Week 13
From 2007.igem.org
(Difference between revisions)
Line 11: | Line 11: | ||
::- I763036; | ::- I763036; | ||
::- p (Lambda)-RFP; | ::- p (Lambda)-RFP; | ||
- | ::- pSB4A3; | + | ::- [http://partsregistry.org/Part:pSB4A3 pSB4A3]; |
::- I763033. | ::- I763033. | ||
*We inoculate (5ml LB, 5ul amp 1000X) straked transformations of: | *We inoculate (5ml LB, 5ul amp 1000X) straked transformations of: |
Revision as of 10:10, 8 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 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.
- 1. pLac-GFP:
- 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.
- 2. pTetR-LacI-pLac-cI-LacY-GFP
- 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.
- 1. pLac-GFP:
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.