Team:UNIPV-Pavia/Calendar/September

71° Bio-Lab - PCR from colony for I47 ligation.Tecan Test was performed on prepared samples.

72° Bio-Lab - Again PCR from colony for I47 ligation: I47-10..26.Tecan Test was performed on prepared samples.

73° Bio-Lab - Today we got our yeast strains.

74° Bio-Lab - Results of genomic integration of samples inoculated yesterday

75° Bio-Lab - Plates were checked and new colonies were picked.

76° Bio-Lab - Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN experiment.

Inoculum of I44-2 and I51-3 into 5 ml LB+Cm34 to evaluate MiniPrep yields.

Inoculum of I44-2 and I51-3 into 5 ml LB+Cm12,5 to evaluate MiniPrep yields and for sequencing.

Inoculum for MiniPrep and sequencing of: <partinfo>BBa_J23100</partinfo>_4C5, <partinfo>BBa_J23101</partinfo>_4C5, <partinfo>BBa_J23105</partinfo>_4C5, <partinfo>BBa_J23106</partinfo>_4C5, <partinfo>BBa_J23110</partinfo>_4C5, <partinfo>BBa_J23114</partinfo>_4C5, <partinfo>BBa_J23116</partinfo>_4C5, <partinfo>BBa_J23118</partinfo>_4C5, I7-A and I8-5-D.

Inoculum of cultures for TECAN test on self-inducible promoters.

Glycerol stocks of MG43, MC42, MC43. Static TECAN test (absorbance, green fluorescence and red fluorescence) of these cultures.

Liquid PCR was performed on MG43, MC42, MC43 and MC1061. MiniPreps of I44-1:5 and I51-1:5 were performed.

77° Bio-Lab -MiniPreps of <partinfo>BBa_J23100</partinfo>_4C5, <partinfo>BBa_J23101</partinfo>_4C5, <partinfo>BBa_J23105</partinfo>_4C5, <partinfo>BBa_J23106</partinfo>_4C5, <partinfo>BBa_J23110</partinfo>_4C5, <partinfo>BBa_J23114</partinfo>_4C5, <partinfo>BBa_J23116</partinfo>_4C5, <partinfo>BBa_J23118</partinfo>_4C5, I7-A, I8-5-D, I44-2, I51-3, I44-2, I51-3 and I47 were performed. Trasformation of I42 and I43. Integration protocol's first day were performed.
13° Meeting - Updating about Bio-Lab activity.

78° Bio-Lab - Inoculum of: I31, I32, I35, I36, I37, I38, I40, I41 and I20 for second step of ligation of phasins/intein.

79° Bio-Lab - Minipreps of I31, I32, I35, I36, I37, I38, I40, I41 and I20 were performed.

80° Bio-Lab - Screening digestion of I44-2 and I51-3.

Gel run/cut for samples of previous day (I31, I32, I35, I36, I37, I38, I40, I41 and I20). Digestions were ligated in order to create: I52, I53, I54, I55, I56, I57, I58.

81° Bio-Lab - MC42 and MC43 plates were transfered.

I42 and I43 have been transformed in MG008 strain, plated and incubated.

82° Bio-Lab - Trasformation of ligations I52, I54, I55, I56, I57 and I58. into E. coli DH5-alpha.

Inoculum of I26, I31 and I34.

Inoculum from single colony of MG42 and MC43.

83° Bio-Lab - Agar plates of I52, I54, I55, I56, I57 and I58 were checked.

Miniprep and quanfification of I26, I31 and I34. After 3 hour digestion, gel run/cut of samples was done. Gel exctraction and ligation was performed in order to obtain I59, I60, I61 and I53.

Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN test of the following day.

Screening PCR on 3 colonies picked from MC42 and MC43 respectively.

Tecan Test was performed on prepared samples, after usual protocol.

84° Bio-Lab - Tecan Test was started to check GFP production by BioBricks in I47, I48, I49 cultures.

Transformation of I 53, I59, I60 and I61.

85° Bio-Lab - Screening through "colony PCR" for ligations I52, I53, I54, I55, I56, I57, I58, I59, I60 and I61 was performed.

Tecan Test was performed on prepared samples, after the usual protocol.

86° Bio-Lab - Miniprep and Nanodrop quantification for: I58a, I58b, I58c, I59a, I59b, I59c, I60a, I60b, I60c, I61a, I61b and I61c.

Digestion (for screening) and gel extraction for: I58a/b/c, I59a/b/c, I60a/b/c.

Digestion (only screening) for I61a/b/c.

87° Bio-Lab - Gel extraction and quantification of: I58, I59, I60. DNA was than stored at -20°C.

88° Bio-Lab - I55 was transformed again into E. coli DH5-alpha and plated on LB+Amp agar plate, that was let grow ON at 37°C.

89° Bio-Lab - Screening PCR on 6 colonies picked from I55 was performed.

Inoculum of I52, I53, I55, I56 and I57 in LB+Amp for MiniPrep of the following day.

90° Bio-Lab - Miniprep and Nanodrop quantification for: I52, I53, I55, I56, I57; 3 hour digestion and gel run/cut were performed on this samples. Gel extraction was performed on I52, I56, I57, I61.

We already had digested DNA so we could perform new ON ligations: I62, I63, I64, I65, I66.

Tecan Test was performed on prepared samples, after a new protocol.

91° Bio-Lab - Trasformation of ligations I62, I63, I64, I65 and I66 into E.coli TOP10.

MiniPrep and quantification of: MyYeast1, MyYeast2, MyYeast3. Screening digestion was performed on this samples.

Glycerol stocks of MyYeast1, MyYeast2, MyYeast3 was performed.

Inoculum in LB+Amp+Cm12,5 from glycerol stocks and colony for antibiotic resistance testing of I47_S1, I48_S1, I49_S1 and I13522.

92° Bio-Lab - Digestion of yesterday are repeted (because none of them were postitive). This time all samples seems to be positive.

Inoculum for three colonies from I62, I63, I64, I65 agar plates into 5ml LB+Amp.

93° Bio-Lab - Glycerol stocks was performed for I62, I63, I64, I65, I66.

MiniPrep and Nanodrop quantification : I62-1, I62-2, I62-3, I63-1, I63-2, I63-3, I64-1, I64-2, I64-3, I65-1, I65-2, I65-3, I66-1, I66-2 and I66-3. Digestion for screening and gel run of samples were performed.

Dilution 1:50 of I47, I48, I49, GFP in LB+Amp+Cm12,5 and S1 in LB+Cm12,5.

94° Bio-Lab - Inoculum of <partinfo>BBa_K173000</partinfo>, I47, I48, I49, <partinfo>BBa_B0031</partinfo> into 3 ml LB+Amp for tomorrow TECAN test.

Pick of six colonies from I66 agar plate (I66-A/B/C/D/E/F) and inoculum into 3 ml LB+Amp for screening thorough E-P digest.

95° Bio-Lab - MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f.

DNA PCR for MyYeast 1-2-3

Glycerol stock was performed for I62-e and stored it at -80°C.

Protein electrophoresis for phasins.
14° Meeting - Updating about Bio-Lab activity.

96° Bio-Lab - Tecan Test was performed on prepared samples, after the usual protocol.

Miniprep and quantification for: I1, I4, I53, <partinfo>BBa_J04450</partinfo>-1, <partinfo>BBa_J04450</partinfo>-2. Digestion of I1, I4, I53, <partinfo>BBa_J04450</partinfo>-1, <partinfo>BBa_J04450</partinfo>-2, MyYeast-3 (DNA already available) and <partinfo>pSB4C5</partinfo>.

97° Bio-Lab - Colony PCR for 6 newly picked colonies. Gel run for all digested parts and cut/gel purification for:4C5 , MyYeast-3, I1, I4, I53, <partinfo>BBa_J04450</partinfo> and <partinfo>pSB4C5</partinfo>.

Ligation of:I70=MyYeast 3+I4, I71=MyYeast 3+I1, I72=BBa_J04450+pSB4C5 and I73= I38+I53.

98° Bio-Lab - Tecan Test was performed on prepared samples, after the usual protocol.

Transformation of I73 (1ul) into E. coli TOP10. It was plated on LB+Amp 100 ug/ml agar plate.

Trasformation of: I70 in TOP10 and plated on LB+Amp, I71 in TOP10 and plated on LB+Amp and I72 in TOP10 and plated on LB+Cm 12,5. All plates were incubated at 37°C ON.

Miniprep and quantification of I20 was performed. Gel run/cut/extraction/quantification for I20. We repeated I55 ligation (I38+I20).

Inoculum into 5ml LB+Amp of: I31, I35, I37,I57,I60, INTEIN, 3x BBa_J04450 (to take pSB1C3, so pSB1C3 from now on). Cultures were let grow ON at 37°C, 220 rpm.

Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!

Inoculum of: S1 in 500ul M9+Cm 12,5; I47-S1 in 500ul M9+Amp+Cm 12,5; GFP-S1 in 500ul M9+Amp+Cm 12,5; RBS32 in 500ul M9+Amp. Cultures were grown ON at 37°C 220 rpm.

Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.