Week 4

From 2007.igem.org

Latest revision as of 15:50, 26 October 2007

07/23/07

• On the plates we observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051].
• Transformation again for [http://partsregistry.org/Part:BBa_R0051 R0051] (2ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];

• [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test ligation protocol. We try with vector and insert 2ul, vector and insert 2-4ul. We leave it at 25°C for 1 h and at 65°C for 10 minutes.
• Then we transform 4ul from the ligation reaction.

07/24/07

• We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50ml 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 5ml. 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 5ml without IPTG to see the protein extinction time.
• We measure OD every 20 minutes.

• We go along with another GFP induction: after 10 minutes the cells are already green.
• We transform bacteria with 4ul of the [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10ul from [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligations.
• We strake on plates.

07/25/07

• Midiprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:
• Midiprep Quantification: conc. 65ng/ul;

• We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50ml.
• We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051].
• We transform again with [http://partsregistry.org/Part:BBa_R0051 R0051] and with [http://partsregistry.org/Part:BBa_R1051 R1051].

07/26/07

• We let colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] grow at 37°C during the day. We inoculate in 5ml in the evening and we leave them O/N.
• We perform a fluorescence test of [http://partsregistry.org/Part:BBa_J04431 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 protocol.
• Then we inoculate in 5ml O/N.

07/27/07

• Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectrophotometer 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] ([http://partsregistry.org/Part:BBa_I763004 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. In fact 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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