Week 13

From 2007.igem.org

(Difference between revisions)
Line 9: Line 9:
::'''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.   
Line 54: Line 54:
::::::::-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.
Line 63: Line 63:
::-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]]

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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