Team:UNIPV-Pavia/Calendar/August

44° Bio-Lab - I20 and I21 plates showed colonies. Some were picked, inoculated and grown in proper medium.

45° Bio-Lab - I20-1/2/3 and I21-1/2/3 ligations sent sequencing.

<partinfo>BBa_R0062</partinfo> and <partinfo>BBa_K081009</partinfo> resuspended from iGEM 2010 Distribution Kit.

Trasformation of pAH123 and <partinfo>BBa_J72008</partinfo> helper into MC1061 and MG1655.

46° Bio-Lab - <partinfo>BBa_R0062</partinfo> and <partinfo>BBa_K081009</partinfo> single colony pick from plates.

MC1061 and MG1655 colonies transformed with helpers were picked from plates and inoculated and and incubated in proper medium.

PCR from colony (MG1655 and MC1061) using synthesized primers to check them and to verify a negative control for integration.

47° Bio-Lab - Glycerol stocks for MC1061 and MG1655 strains transformed with pAH123 or <partinfo>BBa_J72008</partinfo> helper plasmids. Miniprep of MC1061 and MG1655 to check again the presence of helper plasmids. Digestion with SpeI and gel run. We got positive samples but a bad run. So we picked again from plates, this time single colonies.

48° Bio-Lab - Screening for MG1655 and MC1061 carrying <partinfo>BBa_J72008</partinfo> or pAH123 helpers: miniprep of new picked colonies MC1061 and MG1655, digestion with SpeI and gel run.

New ligation cycle for phasins (I20-new, I21-new).

49° Bio-Lab - Transformation of I20-new and I21-new ligations into E. coli Dh5-alpha.

Competentization of MG008/123 and MC008/123 strains selected previous day.

50° Bio-Lab -

51° Bio-Lab -

52° Bio-Lab - Phasins plates showed very few colonies (17 for I20-new and 12 for I21-new): they were all picked and let grow in LB+Amp 100ug/ml. In the evening we made glycerol stocks and re-filled the tubes for the screening of the following day. Not to loose time we performed again PCR-amplification/modification of <partinfo>BBa_K208001</partinfo>.

We checked LB+Cm 6 ug/ml agar plates with MC1061, MG1655, MC008, MG008, MC123, MG123 (none of them should grow).

Check for transformation efficiency of MC123/008 and MG123/008.

53° Bio-Lab -

54° Bio-Lab -

55° Bio-Lab -

56° Bio-Lab -

57° Bio-Lab -

58° Bio-Lab -

59° Bio-Lab -

60° Bio-Lab -

61° Bio-Lab -

62° Bio-Lab -

63° Bio-Lab -

64° Bio-Lab -