From 2007.igem.org

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 for [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 transform 4 ul from the ligation reaction.

07/24/07

• We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. 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],[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 inoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.

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

• We get along with another GFP induction: yet after 10 minutes the cells are green.
• We transform bacteria with 4 ul from [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul from [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 colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50 ml.
• We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051].
• We transform again with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and with [http://partsregistry.org/Part:BBa_R1051 R1051].

07/26/07

• We leave colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml in the 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) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)

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