Week 13

From 2007.igem.org

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Line 2: Line 2:
*Ligation for [http://partsregistry.org/Part:BBa_I763031 I763031].
*Ligation for [http://partsregistry.org/Part:BBa_I763031 I763031].
*Transformation of [http://partsregistry.org/Part:BBa_I763031 I763031].
*Transformation of [http://partsregistry.org/Part:BBa_I763031 I763031].
-
*We inoculate I763036, Plamba-RFP, PSB4A3 in 5ml of LB.
+
*We inoculate [http://partsregistry.org/Part:BBa_I763036 I763036], [http://partsregistry.org/Part:BBa_I763007 I763007], [http://partsregistry.org/Part:pSB4A3 pSB4A3] in 5ml of LB.
-
*Transformation of Ptet-LacI-pLac-CI-LacY-GFP.
+
*Transformation of [http://partsregistry.org/Part:BBa_I763028 I763028].
Line 9: Line 9:
::'''09/25/07'''
::'''09/25/07'''
*Miniprep of:
*Miniprep of:
-
::- I763036;
+
::- [http://partsregistry.org/Part:BBa_I763036 I763036];
-
::- p (Lambda)-RFP;
+
::- [http://partsregistry.org/Part:BBa_I763007 I763007];
-
::- pSB4A3;
+
::- [http://partsregistry.org/Part:pSB4A3 pSB4A3];
-
::- I763033.   
+
::- [http://partsregistry.org/Part:BBa_I763033 I763033].   
*We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:
*We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:
-
::- I763033;   
+
::- [http://partsregistry.org/Part:BBa_I763033 I763033];   
-
::- pTet-LacI-pLac-cI-LacY-GFP (vitality test).   
+
::- [http://partsregistry.org/Part:BBa_I763028 I763028] (vitality test).   
   
   
*Transformations of:
*Transformations of:
-
::-I763031.   
+
::-[http://partsregistry.org/Part:BBa_I763031 I763031].   
Line 24: Line 24:
::'''09/26/07'''
::'''09/26/07'''
-
'''Fluorescence test according to [[Bologna University/Fluorescence Test | fluorescence test protocol]]'''  of:
+
'''Fluorescence test according to [[Bologna University/Fluorescence Test1 | Fluorescence Test Protocol]]'''  of:
-
::1. ''pLac-GFP'':
+
::1. ''[http://partsregistry.org/Part:BBa_I763004 I763004]'':
::::-bacteria use to have the PMT output maximum value;
::::-bacteria use to have the PMT output maximum value;
::::-normalization of OD value at 0.5;
::::-normalization of OD value at 0.5;
Line 37: Line 37:
::::-thus we decide to grow the OD value to have higher output value.
::::-thus we decide to grow the OD value to have higher output value.
-
::2. ''pTetR-LacI-pLac-cI-LacY-GFP''
+
::2. ''[http://partsregistry.org/Part:BBa_I763028 I763028]''
-
::::- 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).
+
::::- 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.
::::- thus we decide to use lactose.
Line 45: Line 45:
::'''09/27/07'''
::'''09/27/07'''
-
'''Fluorescence test according to [[Bologna University/Fluorescence Test | Fluorescence test protocol]]'''  of:
+
'''Fluorescence test according to [[Bologna University/Fluorescence Test1 | Fluorescence Test Protocol]]'''  of:
-
::1. ''pLac-GFP'':
+
::1. ''[http://partsregistry.org/Part:BBa_I763004 I763004]'':
::::-output value like to value with OD=0.5.
::::-output value like to value with OD=0.5.
-
::2. ''pTetR-LacI-pLac-cI-LacY-GFP'':
+
::2. ''[http://partsregistry.org/Part:BBa_I763028 I763028]'':
::::-growth in LB medium;
::::-growth in LB medium;
::::-pellet in M9 with lactose;
::::-pellet in M9 with lactose;
Line 54: Line 54:
::::::::-bacteria vitality confirmed;
::::::::-bacteria vitality confirmed;
::::::::-bacteria don’t beam fluorescent.  
::::::::-bacteria don’t beam fluorescent.  
-
::3. ''I763031'':
+
::3. ''[http://partsregistry.org/Part:BBa_I763031 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.
-
::4. ''pLac-cI-LacY-GFP'':
+
::4. ''[http://partsregistry.org/Part:BBa_I763019 I763019]'':
::::-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;
-
::-I763032 and I763034 fluorescent test.
+
::-[http://partsregistry.org/Part:BBa_I763032 I763032] and [http://partsregistry.org/Part:BBa_I763034 I763034] fluorescent test.
-
*We inoculate I763033 and pTet-LacI-pLac-cI-LacY-GFP.
+
*We inoculate [http://partsregistry.org/Part:BBa_I763033 I763033] and [http://partsregistry.org/Part:BBa_I763028 I763028].
      
      
::'''09/28/07'''
::'''09/28/07'''
-
*Digestion of : I763033 with Eco/Spe.
+
*Digestion of : [http://partsregistry.org/Part:BBa_I763033 I763033] with Eco/Spe.
-
*Ligation with JO4431 (Eco/Xba).  
+
*Ligation with [http://partsregistry.org/Part:BBa_J04431 J04431] (Eco/Xba).  
*We transform and strake on plates.
*We transform and strake on plates.
-
[[Bologna | Back]]
+
[[Bologna#Diary | Back]]

Latest revision as of 15:51, 26 October 2007

09/24/07
  • Ligation for [http://partsregistry.org/Part:BBa_I763031 I763031].
  • Transformation of [http://partsregistry.org/Part:BBa_I763031 I763031].
  • We inoculate [http://partsregistry.org/Part:BBa_I763036 I763036], [http://partsregistry.org/Part:BBa_I763007 I763007], [http://partsregistry.org/Part:pSB4A3 pSB4A3] in 5ml of LB.
  • Transformation of [http://partsregistry.org/Part:BBa_I763028 I763028].


09/25/07
  • Miniprep of:
- [http://partsregistry.org/Part:BBa_I763036 I763036];
- [http://partsregistry.org/Part:BBa_I763007 I763007];
- [http://partsregistry.org/Part:pSB4A3 pSB4A3];
- [http://partsregistry.org/Part:BBa_I763033 I763033].
  • We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:
- [http://partsregistry.org/Part:BBa_I763033 I763033];
- [http://partsregistry.org/Part:BBa_I763028 I763028] (vitality test).
  • Transformations of:
-[http://partsregistry.org/Part:BBa_I763031 I763031].



09/26/07

Fluorescence test according to Fluorescence Test Protocol of:

1. [http://partsregistry.org/Part:BBa_I763004 I763004]:
-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. [http://partsregistry.org/Part:BBa_I763028 I763028]
- 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. [http://partsregistry.org/Part:BBa_I763004 I763004]:
-output value like to value with OD=0.5.
2. [http://partsregistry.org/Part:BBa_I763028 I763028]:
-growth in LB medium;
-pellet in M9 with lactose;
-we observe them every 30min:
-bacteria vitality confirmed;
-bacteria don’t beam fluorescent.
3. [http://partsregistry.org/Part:BBa_I763031 I763031]:
-Output value without IPTG:0.05V;
-Output value with IPTG: about 0V.
4. [http://partsregistry.org/Part:BBa_I763019 I763019]:
-Output value without IPTG:0.05V;
-Output value with IPTG: about 0V.

Conclusions:

-using of low copy plasmid;
-higher OD;
-[http://partsregistry.org/Part:BBa_I763032 I763032] and [http://partsregistry.org/Part:BBa_I763034 I763034] fluorescent test.
  • We inoculate [http://partsregistry.org/Part:BBa_I763033 I763033] and [http://partsregistry.org/Part:BBa_I763028 I763028].


09/28/07
  • Digestion of : [http://partsregistry.org/Part:BBa_I763033 I763033] with Eco/Spe.
  • Ligation with [http://partsregistry.org/Part:BBa_J04431 J04431] (Eco/Xba).
  • We transform and strake on plates.

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