Week 11

From 2007.igem.org

(Difference between revisions)
Line 65: Line 65:
*Control digestion for I763028.
*Control digestion for I763028.
*M9 Medium and lactose stocks preparation.
*M9 Medium and lactose stocks preparation.
 +
 +
*'''Testing our devices'''
 +
 +
We take 5 ml of LB medium in which we put a colony of bacteria which contain Ptet-LacI Plac-cI-LacY-GFP plasmid.
 +
We examine a drop of it using our fluorescence microscopy setup. In these conditions, the bacteria don’t beam fluorescence.
 +
Starting from this moment, we put into the LB medium 1 mM of IPTG every 30 minutes until it is 4 mM , checking each time any eventual fluorescence, with negative results. After diluting 1 ml of the previous one with 4 ml of LB, we add IPTG until 1 ml of our new solution is 10 mM, then 20 mM and finally 40 mM. Despite that, no fluorescence is visible.
 +
For this reason now we test single parts of  the plasmid. We take three populations of Plac-cI-LacY-GFP  bacteria and one of Plac-cI-GFP bacteria. We add 1mM of IPTG to every population to check for fluorescence. We see the three Plac-cI-LacY-GFP populations produce a photomultiplier output between 5.55 and 5.61 Volts, while the Plac-cI-GFP population results more fluorescent giving a 5.91 Volts output.
 +
Every time we check for fluorescence, we use, as said, not only the photo camera but also the photomultiplier. Every measurement shows a 0.18 Volts offset due to environment light and a 1.30 Volts offset due to the excitation light at 501 nm. Since we know GFP gives a maximum of fluorescence when excited at 501 nm, we decide to go on with this wavelength despite the offset (a change of excitation wavelength reduces both the offset and the signal).
 +

Revision as of 10:23, 20 September 2007

09/10/07
  • Miniprep for:

-[http://partsregistry.org/Part:BBa_I763027 I763027];

-[http://partsregistry.org/Part:BBa_I763028 I763028];

-[http://partsregistry.org/Part:BBa_I763019 I763019];

-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_P0412 P0412];

-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S03520 S03520];

-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S0100 S0100];

Digestion for:

-[http://partsregistry.org/Part:BBa_I763027 I763027] with Xba/Spe;

-[http://partsregistry.org/Part:BBa_I763028 I763028] with Spe/Pst1 and with Xba/Spe;

-[http://partsregistry.org/Part:BBa_I763019 I763019] with Xba/Spe;

-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_P0412 P0412] with Eco/Spe;

-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S03520 S03520] with Eco/Spe;

-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S0100 S0100] with Eco/Spe;

  • Band extraction from gel for all digestion;
  • We have problems with [http://partsregistry.org/Part:BBa_J52034 J52034] and with [http://partsregistry.org/Part:BBa_I763019 I763019].
  • Ligations for:

-[http://partsregistry.org/Part:BBa_I763028 I763028] + [http://partsregistry.org/Part:BBa_I763007 I763007]

-[http://partsregistry.org/Part:BBa_S0100 S0100] + [http://partsregistry.org/Part:BBa_J04431 J04431], because we want to understand if LacI operates well;

-[http://partsregistry.org/Part:BBa_J06550 J06550] + [http://partsregistry.org/Part:BBa_J04631 J04631], with this ligation we want to understand if it operates well, because it doesn't leak.

09/11/07
  • Digestion for:

-[http://partsregistry.org/Part:BBa_I763020 I763020] with Xba/Pst1;

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


09/12/07
  • Control digestion before fluorescence tests. (photos)
  • Ligations for:

-I7633005(Spe/Pst1) + J04031(Xba/Pst1);

-S0100(Eco/Spe) + J04031(Eco/Xba);

-I763015 (Eco/Spa) + J04031(Eco/Xba).


09/13/07
  • We have find any colonies for I763015 + J04031;
  • Miniprep for:

- I7633005 + J04031;

-S0100 + J04031;

-I763028.

  • Control digestion for I763028.
  • M9 Medium and lactose stocks preparation.
  • Testing our devices

We take 5 ml of LB medium in which we put a colony of bacteria which contain Ptet-LacI Plac-cI-LacY-GFP plasmid. We examine a drop of it using our fluorescence microscopy setup. In these conditions, the bacteria don’t beam fluorescence. Starting from this moment, we put into the LB medium 1 mM of IPTG every 30 minutes until it is 4 mM , checking each time any eventual fluorescence, with negative results. After diluting 1 ml of the previous one with 4 ml of LB, we add IPTG until 1 ml of our new solution is 10 mM, then 20 mM and finally 40 mM. Despite that, no fluorescence is visible. For this reason now we test single parts of the plasmid. We take three populations of Plac-cI-LacY-GFP bacteria and one of Plac-cI-GFP bacteria. We add 1mM of IPTG to every population to check for fluorescence. We see the three Plac-cI-LacY-GFP populations produce a photomultiplier output between 5.55 and 5.61 Volts, while the Plac-cI-GFP population results more fluorescent giving a 5.91 Volts output. Every time we check for fluorescence, we use, as said, not only the photo camera but also the photomultiplier. Every measurement shows a 0.18 Volts offset due to environment light and a 1.30 Volts offset due to the excitation light at 501 nm. Since we know GFP gives a maximum of fluorescence when excited at 501 nm, we decide to go on with this wavelength despite the offset (a change of excitation wavelength reduces both the offset and the signal).



09/14/07
  • Glicerol stocks for:

-I763028;

-pLac-cI-GFP;

-I763020;

-I763026.

  • Fluorescence test for:

-I763028;

-pLac-cI-GFP;

-I763028(midi).

  • Miniprep for:

-I763028;

-pLac-cI-GFP;

-I763026;

-I763020.

  • Digestion for:

-I763028 with Eco/Xba;

-I763026 with Eco/Spa;

-S0100 with Spa/Pst1;

-S0100 with Eco/Spa;

-I763020 with Xba/Pst1;

-pLac-cI-GFP with Xba/Pst1.

  • Band extraction from gel for all digestion except for pLac-cI-GFP. (photos)



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