Week 12

From 2007.igem.org

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Line 18: Line 18:
-[http://partsregistry.org/Part:BBa_I763028 I763028];
-[http://partsregistry.org/Part:BBa_I763028 I763028];
-
-Ptet-LacI-T-PLac-GFP;
+
-[http://partsregistry.org/Part:BBa_I763027 I763027];
-
-Ptet-LacI-GFP(Spe/Pst1), (Xba/Pst1).
+
-[http://partsregistry.org/Part:BBa_I763035 I763035] (Spe/Pst1), (Xba/Pst1).
*Digestion for:
*Digestion for:
-[http://partsregistry.org/Part:BBa_I763028 I763028] with Eco/Spe;
-[http://partsregistry.org/Part:BBa_I763028 I763028] with Eco/Spe;
-
-Ptet-LacI-T-PLac-GFP with Eco/Spe;
+
-[http://partsregistry.org/Part:BBa_I763027 I763027] with Eco/Spe;
-
-Ptet-LacI-GFP with Eco/Spe;
+
-[http://partsregistry.org/Part:BBa_I763035 I763035] with Eco/Spe;
-[http://partsregistry.org/Part:BBa_I763007 I763007] with Eco/Xba;
-[http://partsregistry.org/Part:BBa_I763007 I763007] with Eco/Xba;
-
-Plac-cI-LacY with Eco/Spe1;
+
-[http://partsregistry.org/Part:BBa_I763036 I763036] with Eco/Spe1;
*Band extraction from gel for all digestion and then we observe:
*Band extraction from gel for all digestion and then we observe:
-
-[http://partsregistry.org/Part:BBa_I763028 I763028], Ptet-LacI-T-PLac-GFP are died;
+
-[http://partsregistry.org/Part:BBa_I763028 I763028], [http://partsregistry.org/Part:BBa_I763027 I763027] are died;
-
-Ptet-LacI-GFP is correct;
+
-[http://partsregistry.org/Part:BBa_I763035 I763035] is correct;
-[http://partsregistry.org/Part:BBa_I763007 I763007] not found;
-[http://partsregistry.org/Part:BBa_I763007 I763007] not found;
-
-Plac-CI-LacY is correct.
+
-[http://partsregistry.org/Part:BBa_I763036 I763036] is correct.
Line 47: Line 47:
We wanted to test if our newly-constructed devices work, that is if they beam fluorescence when inducted with IPTG. To do so we performed 3 tests in parallel in 3 different tubes, that we name here A, B and C.
We wanted to test if our newly-constructed devices work, that is if they beam fluorescence when inducted with IPTG. To do so we performed 3 tests in parallel in 3 different tubes, that we name here A, B and C.
-
::1. In tube A e in tube B we add 5ml of LB medium and 2 different colonies of  I763019 plasmid.  
+
::1. In tube A e in tube B we add 5ml of LB medium and 2 different colonies of  [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid.  
-
::2. In tube C we add 5ml of LB medium and a colony of PLac-cI-GFP plasmid.  
+
::2. In tube C we add 5ml of LB medium and a colony of [http://partsregistry.org/Part:BBa_I763031 I763031] plasmid.  
::3. After 2 hours we measure the OD value of  tubes A, B, C and it is around 0.5.  
::3. After 2 hours we measure the OD value of  tubes A, B, C and it is around 0.5.  
::4. For tubes A, B, C we divided the bacteria fluid in two different tubes A1, A2; B1, B2; C1, C2.  
::4. For tubes A, B, C we divided the bacteria fluid in two different tubes A1, A2; B1, B2; C1, C2.  
Line 54: Line 54:
* The analyzed fluid without IPTG (tubes A1, B1, C1) includes:  
* The analyzed fluid without IPTG (tubes A1, B1, C1) includes:  
::-2.5ml of original tube fluid;  
::-2.5ml of original tube fluid;  
-
::--2.5ml of LB medium;  
+
::-2.5ml of LB medium;  
::-2.5ul of kanamicin.  
::-2.5ul of kanamicin.  
*The analyzed fluid with IPTG (tube A2, B2, C2) includes:  
*The analyzed fluid with IPTG (tube A2, B2, C2) includes:  
Line 60: Line 60:
::-2.5ml of LB medium;  
::-2.5ml of LB medium;  
::-2.5ul of kanamicin;  
::-2.5ul of kanamicin;  
-
-50ul of 100mM IPTG.  
+
::-50ul of 100mM IPTG.  
::6. We examine a bacteria fluid drop of tubes A1, A2, B1, B2, C1, C2  with our fluorescence microscope.  
::6. We examine a bacteria fluid drop of tubes A1, A2, B1, B2, C1, C2  with our fluorescence microscope.  
-
::7. The bacteria with I763019 plasmid doesn’t beam fluorescence with (A2, B2) nor without (A1, B1)  IPTG;  
+
::7. The bacteria with [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid doesn’t beam fluorescence with (A2, B2) nor without (A1, B1)  IPTG;  
-
::8. Very few bacteria with PLac-cI-GFP plasmid with (C2) and without (C1) IPTG beam fluorescence.  
+
::8. Very few bacteria with [http://partsregistry.org/Part:BBa_I7630231 I763031] plasmid with (C2) and without (C1) IPTG beam fluorescence.  
-
*In conclusion, we see that bacteria with I763019 plasmid (tubes A1, B1, A2, B2) don't seem to work at all, and bacteria with PLac-cI-GFP plasmid (tube C1, C2) don't seem to work properly either. We plan other experiments for next week.
+
*In conclusion, we see that bacteria with [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid (tubes A1, B1, A2, B2) don't seem to work at all, and bacteria with [http://partsregistry.org/Part:BBa_I763031 I763031] plasmid (tube C1, C2) don't seem to work properly either. We plan other experiments for next week.
Line 70: Line 70:
      
      
::'''09/21/07'''
::'''09/21/07'''
 +
Meeting: definition of fluorescence test protocol.
-
 
+
[[Bologna#Diary | Back]]
-
[[Bologna | Back]]
+

Latest revision as of 15:52, 26 October 2007

09/17/07
  • Ligations for:

-I763026 + I763019;

-I763026 + I763004;

-I763025 + J04031.

  • We transform ligations and strake them on plates.


09/18/07
  • We inoculate a colony for yesterday ligations in 5ml of LB medium O/N.


09/19/07
  • Miniprep for:

-I763028;

-I763027;

-I763035 (Spe/Pst1), (Xba/Pst1).

  • Digestion for:

-I763028 with Eco/Spe;

-I763027 with Eco/Spe;

-I763035 with Eco/Spe;

-I763007 with Eco/Xba;

-I763036 with Eco/Spe1;

  • Band extraction from gel for all digestion and then we observe:

-I763028, I763027 are died;

-I763035 is correct;

-I763007 not found;

-I763036 is correct.


09/20/07

Testing our devices

We wanted to test if our newly-constructed devices work, that is if they beam fluorescence when inducted with IPTG. To do so we performed 3 tests in parallel in 3 different tubes, that we name here A, B and C.

1. In tube A e in tube B we add 5ml of LB medium and 2 different colonies of I763019 plasmid.
2. In tube C we add 5ml of LB medium and a colony of I763031 plasmid.
3. After 2 hours we measure the OD value of tubes A, B, C and it is around 0.5.
4. For tubes A, B, C we divided the bacteria fluid in two different tubes A1, A2; B1, B2; C1, C2.
5. We add 1mM IPTG to the solution into tubes A2, B2, C2.
  • The analyzed fluid without IPTG (tubes A1, B1, C1) includes:
-2.5ml of original tube fluid;
-2.5ml of LB medium;
-2.5ul of kanamicin.
  • The analyzed fluid with IPTG (tube A2, B2, C2) includes:
-2.5ml of original tube fluid;
-2.5ml of LB medium;
-2.5ul of kanamicin;
-50ul of 100mM IPTG.
6. We examine a bacteria fluid drop of tubes A1, A2, B1, B2, C1, C2 with our fluorescence microscope.
7. The bacteria with I763019 plasmid doesn’t beam fluorescence with (A2, B2) nor without (A1, B1) IPTG;
8. Very few bacteria with I763031 plasmid with (C2) and without (C1) IPTG beam fluorescence.
  • In conclusion, we see that bacteria with I763019 plasmid (tubes A1, B1, A2, B2) don't seem to work at all, and bacteria with I763031 plasmid (tube C1, C2) don't seem to work properly either. We plan other experiments for next week.



09/21/07

Meeting: definition of fluorescence test protocol.


Back