Week 4

From 2007.igem.org

  • J04500+J04631 (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.

  • We observe colonies for C0051 plate. We inoculate in 50ml and we leave at 37°C O/N.
  • We don’t observe any colony for R0051,I13507 and for the 2 ligations.

GFP induction test in 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 I13507 and R0051 and with 10ul from J04500 + J04631 (I763004) ligations.
  • We strake on plates.

  • Midiprep for C0051:
  • Midiprep Quantification: conc. 65ng/ul;
  • We observe colonies for I13507 and ligation. We inoculate in 50ml.
  • We don’t find any colony for R0051.
  • We transform again with R0051 and with R1051.

  • We let colonies from R0051 and fromR1051 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 J04431 and J04500+J04631 (I763004) ligation to test the ligation protocol.
  • Then we inoculate in 5ml O/N.

  • Miniprep for R0051 and R1051. We don’t try to quantificate with spectrophotometer because it isn’t sensible enough. Midi for I13507:

-Quantification: conc.:56 ng/ul.

  • Digestion for:

-I13507 with XbaI/PstI;

-R0051 with SpeI/Pst1;

-C0051 with XbaI/PstI.

  • Then we extract from gel.
  • We observe green fluorescence in J04500+J04631 (I763004) and 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)