Team:UNIPV-Pavia/Calendar/August

45° Bio-Lab - I20 and I21 plates showed colonies. Some were picked, inoculated and grown in proper medium.

46° 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.

47° 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.

48° 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.

49° 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).

50° 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.

51° Bio-Lab - Plates of transformed cells were stored at +4°C. A Tecan test was performed with usual protocol.

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 - Glycerol stock was prepared for: linker (<partinfo>BBa_K105012</partinfo>), I22-1, I22-2, I22-3, I23-1, I23-2, I23-3, I8-5 D, I8-5 E and I8-5 F.

I8-5 D, I8-5 E, I8-5 F, I74C5, I84C5, I104C5, I124C5, <partinfo>BBa_B0032</partinfo>, A2 were diluited for Tecan Test.

MiniPrep and digestion was performed for following cultures: I20-1, I20-2, I20-3, I20-4, I20-5, I20-6, I20-7, I20-8, I20-9, I20-10, I20-11, I20-12, I21-1, I21-2, I21-3, I21-4, I21-5, I21-6, I21-7, I21-8, I21-9, I21-10, I21-11, I21-12, I21-13, I21-14, I21-15, I21-16, I21-17, I22-1, I22-2, I22-3, I23-1, I23-2, I23-3, I15-1, Ent4C5, <partinfo>BBa_B0034</partinfo>, linker (<partinfo>BBa_K105012</partinfo>) and plux (<partinfo>BBa_R0062</partinfo>). Samples were gel ran/cut. After purification, digested DNA was quantified.

Ligation of: I15_4C5, I24, I26, I20_M, I21_M, I20_A, I21_A, I20_C, I21_C.

54° Bio-Lab - Trasformation of ligations of yestrday.

The following plates were checked: F2620-4C5 in MC123, MC008, MG123, MG008;RING in MC123, MC008, MG123, MG008; NOTHING in MC123, MC008, MG123, MG008

55° Bio-Lab - A massive screening for phasins was performed on I20_M-1, I21_M-2, I20_M-3, I20_M-4, I21_M-1, I21_M-2, I21_M-3, I21_M-4, I20_A-1, I20_A-2, I20_A-3, I20_A-4, I20_A-5, I21_A-6, I20_A-7, I21_A-1, I21_A-2, I21_A-3, I21_A-4, I21_A-5,I21_A-6, I21_A-7, I20_C-1, I20_C-2, I20_C-3, I20_C-4, I21_C-1, I21_C-2, I21_C-3, I21_C-4, I21_C-5, I26-1, I26-2, I26-3, I26-4 and I26-5.

For the cultures that Screening showed positive (colonies bearing the insert) an inoculum was performed in 6ml LB+Amp.

Glycerol stock was prepared for: I15_4C5-1, I15_4C5-2, I15_4C5-3, I24-1, I24-2 and I24-3.

56° Bio-Lab - Intein and CREAM were stored +4°C.

Efficiency of our home-made competent cells MC123/008 and MG123/008 wwas calculated.

Glycerol stock was prepared for: I20_M-1, I21_M-3, I20_A-1, I20_A-2, I20_A-3, I20_A-4, I20_A-6, I21_A-2, I21_A-3, I21_A-4, I21_A-5, I21_A-6, I21_A-7 and I26-2.

MiniPrep and digestion was performed for the following cultures: I15_4C5-1, I15_4C5-2, I15_4C5-3, I24-1, I24-2, I24-3, I20_M-1, I21_M-3, I20_A-1, I20_A-2, I20_A-3, I20_A-4, I20_A-6, I21_A-2, I21_A-3, I21_A-4, I21_A-5, I21_A-6, I21_A-7 and I26-2. Cultures were digested and gel run.

57° Bio-Lab - Resuspension from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> and <partinfo>BBa_I13522</partinfo>.

Transformation (1 ul) of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM (Mr.Gene) and 15_4C5.

Transfomants were plated on LB+Amp 100 ug/ml agar plates or on LB+Amp100+Cm12,5 agar plates and were let grow.

58° Bio-Lab - Pick of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM, co-trasformed <partinfo>BBa_T9002</partinfo>.

Inoculum of I5, I26, <partinfo>BBa_K105012</partinfo>, <partinfo>BBa_B0034</partinfo>, I20M-1, I20A-1, I21M-3, I21-4, I21A-7 and I0.

I7-A, I8-5 D, I9-1, I10-B, I12-2, A2, BBa_B0032, I144C5-T9002, I164C5-T9002, I174C5-T9002, I184C5-T9002, I194C5-T9002 and ENTERO4C5-T9002 were diluted for Tecan Test.

59° Bio-Lab - I7-A, I8-5 D, I9-1, I10-B, I12-2, A2, BBa_B0032, I144C5-T9002, I154C5-T9002, I164C5-T9002, I174C5-T9002, I184C5-T9002, I194C5-T9002 and ENTERO4C5-T9002 were diluted for Tecan Test.

The following cultures were miniprepped and after digested: I26, BBa_K105012, BBa_B0034, BBa_F2620, I20M-1, I20A-1, I21M-3, I21-4, I0, INTEIN and BBa_J13002. Two medium gel were prepared and samples were loaded and gel run/cut. After gel extraction they were stored at -20°.

60° Bio-Lab - After gel exctraction were performed the following ligations: I31, I32, I33, I34, I35, I36, I37, I38, I39, I40 and I41.

61° Bio-Lab - Ligations of previous day were transformed and than plated on agar plates and incubated.

A Tecan test was performed with the usual protocol.

62° Bio-Lab - Plates with ligations I31..I41 were stored at +4°C.

63° Bio-Lab - Colony PCR as screening for ligations I31..I41. For each plate three colonies were picked and PCR amplification was performed.

MyCrim-9, BBa_K173001, BBa_J23101, pSB4C5 were digested, after that gel exctraction was performed.

64° Bio-Lab - The following cultures :I32 (I32-4/5/6/7), I36 (I36-4/5/6/7) and I39 (I39/4/5/6/7) were screened.

Inoculum of <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23114</partinfo> and <partinfo>BBa_J23116</partinfo>.

65° Bio-Lab -Miniprep was performed for I40-1/4/5/6/7 to make the screening of ligations. Samples were digested and than gel run to check the length of ligations.

MiniPrep was performed for <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo> and <partinfo>BBa_J23118</partinfo>

Ligation of <partinfo>BBa_J23100</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 and <partinfo>BBa_J23118</partinfo>_4C5.

Preparation of samples for BioPlastic screening.

66° Bio-Lab - Transformation of ligations: <partinfo>BBa_J23100</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 and <partinfo>BBa_J23118</partinfo>_4C5.

Inoculum of self inducible promoters for TECAN test.

Inoculum of <partinfo>BBa_J13002</partinfo>, I33, I34, I39 and I41.

67° Bio-Lab - Inoculated strains (<partinfo>BBa_J13002</partinfo>, I33, I34, I39 and I41) were MiniPrepped and quantified.After digestion they were gel run and cut.Gel extraction was quantified nd ligation of I-47, I-48 and I-49 were performed. Colony PCR was performed to screen if the length of insert was correct on <partinfo>BBa_J23100</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 and <partinfo>BBa_J23118</partinfo>_4C5.

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

Transformation of I29 in E. coli TOP10.

68° Bio-Lab - Ligations I47, I48, I49 and I29 plate were stored at +4°C.

69° Bio-Lab - Colony PCR was performed on <partinfo>BBa_J23116</partinfo>_4C5 (3 colonies) and <partinfo>BBa_J23118</partinfo>_4C5 (3 colonies).

Transformation of ligations I47, I48, I49 into E. coli DH5-alpha.

70° Bio-Lab - Glycerol stocks were prepared for I29_1, I29_2 and I29_3.

I29_1, I29_2 and I29_3 was MiniPrepped to purify DNA, and than gel was run to check the length of ligations.

Sudan Black staining protocol for BioPlastic was performed.

PCR from colony for I47, I48, I49 ligations was performed.