Week 11

From 2007.igem.org

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Line 1: Line 1:
::'''09/10/07'''
::'''09/10/07'''
-
*Miniprep for
+
*Miniprep for:
-[http://partsregistry.org/Part:BBa_I763027 I763027];
-[http://partsregistry.org/Part:BBa_I763027 I763027];
Line 42: Line 42:
-[http://partsregistry.org/Part:BBa_J22101 J22101] with Xba/Pst1;
-[http://partsregistry.org/Part:BBa_J22101 J22101] with Xba/Pst1;
-
* '''Testing our devices'''
 
-
 
-
We take 5 ml of LB culture ground 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 culture ground 1 mM of IPTG every 30 minutes until it is 4 millimolar, 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.
 
-
Every time we checked for fluorescence, we used not only the photo camera but also the photomultiplier; every measurement showed a 0.16-0.18 volts offset, despite the bacteria being non-fluorescent: at least, we regulated that offset!
 
::'''09/12/07'''
::'''09/12/07'''
 +
* Control digestion before fluorescence tests. [[(photos)]]
 +
* Ligations for:
 +
-[http://partsregistry.org/Part:BBa_I763005 I763005] (Spe/Pst1) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Xba/Pst1);
 +
-[http://partsregistry.org/Part:BBa_I763025 I763025] (Eco/Spe) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Eco/Xba);
 +
-[http://partsregistry.org/Part:BBa_I763015  I763015] (Eco/Spe) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Eco/Xba).
::'''09/13/07'''
::'''09/13/07'''
 +
 +
*We have find any colonies for [http://partsregistry.org/Part:BBa_I763015  I763015] + [http://partsregistry.org/Part:BBa_J04031 J04031].
 +
*Miniprep for:
 +
-[http://partsregistry.org/Part:BBa_I763005 I763005] + [http://partsregistry.org/Part:BBa_J04031 J04031];
 +
 +
-[http://partsregistry.org/Part:BBa_I763025 I763025] + [http://partsregistry.org/Part:BBa_J04031 J04031];
 +
 +
-[http://partsregistry.org/Part:BBa_I763019 I763019].
 +
*Control digestion for [http://partsregistry.org/Part:BBa_I763019 I763019].
 +
*M9 medium and lactose stocks preparation.
 +
 +
*'''Testing our devices'''
 +
 +
We take 5ml of LB medium in which we put a colony of bacteria which contain the  [http://partsregistry.org/Part:BBa_I763028 I763028] plasmid.
 +
We examine a drop of it with our fluorescence microscopy. In these conditions, the bacteria don’t beam fluorescence.
 +
Starting from this moment, we add into the LB medium 1mM of IPTG every 30 minutes until it reaches 4mM , checking each time for any possible fluorescence, with negative results. We then try another option: after diluting 1ml of the original solution with 4ml of LB, we add IPTG until this solution reaches 10mM, then 20mM and finally 40mM. Despite that, no fluorescence is visible.
 +
For this reason we then test single parts of  the plasmid. We take three populations of [http://partsregistry.org/Part:BBa_I763019 I763019]  bacteria and one of Plac-cI-GFP bacteria. We add 1mM of IPTG to every population to check for fluorescence. We see the three [http://partsregistry.org/Part:BBa_I763019 I763019] populations produce a photomultiplier output between 5.55 and 5.61Volts, while the Plac-cI-GFP population results more fluorescent giving a 5.91Volts output.
 +
Every time we check for fluorescence, we use, as said, not only the photo camera but also the photomultiplier. Every measurement shows a 0.18Volts offset due to environment light and a 1.30Volts offset due to the excitation light at 501nm. Since we know GFP gives a maximum of fluorescence when excited at 501nm, we decide to go on with this wavelength despite the offset (a change of excitation wavelength reduces both the offset and the signal).
Line 61: Line 78:
      
      
::'''09/14/07'''
::'''09/14/07'''
 +
 +
*Glicerol stocks for:
 +
-[http://partsregistry.org/Part:BBa_I763019 I763019];
 +
 +
-[http://partsregistry.org/Part:BBa_I763031 I763031];
 +
 +
-[http://partsregistry.org/Part:BBa_I763020 I763020];
 +
 +
-[http://partsregistry.org/Part:BBa_I763026 I763026].
 +
*Fluorescence test for:
 +
-[http://partsregistry.org/Part:BBa_I763019 I763019];
 +
 +
-[http://partsregistry.org/Part:BBa_I763031 I763031].
 +
 +
*Miniprep for:
 +
-[http://partsregistry.org/Part:BBa_I763019 I763019];
 +
 +
-[http://partsregistry.org/Part:BBa_I763031 I763031];
 +
 +
-[http://partsregistry.org/Part:BBa_I763026 I763026];
 +
 +
-[http://partsregistry.org/Part:BBa_I763020 I763020].
 +
*Digestion for:
 +
-[http://partsregistry.org/Part:BBa_I763019 I763019] with Eco/Xba;
 +
 +
-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Spe;
 +
 +
-[http://partsregistry.org/Part:BBa_I763026 I763026] with Spe/Pst1;
 +
 +
-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Spe;
 +
 +
-[http://partsregistry.org/Part:BBa_I763020 I763020] with Xba/Pst1;
 +
 +
-[http://partsregistry.org/Part:BBa_I763031 I763031] with Xba/Pst1.
 +
*Band extraction from gel for all digestion except for [http://partsregistry.org/Part:BBa_I763031 I763031]. [[(photos2)]]
-
[[Bologna | Back]]
+
[[Bologna#Diary | Back]]

Latest revision as of 15:53, 26 October 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:

-[http://partsregistry.org/Part:BBa_I763005 I763005] (Spe/Pst1) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Xba/Pst1);

-[http://partsregistry.org/Part:BBa_I763025 I763025] (Eco/Spe) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Eco/Xba);

-[http://partsregistry.org/Part:BBa_I763015 I763015] (Eco/Spe) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Eco/Xba).


09/13/07
  • We have find any colonies for [http://partsregistry.org/Part:BBa_I763015 I763015] + [http://partsregistry.org/Part:BBa_J04031 J04031].
  • Miniprep for:

-[http://partsregistry.org/Part:BBa_I763005 I763005] + [http://partsregistry.org/Part:BBa_J04031 J04031];

-[http://partsregistry.org/Part:BBa_I763025 I763025] + [http://partsregistry.org/Part:BBa_J04031 J04031];

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

  • Control digestion for [http://partsregistry.org/Part:BBa_I763019 I763019].
  • M9 medium and lactose stocks preparation.
  • Testing our devices

We take 5ml of LB medium in which we put a colony of bacteria which contain the [http://partsregistry.org/Part:BBa_I763028 I763028] plasmid. We examine a drop of it with our fluorescence microscopy. In these conditions, the bacteria don’t beam fluorescence. Starting from this moment, we add into the LB medium 1mM of IPTG every 30 minutes until it reaches 4mM , checking each time for any possible fluorescence, with negative results. We then try another option: after diluting 1ml of the original solution with 4ml of LB, we add IPTG until this solution reaches 10mM, then 20mM and finally 40mM. Despite that, no fluorescence is visible. For this reason we then test single parts of the plasmid. We take three populations of [http://partsregistry.org/Part:BBa_I763019 I763019] bacteria and one of Plac-cI-GFP bacteria. We add 1mM of IPTG to every population to check for fluorescence. We see the three [http://partsregistry.org/Part:BBa_I763019 I763019] populations produce a photomultiplier output between 5.55 and 5.61Volts, while the Plac-cI-GFP population results more fluorescent giving a 5.91Volts output. Every time we check for fluorescence, we use, as said, not only the photo camera but also the photomultiplier. Every measurement shows a 0.18Volts offset due to environment light and a 1.30Volts offset due to the excitation light at 501nm. Since we know GFP gives a maximum of fluorescence when excited at 501nm, 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:

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

-[http://partsregistry.org/Part:BBa_I763031 I763031];

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

-[http://partsregistry.org/Part:BBa_I763026 I763026].

  • Fluorescence test for:

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

-[http://partsregistry.org/Part:BBa_I763031 I763031].

  • Miniprep for:

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

-[http://partsregistry.org/Part:BBa_I763031 I763031];

-[http://partsregistry.org/Part:BBa_I763026 I763026];

-[http://partsregistry.org/Part:BBa_I763020 I763020].

  • Digestion for:

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

-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Spe;

-[http://partsregistry.org/Part:BBa_I763026 I763026] with Spe/Pst1;

-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Spe;

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

-[http://partsregistry.org/Part:BBa_I763031 I763031] with Xba/Pst1.

  • Band extraction from gel for all digestion except for [http://partsregistry.org/Part:BBa_I763031 I763031]. (photos2)



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