Week 4

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Revision as of 10:23, 3 September 2007

07/23/07

• We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051].
• We get along with:

- another transformation of [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];

- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes.

• Then we transforme 4 ul of ligate.

07/24/07

• We observe colony for [http://partsregistry.org/Part:BBa_C0051 C0051] on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.
• We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.

GFP induction test in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.

• We harvest them and we put them in 5 ml without IPTG to see the extinction time of the protein.
• We read every 20 minutes.

• We get along with another GFP induction: yet after 10 minutes the cells are green.
• We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.
• We strake on plates.

07/25/07

• We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:

-Quantification: conc. 65 ng/ul;

-Cleanness n.:1.8.

• We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.
• We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051].
• We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].

07/26/07

• We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.
• We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.
• Then we inoculate in 5 ml O/N.

07/27/07

• We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:

-Quantification: conc.:56 ng/ul.

• Digestion for :

-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;

-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;

-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.

• Then we extract from gel.
• We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) cells and in IPTG not yet inducted [http://partsregistry.org/Part:BBa_J04431 J04431] cells. To explain this unespected observation, we decide to identify the istant during the bacterial growth when the fluorescence appears to understand if fluorescence appearance is determinated by glucose depletion in the culture medium and/or by a deficient LacI quantity inside the cell. Infact the indogenous LacI in coli is sufficient for an only Plac and we suspect it can’t repress the high copy number plasmid Plac we have used. (We will perform this test on 31/07)

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