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JUNE
| DATE | ACTIVITY |
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| June, 3th | 9° Meeting - Discussion of final and technical details about The Project. Planning of Bio-Lab activity. |
| June, 7th | 1° Bio-Lab - Susanna, Nicolò and Federica.
<partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo> were resuspended from Spring 2010 DNA distribution. <partinfo>BBa_P1004</partinfo> was resuspended from Spring 2009 DNA distribution. All BioBricks were transformed in E. coli DH5alpha.
Liquid LB+Amp and LB agar plates +Amp were prepared according with "protocols". |
| June, 8th | 2° Bio-Lab - Susanna Z., Alessandro and Chiara.
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| June, 9th | 3° Bio-Lab - Susanna Z., Nicolò and Sara.
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| June, 10th | 4° Bio-Lab - Susanna Z., Manuel Z. and Riccardo.
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| June, 11th | 5° Bio-Lab - Susanna Z. and Nicolò.
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| June, 12th | iGEM EU workshop
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| June, 13th | iGEM EU workshop
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| June, 14th | 6° Bio-Lab - Lorenzo and Nicolò.
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| June, 15th | 7° Bio-Lab - Lorenzo and Manuel L., Sara and Alessandro.
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| June, 16th | 8° Bio-Lab - Susanna Z., Sara and Nicolò.
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| June, 17th | 9° Bio-Lab - Susanna Z., Nicolò and Riccardo.
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| June, 18th | 10° Bio-Lab - Susanna Z., Susanna S. and Federica.
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June, 7th
These BioBrick were resuspended in 15ul ddH20:
- BBa_E2050 (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
- BBa_K165018 (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
- BBa_K165037 (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
- BBa_P1004 (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
- BBa_J61001 (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
- BBa_K081008 (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
- BBa_K125500 (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2
1,5 ul of each culture was transformed in 100 ul of home-made competent E. coli DH5alpha.
Tranformants were all plated on LB agar plates added with Ampicillin, except for BBa_E2050 (Kanamycin).
500 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).
June, 8th
All plates grown overnight at 37°C show colonies!
In particular, <partinfo>BBa_E2050</partinfo> (gorwn on LB+Kan), <partinfo>BBa_K125500</partinfo> (gorwn on LB+Amp), <partinfo>BBa_K081008</partinfo> (gorwn on LB+Amp) and <partinfo>BBa_K165018</partinfo> (gorwn on LB+Amp) showed big colonies, well separated on the plate.
<partinfo>BBa_P1004</partinfo> (gorwn on LB+Amp) and <partinfo>BBa_J61001</partinfo> (gorwn on LB+Amp) showed many colonies, with big colonies surrounded by small colonies.
<partinfo>BBa_K165037</partinfo> (gorwn on LB+Amp) showed only 11 big colonies.
A colony was peaked for each plate and inoculated in 1ml LB+antibiotic.
| <partinfo>BBa_E2050</partinfo> | 1ml LB+Kan
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| <partinfo>BBa_K165037</partinfo> | 1ml LB+Amp
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| <partinfo>BBa_P1004</partinfo> | 1ml LB+Amp
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| <partinfo>BBa_K125500</partinfo> | 1ml LB+Amp
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| <partinfo>BBa_K081008</partinfo> | 1ml LB+Amp
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| <partinfo>BBa_J61001</partinfo> | 1ml LB+Amp
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| <partinfo>BBa_K165018</partinfo> | 1ml LB+Amp
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<partinfo>BBa_K165037</partinfo> in pSB1AK3 plasmid was inocultaed both in 1ml LB+Amp and in 1ml LB+Kan for a phenotipic assay.
Cultures were grown for 8 hours at 37°C 220 rpm.
After this time, cultures were all grown and in saturation phase.
Glycerol stocks were prepared for each culture (250ul saturated culture + 750 ul glycerol 80%) and stored at -80°C.
The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.
June, 9th
Ligation of:
- I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
- I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X)
- I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
- I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
<partinfo>BBa_B0015</partinfo> was accidentally not inoculated yesterday... :(
Luckily, we had a DNA stock for this part stored at -20°C, so we used this stock for ligations.
Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit.
Surprisingly, after the first centrifugation, we observed that <partinfo>BBa_E2050</partinfo> pellet was orange!! This is curious, because this BioBrick expresses mOrange, but it lacks of promoters. Probably, this spurious transcription is due to the promoter of Kanamycin resistance, that is oriented in the same direction.
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After MiniPrep, purified DNA was quantified with NanoDrop.
| <partinfo>BBa_E2050</partinfo> | 139,2 ng/ul
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| <partinfo>BBa_K165037</partinfo> | 181,5 ng/ul
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| <partinfo>BBa_P1004</partinfo> | 132,8 ng/ul
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| <partinfo>BBa_K125500</partinfo> | 124,3 ng/ul
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| <partinfo>BBa_K081008</partinfo> | 138,3 ng/ul
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| <partinfo>BBa_J61001</partinfo> | 122,2 ng/ul
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| <partinfo>BBa_K165018</partinfo> | 166,3 ng/ul
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</table
Digestion of:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 | Enzyme 2 | Buffer H
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| <partinfo>BBa_K125500</partinfo> | Insert | 25 | 16 | 4,5 | 1 EcoRI | 1 SpeI | 2,5
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| <partinfo>BBa_B0015</partinfo> | Vector | 25 | 6,3 | 14,2 | 1 EcoRI | 1 Xba I | 2,5
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| <partinfo>BBa_E2050</partinfo> | Insert | 25 | 14 | 6,5 | 1 EcoRI | 1 Spe I | 2,5
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| <partinfo>BBa_K165018</partinfo> | Vector | 25 | 6 | 14,5 | 1 EcoRI | 1 Xba I | 2,5
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| <partinfo>BBa_P1004</partinfo> | Insert | 25 | 15 | 5,5 | 1 EcoRI | 1 Spe I | 2,5
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| <partinfo>BBa_K081008</partinfo> | Insert | 25 | 14,5 | 6 | 1 EcoRI | 1 Spe I | 2,5
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Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.
Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.
June, 10th
I0, I1, I2 and I3 were tranformed in home made compotent E. coli DH5-alpha and plated on LB+Amp agar plates.
Transformed plates were incubated overnight at 37°C 220 rpm.
June, 11th
June, 14th
June, 15th
June, 16th
June, 17th
June, 18th
</td>
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</tr>
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