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	::'''09/25/07'''		::'''09/25/07'''
	*Miniprep of:		*Miniprep of:
-	::- pLac-cI-LacY-B0015;	+	::- I763036;
	::- p (Lambda)-RFP;		::- p (Lambda)-RFP;
	::- pSB4A3;		::- pSB4A3;
-	::- pTet-RFP.	+	::- I763033.
	*We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:		*We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:
-	::- pTet-RFP;	+	::- I763033;
	::- pTet-LacI-pLac-cI-LacY-GFP (vitality test).		::- pTet-LacI-pLac-cI-LacY-GFP (vitality test).
	*Transformations of:		*Transformations of:
-	::-pLac-cI-GFP.	+	::-I763031.
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	::::::::-bacteria vitality confirmed;		::::::::-bacteria vitality confirmed;
	::::::::-bacteria don’t beam fluorescent.		::::::::-bacteria don’t beam fluorescent.
-	::3. ''pLac-cI-GFP'':	+	::3. ''I763031'':
	::::-Output value without IPTG:0.05V;		::::-Output value without IPTG:0.05V;
	::::-Output value with IPTG: about 0V.		::::-Output value with IPTG: about 0V.
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	::-using of low copy plasmid;		::-using of low copy plasmid;
	::-higher OD;		::-higher OD;
-	::-pLac-LacY-GFP and pTetR-RFP-pLac-GFP fluorescent test.	+	::-I763032 and I763034 fluorescent test.
-	*We inoculate pTet-RFP and pTet-LacI-pLac-cI-LacY-GFP.	+	*We inoculate I763033 and pTet-LacI-pLac-cI-LacY-GFP.
	::'''09/28/07'''		::'''09/28/07'''
-	*Digestion of : pTet-RFP with Eco/Spe.	+	*Digestion of : I763033 with Eco/Spe.
	*Ligation with JO4431 (Eco/Xba).		*Ligation with JO4431 (Eco/Xba).
	*We transform and strake on plates.		*We transform and strake on plates.
	[[Bologna | Back]]		[[Bologna | Back]]

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Revision as of 09:40, 8 October 2007

09/24/07

• Ligation for I763031.
• Transformation of I763031.
• We inoculate I763036, Plamba-RFP, PSB4A3 in 5ml of LB.
• Transformation of Ptet-LacI-pLac-CI-LacY-GFP.

09/25/07

• Miniprep of:

- I763036;

- p (Lambda)-RFP;

- pSB4A3;

- I763033.

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

- I763033;

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

• Transformations of:

-I763031.

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. I763031:

-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;

-I763032 and I763034 fluorescent test.

• We inoculate I763033 and pTet-LacI-pLac-cI-LacY-GFP.

09/28/07

• Digestion of : I763033 with Eco/Spe.
• Ligation with JO4431 (Eco/Xba).
• We transform and strake on plates.

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