Week 13

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09/24/07
  • Ligation for [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.
  • Transformation of Ptet-LacI-pLac-CI-LacY-GFP.


09/25/07
  • Miniprep of:
- I763036;
- p (Lambda)-RFP;
- [http://partsregistry.org/Part:pSB4A3 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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