Week 11

From 2007.igem.org

  • Miniprep for:







Digestion for:

-I763027 with Xba/Spe;

-I763028 with Spe/Pst1 and with Xba/Spe;

-I763019 with Xba/Spe;

-I763021+P0412 with Eco/Spe;

-I763021+S03520 with Eco/Spe;

-I763021+S0100 with Eco/Spe;

  • Band extraction from gel for all digestion;
  • We have problems with J52034 and with I763019.
  • Ligations for:

-I763028 + I763007

-S0100 + J04431, because we want to understand if LacI operates well;

-J06550 + J04631, with this ligation we want to understand if it operates well, because it doesn't leak.

  • Digestion for:

-I763020 with Xba/Pst1;

-J22101 with Xba/Pst1;

  • Control digestion before fluorescence tests. (photos)
  • Ligations for:

-I763005 (Spe/Pst1) + J04031 (Xba/Pst1);

-I763025 (Eco/Spe) + J04031 (Eco/Xba);

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


-I763005 + J04031;

-I763025 + J04031;


  • Control digestion for 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 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 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 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).

  • Glicerol stocks for:





  • Fluorescence test for:



  • Miniprep for:





  • Digestion for:

-I763019 with Eco/Xba;

-I763026 with Eco/Spe;

-I763026 with Spe/Pst1;

-I763026 with Eco/Spe;

-I763020 with Xba/Pst1;

-I763031 with Xba/Pst1.