion:'''  
 +
 
 +
pLac E/X
 +
 
 +
ssOmpA E/S
 +
 
 +
Jun E/X and E/P
 +
 
 +
PSB1A3 E/P
 +
 
 +
|bgcolor="#E0E3FE"|'''Digestion:'''
 +
none
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
-
TatABCE
 
-
|bgcolor="#E0E3FE"|'''digestion:'''
 
-
|bgcolor="#CBD1FD"|'''digestion:'''
 
-
|bgcolor="#BCCDFD"|'''digestion:'''
 
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|bgcolor="#F8F8F8"|'''verification digestion:'''
+
|bgcolor="#F8F8F8"|'''Digestion checking:'''
ok
ok
-
|bgcolor="#E0E3FE"|'''verification digestion:'''
+
|bgcolor="#E0E3FE"|'''Digestion checking:'''
-
-
+
none
-
 
+
-
|bgcolor="#CBD1FD"|'''verification digestion:'''
+
 +
|bgcolor="#CBD1FD"|
-
-
-
|bgcolor="#BCCDFD"|'''verification digestion:'''
+
|bgcolor="#BCCDFD"|
-
 
+
-
-
-
 
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|bgcolor="#F8F8F8"|'''ligation:'''  
+
|bgcolor="#F8F8F8"|'''Ligation:'''  
 +
 
 +
pLac on PSB2K3 E/X :: ssOmpA E/S
 +
 
 +
Jun E/P :: PSB1A3 E/P
 +
 
 +
|bgcolor="#E0E3FE"|'''Ligation:'''
 +
none
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|'-
-
TatABCE
 
-
|bgcolor="#E0E3FE"|'''ligation:'''
 
-
|bgcolor="#CBD1FD"|'''ligation:'''
 
-
|bgcolor="#BCCDFD"|'''ligation:'''
 
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|bgcolor="#F8F8F8"|'''PCR colo :'''
+
|bgcolor="#F8F8F8"|'''Colony PCR:'''
ok
ok
-
|bgcolor="#E0E3FE"|'''PCR colo :'''
+
|bgcolor="#E0E3FE"|'''Colony PCR:'''
-
 
+
none
-
-
+
-
|bgcolor="#CBD1FD"|'''PCR colo :'''
+
|bgcolor="#CBD1FD"|-
-
-
-
|bgcolor="#BCCDFD"|'''PCR colo :'''
+
|bgcolor="#BCCDFD"|-
-
 
+
-
-
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|bgcolor="#F8F8F8"|'''miniprep:'''
+
|bgcolor="#F8F8F8"|'''Miniprep:'''
-
STOPPED
+
ok
 +
|bgcolor="#E0E3FE"|'''Miniprep:'''
-
|bgcolor="#E0E3FE"|'''miniprep:'''
+
none
-
-
+
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
-
|bgcolor="#CBD1FD"|'''miniprep:'''
+
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Sequencing :'''
-
-
+
-ok
-
|bgcolor="#BCCDFD"|'''miniprep:'''
+
|bgcolor="#E0E3FE"|'''Sequencing :'''
-
-
+
-none
-
|- style="background: #bebebe; text-align: center;" 
+
|bgcolor="#CBD1FD"|'-
-
|bgcolor="#F8F8F8"|'''sequencing :'''
+
|bgcolor="#BCCDFD"|-
-
-
+
|- style="background: #d8d8d8; text-align: center;" 
 +
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
 +
none
-
|bgcolor="#E0E3FE"|'''sequencing :'''
+
unecessary
 +
|bgcolor="#E0E3FE"|'''Stock glycerol'''
-
-
+
none
-
 
+
|bgcolor="#CBD1FD"|-
-
|bgcolor="#CBD1FD"|'''sequencing :'''
+
|bgcolor="#BCCDFD"|-
-
-
-
|bgcolor="#BCCDFD"|'''sequencing :'''
 
-
 
-
-
 
-
 
|}
|}
Line 423: Line 460:
-
the transduction system of the fec operon (FecA, FecR, FecI).
+
The transduction system of the fec operon (FecA, FecR, FecI).
-
[[Image:Paris_construc_6_pfec.jpg  |700px|center|plasmid = ]]
 
 +
<center><font style="color:green;font-weight:bold; font-size:16px">The green color means the experiment was a success</font></center>
-
[[Image:Paris_construc_2_feca.jpg  |700px|center|plasmid = ]]
 
 +
[[Image:Paris_construct_9_pFec-RFP.jpg  |800px|center|plasmid = ]]
Line 439: Line 476:
 +
<font style="color:black;font-weight:bold; font-size:16px"><I>'''Experiments ran :'''</I></font>
-
Time required : A lOT !!!!!
 
 +
{|
 +
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold;font-size:14px "
 +
|width="300px" ; bgcolor="#F8F8F8"| Column 1
 +
|width="300px" ; bgcolor="#E0E3FE"| Column 2
 +
|width="300px" ; bgcolor="#CBD1FD"| Column 3
 +
|width="300px" ; bgcolor="#BCCDFD"| Column 4
 +
|- style="background: #bebebe; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''PCR :'''
 +
pFec matrix: K12 DNA
-
Experiments ran :
+
Oligo:   TM
-
{|
+
FecI matrix: K12 DNA
-
|- style="background: #0a3585; text-align: center; color:white; font-weight:bold; "
+
-
|width= 300px| row 1
+
-
|width=300px| row 2
+
-
|width=300px| row 3
+
-
|- style="background: #bebebe; text-align: center;"
+
Oligo:   TM
-
|'''PCR :'''
+
 +
FecR 1-85  matrix: K12 DNA
 +
Oligo:  TM
 +
mRFP matrix: PSB1A3 (previous IGEM plate)
-
|'''PCR :'''
+
Oligo:   TM
-
|'''PCR :'''
+
 +
 +
|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"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Purification on gel :'''
ok
ok
-
|'''Verification on gel :'''
+
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 
 +
 
 +
 
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Digestion:'''
 +
 
 +
Ter E/X
 +
 
 +
mRFP E/S
 +
 
 +
|bgcolor="#E0E3FE"| -
 +
|bgcolor="#CBD1FD"| -
 +
|bgcolor="#BCCDFD"| -
 +
 
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Digestion checking:'''
ok
ok
 +
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 +
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Ligation:'''
 +
 +
mRFP E/S Ter E/X in Ter vector
 +
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"| -
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''Purification on gel :'''
+
|bgcolor="#F8F8F8"|'''Colony PCR:'''
ok
ok
-
|'''Purification on gel :'''
+
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Miniprep:'''
ok
ok
-
|'''Purification on gel :'''
+
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Sequencing :'''
ok
ok
 +
|bgcolor="#E0E3FE"|
 +
-
-
|- style="background: #d8d8d8; text-align: center;"
+
|bgcolor="#CBD1FD"|
-
|'''digestion:'''  
+
-
 +
|bgcolor="#BCCDFD"|
 +
-
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
 +
-
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
|}
-
|'''digestion:'''
 
-
|'''digestion:'''
 
-
|- style="background: #bebebe; text-align: center;" 
 
-
|'''verification digestion:'''
 
-
ok
+
<center><font style="color:green;font-weight:bold; font-size:16px">The green color means the experiment was a success</font></center>
-
|'''verification digestion:'''
+
 
 +
 
 +
[[Image:Paris_construc_6_pfec.jpg  |800px|center|plasmid = ]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<font style="color:black;font-weight:bold; font-size:16px"><I>'''Experiments ran :'''</I></font>
 +
 
 +
 
 +
 
 +
{|
 +
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold;font-size:14px "
 +
|width="300px" ; bgcolor="#F8F8F8"| Column 1
 +
|width="300px" ; bgcolor="#E0E3FE"| Column 2
 +
|width="300px" ; bgcolor="#CBD1FD"| Column 3
 +
|width="300px" ; bgcolor="#BCCDFD"| Column 4
 +
 
 +
|- style="background: #bebebe; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''PCR :'''
 +
 
 +
pFec matrix: K12 DNA
 +
 
 +
Oligo:  TM
 +
 
 +
FecI matrix: K12 DNA
 +
 
 +
Oligo:  TM
 +
 
 +
FecR 1-85  matrix: K12 DNA
 +
 
 +
Oligo:  TM
 +
 
 +
mRFP matrix: PSB1A3 (previous IGEM plate)
 +
 
 +
Oligo:  TM
 +
 
 +
 +
 
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Verification on gel :'''
ok
ok
-
|'''verification digestion:'''
+
|bgcolor="#E0E3FE"|
 +
 
 +
-
 +
 
 +
|bgcolor="#CBD1FD"|
 +
-
 +
|bgcolor="#BCCDFD"|
 +
-
 +
 
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Purification on gel :'''
ok
ok
 +
 +
|bgcolor="#E0E3FE"|
 +
 +
-
 +
 +
|bgcolor="#CBD1FD"|
 +
 +
-
 +
|bgcolor="#BCCDFD"|
 +
 +
-
 +
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''ligation:'''  
+
|bgcolor="#F8F8F8"|'''Digestion:'''  
 +
Ter E/X
 +
mRFP E/S
-
|'''ligation:'''
+
|bgcolor="#E0E3FE"|-
-
|'''ligation:'''
+
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Verification digestion:'''
 +
 +
ok
 +
 +
|bgcolor="#E0E3FE"|
 +
-
 +
 +
|bgcolor="#CBD1FD"|
 +
 +
-
 +
|bgcolor="#BCCDFD"|
 +
 +
-
 +
 +
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Ligation:'''
 +
 +
mRFP E/S Ter E/X in Ter vector
 +
 +
|bgcolor="#E0E3FE"|-
 +
|bgcolor="#CBD1FD"|-
 +
|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"|-
 +
 
 +
-
 +
 
 +
|bgcolor="#CBD1FD"|-
 +
 
 +
-
 +
|bgcolor="#BCCDFD"|
 +
 
 +
-
 +
 
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Miniprep:'''
ok
ok
-
|'''PCR colo :'''
+
|bgcolor="#E0E3FE"|
 +
-
 +
 +
|bgcolor="#CBD1FD"|
 +
 +
-
 +
 +
|bgcolor="#BCCDFD"|
 +
-
 +
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Sequencing :'''
ok
ok
 +
|bgcolor="#E0E3FE"|
 +
 +
-
 +
 +
|bgcolor="#CBD1FD"|
 +
-
 +
|bgcolor="#BCCDFD"|
 +
-
 +
|- style="background: #d8d8d8; text-align: center;" 
 +
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
 +
 +
-
 +
|bgcolor="#E0E3FE"|
 +
 +
-
 +
 +
|bgcolor="#CBD1FD"|
 +
-
 +
|bgcolor="#BCCDFD"|
 +
 +
-
 +
|}
 +
 +
 +
 +
 +
 +
 +
<center><font style="color:green;font-weight:bold; font-size:16px">The green color means the experiment was a success</font></center>
 +
 +
 +
[[Image:Paris_construc_2_feca.jpg  |800px|center|plasmid = ]]
 +
 +
 +
 +
 +
 +
 +
<font style="color:black;font-weight:bold; font-size:16px"><I>'''Experiments ran :'''</I></font>
 +
 +
 +
 +
{|
 +
|- style="background: #0a3585; text-align: center; color:black; font-weight:bold;font-size:14px "
 +
|width="300px" ; bgcolor="#F8F8F8"| Column 1
 +
|width="300px" ; bgcolor="#E0E3FE"| Column 2
 +
|width="300px" ; bgcolor="#CBD1FD"| Column 3
 +
|width="300px" ; bgcolor="#BCCDFD"| Column 4
 +
 +
|- style="background: #bebebe; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''PCR :'''
 +
 +
FecA matrix:  K12
 +
 +
pBAD:
 +
 +
OmpAs: pINR2
 +
 +
|bgcolor="#E0E3FE"|'-
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
|- style="background: #d8d8d8; text-align: center;"
|- style="background: #d8d8d8; text-align: center;"
-
|'''miniprep:'''
+
|bgcolor="#F8F8F8"|'''Verification on gel :'''
-
clone :
+
ok
-
|'''miniprep:'''
+
|bgcolor="#E0E3FE"|
-
clone :
+
-
-
|'''miniprep:'''
+
|bgcolor="#CBD1FD"|
-
clone :
+
-
 +
|bgcolor="#BCCDFD"|-
 +
-
|- style="background: #bebebe; text-align: center;"   
|- style="background: #bebebe; text-align: center;"   
-
|'''sequencing :'''
+
|bgcolor="#F8F8F8"|'''Purification on gel :'''
ok
ok
-
|'''sequencing :'''
+
|bgcolor="#E0E3FE"|-
-
ok
+
-
-
|'''sequencing :'''
+
|bgcolor="#CBD1FD"|-
 +
 
 +
-
 +
|bgcolor="#BCCDFD"|-
 +
 
 +
-
 +
 
 +
 
 +
 
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Digestion:'''
 +
 
 +
pBAD  E/S
 +
 
 +
|bgcolor="#E0E3FE"|'''Digestion:'''
 +
none
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Digestion checking'''
ok
ok
 +
|bgcolor="#E0E3FE"|'''Digestion checking'''
 +
 +
none
 +
 +
|bgcolor="#CBD1FD"|
 +
 +
-
 +
|bgcolor="#BCCDFD"|
 +
-
 +
 +
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Ligation:'''
 +
 +
none
 +
 +
|bgcolor="#E0E3FE"|'''Ligation:'''
 +
none
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|-
 +
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Colony PCR :'''
 +
 +
none
 +
 +
|bgcolor="#E0E3FE"|'''Colony PCR :'''
 +
none
 +
 +
|bgcolor="#CBD1FD"|
 +
 +
-
 +
|bgcolor="#BCCDFD"|
 +
-
 +
 +
|- style="background: #d8d8d8; text-align: center;"
 +
|bgcolor="#F8F8F8"|'''Miniprep'''
 +
 +
none
 +
 +
|bgcolor="#E0E3FE"|'''Miniprep'''
 +
 +
none
 +
 +
|bgcolor="#CBD1FD"|
 +
-
 +
 +
|bgcolor="#BCCDFD"|
 +
 +
-
 +
 +
|- style="background: #bebebe; text-align: center;" 
 +
|bgcolor="#F8F8F8"|'''Sequencing'''
 +
none
 +
 +
|bgcolor="#E0E3FE"|'''Sequencing'''
 +
none
 +
 +
|bgcolor="#CBD1FD"|
 +
 +
-
 +
|bgcolor="#BCCDFD"|
 +
-
 +
|- style="background: #d8d8d8; text-align: center;" 
 +
|bgcolor="#F8F8F8"| '''Stock glycerol:'''
 +
none
 +
 +
|bgcolor="#E0E3FE"| '''Stock glycerol:'''
 +
none
 +
 +
|bgcolor="#CBD1FD"|-
 +
|bgcolor="#BCCDFD"|
 +
-
|}
|}
 +

Latest revision as of 03:38, 22 October 2009

iGEM > Paris > WetLab > Reception


Contents

WetLab - Reception

In this section we have two kinds of experiments :


  1. Fusion : the construction required for the fusion of the vesicles that contain the message with the receiver.
  2. Transduction : the construction of the system that will be able de decypher the message.


The first part deals with G3P , OmpAL , Jun, Fos , and AIDA protein. the second with the transduction system of the fec operon (FecA, FecR, FecI).

Fusion

Fusion : the construction required for the fusion of the vesicles that contain the message.

G3P , OmpAL , Jun, Fos , and AIDA protein.



The green color means the experiment was a success


plasmid =




Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

AidA and Linker : Design, synthesize and order in two parts due to the seize.

- - -


Verification on gel :

ok

- - -
Purification on gel :

ok

- - -


Digestion:

AidA CTerm Kpn1/HindIII

AidA NTerm Kpn1/HindIII

Digestion:

AidA complete E/P

PSB1A3 E/P

We did it to obtain a biobrick first

Digestion:

none

-


Digestion checking:

ok

Digestion checking:

ok


Digestion checking:

none


-


Ligation:

AidA CTerm (Kpn1/HindIII):: aidA NTerm (Kpn1/HindIII)


Ligation:

AidA complete E/P :: PSB1A3 E/P

Ligation:

none

-
Colony pCR :

ok

Colony PCR:

ok

Colony PCR :

none

-
Miniprep:

done

Miniprep:

done

Miniprep:

none

-
Sequencing :

unecessary

Sequencing :

none

Sequencing :

none


-
Stock glycerol:

-

Stock glycerol

S75

Stock glycerol

none

-




The green color means the experiment was a success


plasmid =




Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

Jun *: design and order to be a a mutant incapable to form homodimere.

pLac : previously done

SSOmpA : previously done

Fos : previously done

- - -


Verification on gel :

ok

-

-

-
Purification on gel :

ok

-

-



Digestion:

pLac E/X

ssOmpA E/S

Jun E/X and E/P

PSB1A3 E/P

Digestion:

none

- -


Digestion checking:

ok

Digestion checking:

none

-

-

Ligation:

pLac on PSB2K3 E/X :: ssOmpA E/S

Jun E/P :: PSB1A3 E/P

Ligation:

none

- '-


Colony PCR:

ok

Colony PCR:

none

-

-

-

-

Miniprep:

ok

Miniprep:

none

- -
Sequencing :

-ok

Sequencing :

-none

'- -
Stock glycerol:

none

unecessary

Stock glycerol

none

- -

-



G3P

Even though it has not been realized, we planned to do these experiments to verify that G3P is expressed into the bacterial surface and induces the membranal fusion.


  • Surface expression of G3P

Surface expression of G3P could be seen with antibodies.
Culture of bacteria expressing OmpA-linker-G3P construction on a membrane. Adding antibodies anti-G3P. Revelation should shows whether G3P is expressed at the surface of bacteria.


  • Floculation test

"Fusion" could be approached by a flocculation test.
Two separate bacterial population growth on media. A population (F-) expressing the OmpA-linker_G3P construction and a population (F +) which express sexual pilus (by expression of the episome F).
Then we growth bacteria without agitation and we look (compared to culture controls) the speed of sedimentation. Faster sedimentation is due to an interaction between bacterial populations showing an increase of contact time. At vesicle level, it induce membrane fusion.


Transduction

Transduction : the construction of the system that will be able de decypher the message.


The transduction system of the fec operon (FecA, FecR, FecI).



The green color means the experiment was a success


plasmid =




Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

pFec matrix: K12 DNA

Oligo: TM

FecI matrix: K12 DNA

Oligo: TM

FecR 1-85 matrix: K12 DNA

Oligo: TM

mRFP matrix: PSB1A3 (previous IGEM plate)

Oligo: TM


- - -
Verification on gel :

ok

- - -
Purification on gel :

ok

- - -


Digestion:

Ter E/X

mRFP E/S

- - -
Digestion checking:

ok

- - -


Ligation:

mRFP E/S Ter E/X in Ter vector

- - -
Colony PCR:

ok

- - -
Miniprep:

ok

- - -
Sequencing :

ok

-

-

-

Stock glycerol:

-

- - -




The green color means the experiment was a success


plasmid =




Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

pFec matrix: K12 DNA

Oligo: TM

FecI matrix: K12 DNA

Oligo: TM

FecR 1-85 matrix: K12 DNA

Oligo: TM

mRFP matrix: PSB1A3 (previous IGEM plate)

Oligo: TM


- - -
Verification on gel :

ok

-

-

-

Purification on gel :

ok

-

-

-


Digestion:

Ter E/X

mRFP E/S

- - -
Verification digestion:

ok

-

-

-


Ligation:

mRFP E/S Ter E/X in Ter vector

- - -
Colony PCR:

ok

-

-

-

-

-

Miniprep:

ok

-

-

-

Sequencing :

ok

-

-

-

Stock glycerol:

-

-

-

-




The green color means the experiment was a success


plasmid =




Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

FecA matrix: K12

pBAD:

OmpAs: pINR2

'- - -
Verification on gel :

ok

-

-

-

-

Purification on gel :

ok

-

-

-

-

-

-


Digestion:

pBAD E/S

Digestion:

none

- -
Digestion checking

ok

Digestion checking

none

-

-


Ligation:

none

Ligation:

none

- -
Colony PCR :

none

Colony PCR :

none

-

-

Miniprep

none

Miniprep

none

-

-

Sequencing

none

Sequencing

none

-

-

Stock glycerol:

none

Stock glycerol:

none

-

-


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