Paris/October 4
From 2007.igem.org
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.
