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 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 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 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

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