From 2007.igem.org

09/24/07

09/25/07

• Miniprep of:

- pLac-cI-LacY-B0015;

- p (Lambda)-RFP;

- pSB4A3;

- pTet-RFP.

• We inoculate (5ml LB,5ul amp 1000X) straked transformations of:

- pTet-RFP;

- pTet-LacI-pLac-cI-LacY-GFP (vitality test).

• Transformations of:

-pLac-cI-GFP.

09/26/07

Fluorescence test according to fluorescence test protocol of:

1. pLac-GFP:

-bacteria use to have the PMT output maximum value;

-normalization of OD value at 0.5;

-output value without IPTG: [0.22, 0.27]V;

-output value with IPTG:

-after 10min: [0.18,0.28]V;

-after 20min: [0.41,0.46]V;

-after 30min: [0.42,0.70]V;

-after 60min: [0.48,0.80]V;

-thus we decide to grow the OD value to have higher output value.

2. pTetR-LacI-pLac-cI-LacY-GFP

- we observe bacteria vitality, but there isn’t fluorescence after O/N growth with and without progressive induction with IPTG(1mM, 2mM, 3mM, 10mM every 60min).

- thus we decide to use lactose.

09/27/07

Fluorescence test according to Fluorescence test protocol of:

1. pLac-GFP:

-output value like to value with OD=0.5.

2. pTetR-LacI-pLac-cI-LacY-GFP:

-growth in LB medium;

-pellet in M9 with lactose;

-we observe them every 30min:

-bacteria vitality confirmed;

-bacteria don’t beam fluorescent.

3. pLac-cI-GFP:

-Output value without IPTG:0.05V;

-Output value with IPTG: about 0V.

4. pLac-cI-LacY-GFP:

-Output value without IPTG:0.05V;

-Output value with IPTG: about 0V.

Conclusions:

-using of low copy plasmid;

-higher OD;

-pLac-LacY-GFP and pTetR-RFP-pLac-GFP fluorescent test.

• We inoculate pTet-RFP and pTet-LacI-pLac-cI-LacY-GFP.

09/28/07

• Digestion of : pTet-RFP with Eco/Spe.
• Ligation with JO4431 (Eco/Xba).
• We transform and strake on plates.

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