Paris/October 4

From 2007.igem.org

< Paris(Difference between revisions)
(→Minipreps)
(→Transformation of W121 and FR781 with pKD46)
 
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[[Paris/October 3|yesterday]] -- [[Paris/October 5|tomorrow]] <br>
== Minipreps ==
== Minipreps ==
Line 5: Line 6:
== Transformation of W121 and FR781 with pKD46 ==
== Transformation of W121 and FR781 with pKD46 ==
 +
 +
Classic protocol. we used 1µL of Miniprep into 100µL of TSS chemically competent W121 and FR781 bacteria.
== Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance ==
== Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance ==
 +
 +
[Frt-CmR-Frt-pTet>>lox71-gfpTripart] (L58)<br>
 +
<br>
 +
- First, we got an abnormally short length for the PCR with the wanner primers (about 1,7kb instead of 2,1kb; see October 2)<br>
 +
- Then i did a PCR on the same plasmid (L58) with the primers O18&O19. We got the same pattern of length (1,7kb and another one at about 3,5kb)<br>
 +
- To investigate the possibility to have lost 400kb in the CmR gene leading to the loss of resistence to Cm for the strain, i put some clones of L58 on plates with either Ampicilline or Chloramphenicol (10µg/mL).<br>
 +
----> the clones grew on Amp but not on Cm... <br>
 +
it confirms the hypothesis of a loss inside the CmR gene.<br>
 +
----> of course the results for the sequencing will give us the last word. They should come on Friday in late afternoon. We called the sequencing facility.
 +
 +
== Digestion reactions ==
 +
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Digestion Products
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=24%| Product Name
 +
|width=24%| Matrix Name
 +
|width=5%| Enzyme 1
 +
|width=5%| Enzyme 2
 +
|width=5%| Size
 +
|width=34%| Description
 +
|width=1%|
 +
|width=1%|
 +
|width=1%|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D79
 +
|pJ23100>>lox71-B0015(T)
 +
|L44.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D80
 +
|pJ23100>>lox71-B0015(T)
 +
|L44.2
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D81
 +
|pJ23107>>lox71-B0015(T)
 +
|L45.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D82
 +
|pJ23107>>lox71-B0015(T)
 +
|L45.2
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D83
 +
|pTet>>lox71-B0015(T)
 +
|L46.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D84
 +
|araC/pBad>>lox71-B0015(T)
 +
|L47.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D85
 +
|B0015(T)-lox66-RBSDapAcoli
 +
|L53.7
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D86
 +
|attB-lox66-RBSDapAcoli
 +
|L54.3
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D87
 +
|attB-lox66-RBSDapAcoli
 +
|L54.4
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D88
 +
|B0015(T)-lox66-RBSdapAsubtilis
 +
|L56.3
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D89
 +
|attB-lox66-RBSdapAsubtilis
 +
|L57.1
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D90
 +
|attB-lox66-RBSdapAsubtilis
 +
|L57.3
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D91
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.1
 +
|EcoRI
 +
|XbaI
 +
|
 +
|FV - Medium constitutive promoter in J61002 (cl. 1) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D92
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.2
 +
|EcoRI
 +
|XbaI
 +
|
 +
|FV - Medium constitutive promoter in J61002 (cl. 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D93
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.1
 +
|Spe1
 +
|Pst1
 +
|
 +
|BV - Medium constitutive promoter in J61002 (cl. 1) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D94
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.2
 +
|Spe1
 +
|Pst1
 +
|
 +
|BV - Medium constitutive promoter in J61002 (cl. 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|}
 +
 +
== Ligations ==
 +
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Ligations
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=20%| Insert
 +
|width=5%| Insert Volume (µL)
 +
|width=25%| Vector
 +
|width=5%| Vector Volume (µL)
 +
|width=35%| Comments
 +
|width=5%| Number of colonies
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C19
 +
|
 +
|
 +
|D72
 +
|2
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C20
 +
|
 +
|
 +
|D73
 +
|2
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C21
 +
|
 +
|
 +
|D64
 +
|2
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L65
 +
|D56 - araC/pBad>>RBS-Cre BBinsert
 +
|10
 +
|D72 - pSB2K3 BBvector
 +
|2
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L66
 +
|D75 - AraC-pBAD-RBS-CRE FI
 +
|10
 +
|D73 - pSB2K3 ES open vector
 +
|2
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L67
 +
|D59 - pTet>>lox71-gfptripart BI
 +
|10
 +
|D64 - FRT-CmR-FRT BBa_J61025 BV
 +
|2
 +
|
 +
|0
 +
|}
 +
 +
== Culture of S28, strain having the [Frt-CmR-Frt] contruction ==
 +
 +
In order to test the Chloramphenicol resistance of these strains.<br>
 +
- S28.1<br>
 +
- S28.2
 +
 +
== Transformation of MP14.1 and MP14.2 in DH5α ==
 +
 +
In order to test the Chloramphenicol resistance given by MP14.

Latest revision as of 15:08, 11 October 2007

yesterday -- tomorrow

Contents

Minipreps

  • from S49 = pKD46
  • pY-2P-IntC

Transformation of W121 and FR781 with pKD46

Classic protocol. we used 1µL of Miniprep into 100µL of TSS chemically competent W121 and FR781 bacteria.

Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance

[Frt-CmR-Frt-pTet>>lox71-gfpTripart] (L58)

- First, we got an abnormally short length for the PCR with the wanner primers (about 1,7kb instead of 2,1kb; see October 2)
- Then i did a PCR on the same plasmid (L58) with the primers O18&O19. We got the same pattern of length (1,7kb and another one at about 3,5kb)
- To investigate the possibility to have lost 400kb in the CmR gene leading to the loss of resistence to Cm for the strain, i put some clones of L58 on plates with either Ampicilline or Chloramphenicol (10µg/mL).

> the clones grew on Amp but not on Cm...

it confirms the hypothesis of a loss inside the CmR gene.

> of course the results for the sequencing will give us the last word. They should come on Friday in late afternoon. We called the sequencing facility.

Digestion reactions

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D79 pJ23100>>lox71-B0015(T) L44.1 Xba1 Pst1 BI
D80 pJ23100>>lox71-B0015(T) L44.2 Xba1 Pst1 BI
D81 pJ23107>>lox71-B0015(T) L45.1 Xba1 Pst1 BI
D82 pJ23107>>lox71-B0015(T) L45.2 Xba1 Pst1 BI
D83 pTet>>lox71-B0015(T) L46.1 Xba1 Pst1 BI
D84 araC/pBad>>lox71-B0015(T) L47.1 Xba1 Pst1 BI
D85 B0015(T)-lox66-RBSDapAcoli L53.7 EcoRI SpeI FI
D86 attB-lox66-RBSDapAcoli L54.3 EcoRI SpeI FI
D87 attB-lox66-RBSDapAcoli L54.4 EcoRI SpeI FI
D88 B0015(T)-lox66-RBSdapAsubtilis L56.3 EcoRI SpeI FI
D89 attB-lox66-RBSdapAsubtilis L57.1 EcoRI SpeI FI
D90 attB-lox66-RBSdapAsubtilis L57.3 EcoRI SpeI FI
D91 BBa_J61025 MP14.1 EcoRI XbaI FV - Medium constitutive promoter in J61002 (cl. 1)
D92 BBa_J61025 MP14.2 EcoRI XbaI FV - Medium constitutive promoter in J61002 (cl. 2)
D93 BBa_J61025 MP14.1 Spe1 Pst1 BV - Medium constitutive promoter in J61002 (cl. 1)
D94 BBa_J61025 MP14.2 Spe1 Pst1 BV - Medium constitutive promoter in J61002 (cl. 2)

Ligations

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C19 D72 2 0
C20 D73 2 0
C21 D64 2 0
L65 D56 - araC/pBad>>RBS-Cre BBinsert 10 D72 - pSB2K3 BBvector 2 0
L66 D75 - AraC-pBAD-RBS-CRE FI 10 D73 - pSB2K3 ES open vector 2 0
L67 D59 - pTet>>lox71-gfptripart BI 10 D64 - FRT-CmR-FRT BBa_J61025 BV 2 0

Culture of S28, strain having the [Frt-CmR-Frt] contruction

In order to test the Chloramphenicol resistance of these strains.
- S28.1
- S28.2

Transformation of MP14.1 and MP14.2 in DH5α

In order to test the Chloramphenicol resistance given by MP14.

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