Week 9

From 2007.igem.org

08/27/07
  • New [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h at 15°C and 1.2ul Ligase).
  • We transform [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation and new [http://partsregistry.org/Part:BBa_S03520 S03520],[http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] biobricks.



08/28/07
  • Lab meeting on:

- model;

- LacI cloning under the pTetR ([http://partsregistry.org/Part:BBa_R0040 R0040]) promoter control.

  • We transform new biobricks: [http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040]
  • We inoculate [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] colonies in 5ml;
  • There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412]([http://partsregistry.org/Part:BBa_I763010 (I763010)] ligation.


08/29/07
  • Miniprep of [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103].
  • [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] digestions with Xba/Pst1.
  • [http://partsregistry.org/Part:BBa_B0034 B0034] digestion with Spe/Pst1.
  • Run on electrophoresis gel and [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034] band extraction. We have some problems with [http://partsregistry.org/Part:BBa_J06550 J06550] plasmid vector due to its molecular weight (too heavy) and with [http://partsregistry.org/Part:BBa_S0103 S0103] insert for the same reason (too heavy). Thus, we decide to use another biobrick.
  • Ligation for [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] ([http://partsregistry.org/Part:BBa_I763016 I763016]), [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sendbox),[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763015 I763015] ), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763020 I763020]).
  • We transform the ligations.



08/30/07
  • There are colonies in the 07/30/07 plates.
  • We inoculate a [http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040] colony in 5ml.
  • We perform some tests to set up the fluorescence acquisition with the PMT (photomultiplier tube).


08/31/07
  • Miniprep of :

-[http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550]

-[http://partsregistry.org/Part:BBa_I763012 I763012]+ [http://partsregistry.org/Part:BBa_S0103 S0103] [http://partsregistry.org/Part:BBa_I763016 (I763016)];

-[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763015 I763015]);

-[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] [http://partsregistry.org/Part:BBa_I763020 (I763020)];

-[http://partsregistry.org/Part:BBa_E0022 E0022 ];

-[http://partsregistry.org/Part:BBa_R0040 R0040].

  • Digestion for:

-[http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_S0103 S0103] with Spe/Pst1;

-[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] with Xba/Pst1;

-[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] [http://partsregistry.org/Part:BBa_I763020 (I763020)] with Xba/Pst1;

-[http://partsregistry.org/Part:BBa_E0022 E0022] with Xba/Pst1 ;

-[http://partsregistry.org/Part:BBa_R0040 R0040] with Spe/Pst1.

  • Band extraction from gel for:

-[http://partsregistry.org/Part:BBa_I763012 I763012]+ [http://partsregistry.org/Part:BBa_S0103 S0103] [http://partsregistry.org/Part:BBa_I763016 (I763016)];

-[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763015 I763015])

-[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] [http://partsregistry.org/Part:BBa_I763020 (I763020)];

-[http://partsregistry.org/Part:BBa_E0020 E0020 ];

-[http://partsregistry.org/Part:BBa_R0040 R0040].

  • Ligations at 15°C for 3h for:

-[http://partsregistry.org/Part:BBa_I763016 I763016]+[http://partsregistry.org/Part:BBa_I763020 I763020];

-[http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_S0100 S0100];

-[http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_S03520 S03520];

-[http://partsregistry.org/Part:BBa_I763005 I763005]+[http://partsregistry.org/Part:BBa_I763015 I763015];

-[http://partsregistry.org/Part:BBa_J52034 J52034]+[http://partsregistry.org/Part:BBa_E0022 E0022];

  • Transformation of all ligations.
  • We find colonies for all ligations, except for the first one.
  • Photomultiplier adjustements

We decided to go on with our fluorescence measurements using not only the photo camera but also the photomultiplier described in the "Materials and Methods" section on our Homepage.

So, we have to adjust the PMT 814 features. First of all we calibrate this instrument by regulating the output offset to have a 0.00 output in case of photomultiplier in total darkness. Then, we adjust the PMT 814 time constant to 50 ns and its gain to 10^(-3) microA/V.

After that we have to cut the photomultiplier field of view, in order to have it the same size as the microscope's one: we do that by moving the appropiate diaphragms.

Then, we have to adjust the external voltage, a sort of gain which is useful to mantain our output signal below 10 volts as prescribed. As the maximum possible output signal, we take a field of view that contains a fluorescent bacteria population at saturation; then we make it match with a 10.00 volts output by regulating the external gain.


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