From 2007.igem.org

09/17/07

• Ligations for:

-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763019 I763019];

-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763004 I763004];

-[http://partsregistry.org/Part:BBa_I763025 I763025] + [http://partsregistry.org/Part:BBa_J04031 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:

-[http://partsregistry.org/Part:BBa_I763028 I763028];

-Ptet-LacI-T-PLac-GFP;

-Ptet-LacI-GFP(Spe/Pst1), (Xba/Pst1).

• Digestion for:

-[http://partsregistry.org/Part:BBa_I763028 I763028] with Eco/Spe;

-Ptet-LacI-T-PLac-GFP with Eco/Spe;

-Ptet-LacI-GFP with Eco/Spe;

-[http://partsregistry.org/Part:BBa_I763007 I763007] with Eco/Xba;

-Plac-cI-LacY with Eco/Spe1;

• 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;

-Ptet-LacI-GFP is correct;

-[http://partsregistry.org/Part:BBa_I763007 I763007] not found;

-Plac-CI-LacY 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 [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.

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 [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.

• 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 PLac-cI-GFP 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.

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