http://2007.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Giov19842007.igem.org - User contributions [en]2024-03-28T15:00:08ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-26T14:10:52Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|220px|center]][[image:Undici.JPG|220px|center]] [[image:Tre.JPG|200px|center]] <br />
[[image:Otto.JPG|center|200px]] [[image:Quattro.JPG|160px|center]] [[image:P1000006.JPG|230px|center]] [[image:Dieci.JPG|120px|center]] [[image:Cinque.JPG|210px|center]]<br />
[[image:Belle.jpg|240px|center]] [[image:Lu-ve.jpg|120px|center]]<br />
[[image:P1000186.JPG|240px|center]]<br />
[[image:New.JPG|180px|center]]<br />
[[image:New2.JPG|180px|center]]<br />
[[image:Belle2.jpg|180px|center]]<br />
[[image:Belle77.jpg|220px|center]]<br />
[[image:Belle4.jpg|240px|center]]</div>Giov1984http://2007.igem.org/wiki/index.php/File:P1000006.JPGFile:P1000006.JPG2007-10-26T14:09:08Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-26T14:07:37Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|220px|center]][[image:Undici.JPG|220px|center]] [[image:Tre.JPG|200px|center]] <br />
[[image:Otto.JPG|center|200px]] [[image:Quattro.JPG|180px|center]] <br />
[[image:Dieci.JPG|140px|center]] [[image:Cinque.JPG|210px|center]]<br />
[[image:Belle.jpg|240px|center]] [[image:Lu-ve.jpg|120px|center]]<br />
[[image:P1000186.JPG|240px|center]]<br />
[[image:New.JPG|180px|center]]<br />
[[image:New2.JPG|180px|center]]<br />
[[image:Belle2.jpg|180px|center]]<br />
[[image:Belle77.jpg|220px|center]]<br />
[[image:Belle4.jpg|240px|center]]</div>Giov1984http://2007.igem.org/wiki/index.php/File:Lu-ve.jpgFile:Lu-ve.jpg2007-10-26T14:05:50Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/Luciferase_assayLuciferase assay2007-10-26T07:46:48Z<p>Giov1984: </p>
<hr />
<div>We cloned one (1ORE) and two tandem copies of the oleate response elements (2XORE) from the FOX3 gene inserted upstream of a minimal CYC1 promoter. This promoter drives the expression of firefly luciferase.<br />
For luciferase assays and preparation of protein extracts, transformants were grown in rich<br />
medium with 1.5% raffinose, 1.5% glycerol and 1% ethanol as carbon sources.<br />
Cell were induced with different concentrations of oleate overnight. Cells were lysed in 100 mM potassium phosphate pH 7.8, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol using glass beads (diameter 0.45 mm). <br />
Cell debris were removed by centrifugation at 15 000 x g at 4°C for 20 min.<br />
Luciferase activities are expressed in relative light units/pg protein and protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard.<br />
<br />
LucAssay result :<br />
<br />
[[Image:luc.jpg]]<br />
<br />
2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment increasing oleate concentrations.</div>Giov1984http://2007.igem.org/wiki/index.php/Luciferase_assayLuciferase assay2007-10-26T07:46:27Z<p>Giov1984: </p>
<hr />
<div>We cloned one (1ORE) and two tandem copies of the oleate response elements (2XORE) from the FOX3 gene inserted upstream of a minimal CYC1 promoter. This promoter drives the expression of firefly luciferase.<br />
For luciferase assays and preparation of protein extracts, transformants were grown in rich<br />
medium with 1.5% raffinose, 1.5% glycerol and 1% ethanol as carbon sources.<br />
Cell were induced with different concentrations of oleate overnight. Cells were lysed in 100 mM potassium phosphate pH 7.8, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol using glass beads (diameter 0.45 mm). <br />
Cell debris were removed by centrifugation at 15 000 x g at 4°C for 20 min.<br />
Luciferase activities are expressed in relative light units/pg protein and protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard.<br />
<br />
LucAssay result :<br />
<br />
[[Image:luc.jpg]]<br />
<br />
2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment when increasing oleate concentrations.</div>Giov1984http://2007.igem.org/wiki/index.php/Cloning_in_BioBrick_vectorsCloning in BioBrick vectors2007-10-26T07:44:38Z<p>Giov1984: </p>
<hr />
<div>We amplified with [[PCR]] promoters and Pho80 coding sequence and we loaded 1µl on the agarose gel to see if there were amplification products,aspecific products or others.Then we digested our PCR products with XbaI and SpeI.<br />
<br />
[[1ORE-CYCtata]]<br />
<br />
[[2XORE-CYCtata]]<br />
<br />
[[FOX3promoter]]<br />
<br />
<br />
We digested in two differents ways our plasmid to be sure of its identity!<br />
Then we digested [[pSB1A3]][https://2007.igem.org/Biobrick_Vector_choice] with the same enzyme used for the insert.<br />
We chose a plasmid containing one insert of 1,5kb so via gel extraction, we were able to separate the linearized plasmid from the insert.<br />
<br />
[[Image:pBs1A3.jpg]]<br />
<br />
We performed ligations between pSB1A3 and 1ore,2ore and fox3.<br />
After ligations, we transformed plasmid pSB1A3 in competent cells and after mini&maxi-inoculations and MIDI-prep we loaded 1µl on the agarose gel.<br />
<br />
<br />
<br />
[[PHO80cds]]<br />
<br />
We decided to clone Pho80 coding sequence in pSB1A3 plasmid but since its CDS has two XbaI restriction sites in positions<br />
91 and 648 we mutated these sites. We then cloned this gene in pSB1A3 plasmid using XbaI and SpeI enzymes for [[digestion]] of Pho80 mutated and pSB1A3 plasmid.<br />
After this ligation, we used EcoRI and SpeI enzymes to digest pSB1A3-Pho80 cassette and to clone it upstream of Rfp in pBca1020-r0040 vector.</div>Giov1984http://2007.igem.org/wiki/index.php/FOX3promoterFOX3promoter2007-10-25T16:56:14Z<p>Giov1984: </p>
<hr />
<div>FOX3 promoter is 832bp long and this is its sequence : <br />
<br />
GAGTATGTCGTGCTCGAAGTCACTTTCTTTTTCCCCCAATGATTGGAACTACGGATTTTA<br />
CATATCAATAACCATTTCCTTCGTAGAGAGATGTTTTGTAAATTCTTATAAGCAGCCCCC<br />
CAAAAAAAAGTTCCATCTTTTAAAAGCTCATCAAACATTCTTTCGACGAGTGATGCGTCC<br />
TGAGGCATGCACTTCGGATTAAAATCCATTTCCGATCCGTTCACTTCTTCGGAAACCATT<br />
GGTGACGAAGAAACGTCATTAGCTGAGAAATTCTTCTGTTTCATGTATATATGTGATCCT<br />
GTACCTTGTTTCTCTAACGCTACACCGACCAACTGCAAGCCAGCGGCAACCAGCCCATCG<br />
TTCAAATTCAATTCTTCCAATTTACGGGCTTCGCTGACCCTATCACGCGCGTGAAGTGAC<br />
AGCGATCTGCGAGGGTAGCGGTACGTCTTCTTGTTCGGGGAAGAGTCCATCGCTGATGAT<br />
TGTGCTATTTTTTTATCTGCTTTTGTGTCACGATCATCATCACTATGTAATCTTCAAAAG<br />
ATCAAGAATTTGCTAGTTTTGAACCTATGCCACAAATAGCGAAAAAAGAAAAAAAACTTG<br />
ACGCGTCAACATGAGGAGGGTAATGATGTGGTAGCGCCGTGTAAGGCGCTATCAAAGGGA<br />
AACGGGGATAATAGTATTAACACCGCAGCTTTTTTTTCCTTTCTCCCTCTATTGGTTTCA<br />
AATTTATTGGAGTTTTACTAGAAAGAAGAGATATAAATAGGGTATACTTTTGCATCTCAG<br />
TTCTATTGAAATCGAATGGTTATTTCTTGTGGCTCTGAGTACAGAGTGAATATAACACTA<br />
CATAAAAGCA<br />
<br />
[[Image:fox3.jpg]]<br />
<br />
In this picture we can see our fragments of amplification.These are localizated in between bands 0.5 and 1 kbp of 1Kb Dna Marker</div>Giov1984http://2007.igem.org/wiki/index.php/2XORE-CYCtata2XORE-CYCtata2007-10-25T16:55:00Z<p>Giov1984: </p>
<hr />
<div>2ORE-CYCtata is 350bp long and this is its sequence :<br />
<br />
atcagttattaccctcgacctcgaccatctcggtgttaatactattatcccgagatggct<br />
cgaccatctcggtgttaatactattatcccgagatggctcgagcagatccgccaggcgtg<br />
tatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgt<br />
gcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttc<br />
ttcttttctctaaatattctttccttatacattaggtcctttgtagcataaattactatac<br />
ttctatagacacgcaaacaccgcgggaat<br />
<br />
<br />
[[Image:2ore.jpg|200px]]<br />
<br />
In this picture we can see Pcr fragments 350bp long</div>Giov1984http://2007.igem.org/wiki/index.php/PHO80cdsPHO80cds2007-10-25T16:53:49Z<p>Giov1984: </p>
<hr />
<div>GenePho<br />
CODING SEQUENCE<br />
<br />
ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG<br />
AGGATCAAGGGATACCAAAAGTAATTCTGCCCGCTGATTT<br />
TAATAAATGCTCTAGAACTGACCTAGTGGTGCTCATATCA<br />
CGAATGTTAGTATCGCTGATAGCAATCAATGAAAATTCAG<br />
CAACAAAGAAATCTGATGACCAAATTACTTTAACACGATA<br />
CCATTCTAAGATTCCTCCAAACATATCAATCTTCAACTAT<br />
TTCATACGACTGACAAAGTTTTCCTCTTTAGAACATTGTG<br />
TGCTTATGACATCACTCTATTATATCGATTTATTGCAAAC<br />
TGTGTATCCTGATTTTACGCTTAATTCGTTGACTGCCCAT<br />
AGGTTTTTATTAACAGCCACCACAGTCGCAACAAAAGGCT<br />
TATGTGATTCGTTCTCAACAAACGCCCATTATGCAAAAGT<br />
TGGAGGAGTACGATGTCACGAATTGAATATACTGGAGAAC<br />
GATTTTTTAAAGAGAGTAAACTACAGAATCATTCCGCGGG<br />
ATCATAACATTACGTTATGTAGTATAGAGCAAAAACAGAA<br />
AAAGTTTGTCATAGATAAAAACGCATTAGGGTCTCTCGAT<br />
TTGGATTCTTATTCTTACGTTAATCGTCCAAAAAGTGGAT<br />
ATAATGTTCTAGATAAATACTATCGAAGAATAGTTCAGCT<br />
GGTGGGTTCCTTTAACGCTTCACCTGATAAGAGTAGAAAG<br />
GTTGATTATGTTCTCCCGCCAAATATTGATATAGTGAGTG<br />
AAAGTGGCTCCCAAACTACTCAACTAAAGGGGTCGTCATC<br />
ACCCAATTCTCACTCTTCACAAAAGCGATATTCTGAGGCA<br />
AAGGACGCACATATCTATAACAAGCGATCAAAGCCAGATT<br />
AA<br />
<br />
XbaI restriction sequence TCTAGA<br />
cuts in sites<br />
91<br />
648<br />
<br />
<br />
<br />
Pho cds<br />
882bp 293aa<br />
<br />
1)91 bp position 31-32aa SR<br />
S=TCTTCCTCATCG<br />
<br />
R=AGAAGG<br />
<br />
2)648 bp position 216-217-218aa VLD<br />
V=TTGTCGTAGTG<br />
<br />
L=TTATTGCTTCTACTCCTG<br />
<br />
D=GATGAC<br />
<br />
<br />
<br />
Frame 5’-3’<br />
VirtualSequence:<br />
<br />
MESTSGERSENIHEDQGIPKVILPADFNKC[[SR]]TDLVVLISRMLVSLIAINENSATKKSDD<br />
QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH<br />
RFLLTATTVATKGLCDSFSTNAHYAKVGGVRCHELNILENDFLKRVNYRIIPRDHNITLC<br />
SIEQKQKKFVIDKNALGSLDLDSYSYVNRPKSGYN[[VLD]]KYYRRIVQLVGSFNASPDKSRK<br />
VDYVLPPNIDIVSESGSQTTQLKGSSSPNSHSSQKRYSEAKDAHIYNKRSKPD<br />
<br />
<br />
<br />
'''PHO80<br />
Primer sequences:'''<br />
<br />
t93c 5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'<br />
<br />
t93c antisense 5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'<br />
<br />
t648c 5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'<br />
<br />
t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'<br />
<br />
'''<br />
results of pho80 coding sequence'''<br />
<br />
<br />
<br />
''PCR of mutagenesis of pho 80 from topo TA plasmid''<br />
Finally volume is 50μl<br />
{| border="1"<br />
|-<br />
! Acqua, µl<br />
| 40<br />
|-<br />
! Buffer, µl<br />
| 5<br />
|-<br />
! dNTPs, µl<br />
| 1<br />
|-<br />
! Taq (Pfu turbo), µl<br />
| 1 <br />
|-<br />
! PrimerFw (125 ng), µl<br />
| 1<br />
|-<br />
! PrimerRev (125 ng), µl<br />
| 1<br />
|-<br />
! Template (5 ng), µl<br />
| 1<br />
|}<br />
<br />
Cycling Conditions<br />
<br />
<br />
{| border="1"<br />
|-<br />
! I°CYCLE <br />
| 95°c for 30 seconds <br />
|-<br />
! 12 CYCLES <br />
| denaturation 95°C for 30 seconds<br />
| annealing 55°c for 1 minutes<br />
| elongation 68°c for 5 minutes <br />
|-<br />
! LAST CYCLE<br />
| 72°C for 2 minutes<br />
| 4°c <br />
|}<br />
<br />
<br />
PRIMERS<br />
<br />
PHO 80 fw Xba I<br />
5'- GC TCTAGA ATGGAAAGCACATCAGGAG-3'<br />
Tm 65.1°C<br />
<br />
PHO 80 rew Spe I<br />
5'-GG ACTAGT ATCTGGCTTTGATCGCTTG-3'<br />
Tm 65.1°C<br />
<br />
[[PCR]] of pho80 from DNA yeast<br />
<br />
[[Image:geldoc pho 1.jpg]]<br />
<br />
<br />
midi pho80 in topo TA plasmid<br />
<br />
[[Image:geldoc pho 2.jpg]]<br />
<br />
<br />
digestion with Dpn I after first mutation of pho 80<br />
<br />
[[Image:geldoc pho 3.jpg]]<br />
<br />
<br />
digestion with Dpn I after second mutation of pho 80<br />
<br />
[[Image:geldoc pho 4.jpg]]<br />
<br />
<br />
midi pho 80 mutated<br />
<br />
[[Image:geldoc pho 5.jpg]]<br />
<br />
<br />
digestion pho 80 mutated with Spe i and Xba I <br />
<br />
[[Image:geldoc pho 6.jpg]]</div>Giov1984http://2007.igem.org/wiki/index.php/FOX3promoterFOX3promoter2007-10-25T16:52:15Z<p>Giov1984: </p>
<hr />
<div>FOX3 promoter is long 832bp and this is its sequence : <br />
<br />
GAGTATGTCGTGCTCGAAGTCACTTTCTTTTTCCCCCAATGATTGGAACTACGGATTTTA<br />
CATATCAATAACCATTTCCTTCGTAGAGAGATGTTTTGTAAATTCTTATAAGCAGCCCCC<br />
CAAAAAAAAGTTCCATCTTTTAAAAGCTCATCAAACATTCTTTCGACGAGTGATGCGTCC<br />
TGAGGCATGCACTTCGGATTAAAATCCATTTCCGATCCGTTCACTTCTTCGGAAACCATT<br />
GGTGACGAAGAAACGTCATTAGCTGAGAAATTCTTCTGTTTCATGTATATATGTGATCCT<br />
GTACCTTGTTTCTCTAACGCTACACCGACCAACTGCAAGCCAGCGGCAACCAGCCCATCG<br />
TTCAAATTCAATTCTTCCAATTTACGGGCTTCGCTGACCCTATCACGCGCGTGAAGTGAC<br />
AGCGATCTGCGAGGGTAGCGGTACGTCTTCTTGTTCGGGGAAGAGTCCATCGCTGATGAT<br />
TGTGCTATTTTTTTATCTGCTTTTGTGTCACGATCATCATCACTATGTAATCTTCAAAAG<br />
ATCAAGAATTTGCTAGTTTTGAACCTATGCCACAAATAGCGAAAAAAGAAAAAAAACTTG<br />
ACGCGTCAACATGAGGAGGGTAATGATGTGGTAGCGCCGTGTAAGGCGCTATCAAAGGGA<br />
AACGGGGATAATAGTATTAACACCGCAGCTTTTTTTTCCTTTCTCCCTCTATTGGTTTCA<br />
AATTTATTGGAGTTTTACTAGAAAGAAGAGATATAAATAGGGTATACTTTTGCATCTCAG<br />
TTCTATTGAAATCGAATGGTTATTTCTTGTGGCTCTGAGTACAGAGTGAATATAACACTA<br />
CATAAAAGCA<br />
<br />
[[Image:fox3.jpg]]<br />
<br />
In this picture we can see our fragments of amplification.These are localizated in corrispondence of bands of Dna Marker between 0.5 and 1 kbp</div>Giov1984http://2007.igem.org/wiki/index.php/2XORE-CYCtata2XORE-CYCtata2007-10-25T16:51:30Z<p>Giov1984: </p>
<hr />
<div>2ORE-CYCtata is long 350bp and this is its sequence :<br />
<br />
atcagttattaccctcgacctcgaccatctcggtgttaatactattatcccgagatggct<br />
cgaccatctcggtgttaatactattatcccgagatggctcgagcagatccgccaggcgtg<br />
tatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgt<br />
gcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttc<br />
ttcttttctctaaatattctttccttatacattaggtcctttgtagcataaattactatac<br />
ttctatagacacgcaaacaccgcgggaat<br />
<br />
<br />
[[Image:2ore.jpg|200px]]<br />
<br />
In this picture we can see Pcr fragments long 350bp</div>Giov1984http://2007.igem.org/wiki/index.php/1ORE-CYCtata1ORE-CYCtata2007-10-25T16:50:58Z<p>Giov1984: </p>
<hr />
<div>1ORE-CYCtata is 300bp long and this is its sequence:<br />
<br />
ccgagatggctcgaccatctcggtgttaatactattatcccgagatggctcgagcagatccgccaggcgtgtatatagcgtggatggccaggcaactttagtgctgacaca<br />
tacaggcatatatatatgtgtgcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttata<br />
cattaggtcctttgtagcataaattactatacttctatagacacgcaaaca<br />
<br />
<br />
[[Image:1ore promoter .jpg]]<br />
<br />
In this photo you can see the result of our PCR.Our amplification fragments are localizated at the level of the 0.5Kb band of the 1 Kb Dna Marker</div>Giov1984http://2007.igem.org/wiki/index.php/1ORE-CYCtata1ORE-CYCtata2007-10-25T16:50:40Z<p>Giov1984: </p>
<hr />
<div>1ORE-CYCtata is 300bp long and this is its sequence:<br />
<br />
ccgagatggctcgaccatctcggtgttaatactattatcccgagatggctcgagcagatccgccaggcgtgtatatagcgtggatggccaggcaactttagtgctgacaca<br />
tacaggcatatatatatgtgtgcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttata<br />
cattaggtcctttgtagcataaattactatacttctatagacacgcaaaca<br />
<br />
<br />
[[Image:1ore promoter .jpg]]<br />
<br />
In this photo you can see the result of our PCR.Our amplification fragments are localizated at the level of the 0.5Kb band of the Dna Marker</div>Giov1984http://2007.igem.org/wiki/index.php/1ORE-CYCtata1ORE-CYCtata2007-10-25T16:49:52Z<p>Giov1984: </p>
<hr />
<div>1ORE-CYCtata is 300bp long and this is its sequence:<br />
<br />
ccgagatggctcgaccatctcggtgttaatactattatcccgagatggctcgagcagatccgccaggcgtgtatatagcgtggatggccaggcaactttagtgctgacaca<br />
tacaggcatatatatatgtgtgcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttata<br />
cattaggtcctttgtagcataaattactatacttctatagacacgcaaaca<br />
<br />
<br />
[[Image:1ore promoter .jpg]]<br />
<br />
In this photo you can see the result of our PCR.Our amplification fragments are localizated at the level the band of the 0.5Kb Dna Marker</div>Giov1984http://2007.igem.org/wiki/index.php/Cloning_in_BioBrick_vectorsCloning in BioBrick vectors2007-10-25T16:47:12Z<p>Giov1984: </p>
<hr />
<div>We have amplified with [[PCR]] promoters and Pho80 coding sequence and we have loaded 1µl on the agarose gel to see if there were amplification products,aspecific products or others.Then we digested our PCR products with XbaI and SpeI.<br />
<br />
[[1ORE-CYCtata]]<br />
<br />
[[2XORE-CYCtata]]<br />
<br />
[[FOX3promoter]]<br />
<br />
<br />
We digested in two differents ways our plasmid to be sure of its identity!<br />
Then we digested [[pSB1A3]][https://2007.igem.org/Biobrick_Vector_choice] with the same enzyme used for the insert.<br />
We chose a plasmid containing one insert of 1,5kb so via gel extraction, we were able to separate the linearized plasmid from the insert.<br />
<br />
[[Image:pBs1A3.jpg]]<br />
<br />
We performed ligations between pSB1A3 and 1ore,2ore and fox3.<br />
After ligations, we transformed plasmid pSB1A3 in competent cells and after mini&maxi-inoculations and MIDI-prep we loaded 1µl on the agarose gel.<br />
<br />
<br />
<br />
[[PHO80cds]]<br />
<br />
We decided to clone Pho80 coding sequence in pSB1A3 plasmid but since its CDS has two XbaI restriction sites in positions<br />
91 and 648 we mutated these sites. We then cloned this gene in pSB1A3 plasmid using XbaI and SpeI enzymes for [[digestion]] of Pho80 mutated and pSB1A3 plasmid.<br />
After this ligation, we used EcoRI and SpeI enzymes to digest pSB1A3-Pho80 cassette and to clone it upstream of Rfp in pBca1020-r0040 vector.</div>Giov1984http://2007.igem.org/wiki/index.php/Luciferase_assayLuciferase assay2007-10-25T16:41:07Z<p>Giov1984: </p>
<hr />
<div>We have cloned one(1ORE)and two tandem copies of the oleate response elements (2XORE) from the FOX3 gene inserted upstream of a minimal CYC1 promoter.This promoter drives the expression of firefly luciferase.<br />
For luciferase assays and the preparation of protein extracts, transformants were grown in rich<br />
medium with 1.5% raffinose, 1.5% glycerol, and 1% ethanol as carbon sources. Cell were induced with different concentrations of oleate overnight.Cells were lysed in 100 mM potassium phosphate pH 7.8, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol using glass beads (diameter 0.45 mm). Cell debris were removed by centrifugation at 15 000 x g at 4°C for 20 min.Luciferase activities are expressed in relative light units/pg protein and protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard.<br />
<br />
LucAssay result :<br />
<br />
[[Image:luc.jpg]]<br />
<br />
2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment when increasing oleate concentrations.</div>Giov1984http://2007.igem.org/wiki/index.php/Yeast_DNA_extractionYeast DNA extraction2007-10-25T16:33:46Z<p>Giov1984: </p>
<hr />
<div>YEAST DNA EXTRACTION<br />
<br />
-centrifuge 5ml of inoculo 3 minutes 3000 rpm<br />
<br />
-add 1ml distiled water <br />
<br />
-vortex<br />
<br />
-spin 20 minutes full speed<br />
<br />
-risospend in 200µl SCE/zymolase<br />
<br />
-vortex<br />
<br />
-leave 1 hour 37°C<br />
<br />
-add 200 µl SDS<br />
<br />
-leave 5 minutes 65°C<br />
<br />
-add 200µl 5M-Koac and to mix<br />
<br />
-leave 30 minutes in ice<br />
<br />
-spin 5 minutes 14000 rpm 4°C<br />
<br />
-add 200µl 5M NH4OAc and 1ml isopropanol at 500 µl surnatante<br />
<br />
-spin 30 minutes 3000 rpm<br />
<br />
-dissolve with 90µl TE<br />
<br />
-add 10µl NH4OAc and 200µl isopropanol<br />
<br />
-centrifuge 1 minute 3000 rpm<br />
<br />
-add 250µl EtOH 80%<br />
<br />
-spin 1 minute 3000 rpm<br />
<br />
-leave 10 minutes 50°C<br />
<br />
-dissolve in 200µl TE</div>Giov1984http://2007.igem.org/wiki/index.php/Images_from_NaplesImages from Naples2007-10-25T13:43:15Z<p>Giov1984: </p>
<hr />
<div>[[Image:Panorama.jpg|220px]] Naples by day<br />
<br />
[[Image:castello2.jpg|230px]] Maschio Angioino<br />
<br />
[[Image:galleria umberto.jpg|200px]] Umberto I Gallery<br />
<br />
[[Image:cristo velato.jpg|200px]] San Carlo Theater<br />
<br />
[[Image:Santakiara.jpg|200px]] The Monaster of S.Chiara<br />
<br />
[[Image:Plebiscito.jpg|200px]] Plebiscito Square<br />
<br />
[[Image:Casteldell'ovo.jpg|230px]] Castel dell'Ovo<br />
<br />
[[Image:shopping.jpg|230px]] Presepi<br />
<br />
[[Image:viaCaracciolo.jpg|230px]] Ischia island<br />
<br />
[[Image:LunaRossa.jpg|210px]] Luna Rossa<br />
<br />
[[Image:Totò.jpg|170px]] Totò, the "Prince"</div>Giov1984http://2007.igem.org/wiki/index.php/Images_from_NaplesImages from Naples2007-10-25T13:41:27Z<p>Giov1984: </p>
<hr />
<div>[[Image:Panorama.jpg|220px]] Naples by day<br />
<br />
[[Image:castello2.jpg|230px]]Maschio Angioino<br />
<br />
[[Image:galleria umberto.jpg|200px]]Umberto Gallery<br />
<br />
[[Image:cristo velato.jpg|200px]] San Carlo Theater<br />
<br />
[[Image:Santakiara.jpg|200px]] The Monaster of S.Chiara<br />
<br />
[[Image:Plebiscito.jpg|200px]] Plebiscito Square<br />
<br />
[[Image:Casteldell'ovo.jpg|230px]] Castel dell'Ovo<br />
<br />
[[Image:shopping.jpg|230px]] Presepi<br />
<br />
[[Image:viaCaracciolo.jpg|230px]] Ischia island<br />
<br />
[[Image:LunaRossa.jpg|210px]] Luna Rossa<br />
<br />
[[Image:Totò.jpg|170px]] Totò, the "Prince"</div>Giov1984http://2007.igem.org/wiki/index.php/Images_from_NaplesImages from Naples2007-10-25T13:40:49Z<p>Giov1984: </p>
<hr />
<div>[[Image:Panorama.jpg|210px]] Naples by day<br />
<br />
[[Image:castello2.jpg|230px]]Maschio Angioino<br />
<br />
[[Image:galleria umberto.jpg|200px]]Umberto Gallery<br />
<br />
[[Image:cristo velato.jpg|200px]] San Carlo Theater<br />
<br />
[[Image:Santakiara.jpg|200px]] The Monaster of S.Chiara<br />
<br />
[[Image:Plebiscito.jpg|200px]] Plebiscito Square<br />
<br />
[[Image:Casteldell'ovo.jpg|220px]] Castel dell'Ovo<br />
<br />
[[Image:shopping.jpg|210px]] Presepi<br />
<br />
[[Image:viaCaracciolo.jpg|230px]] Ischia island<br />
<br />
[[Image:LunaRossa.jpg|210px]] Luna Rossa<br />
<br />
[[Image:Totò.jpg|170px]] Totò, the "Prince"</div>Giov1984http://2007.igem.org/wiki/index.php/File:Castellovo.jpgFile:Castellovo.jpg2007-10-25T13:00:36Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-25T11:58:37Z<p>Giov1984: /* '''Our Project - YeSOil: A Yeast Sensor for real Extra Virgin Olive oil''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
[[images from Naples]]<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== <br />
[[image:logo2.jpg|center|160px]]<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast. This system will help in evaluating the quality of olive oil, one of the wordly famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some brainstorming we had this idea!!!<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
The whole circuit is based on the reaction of the transcription factor for PHO4p which is activated when there is a low oleic acid concentration, <br />
i.e. ''extra virgin olive oil'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. ''not edible oil''. <br />
When PHO4p is activated PHO8, which is integrated with GFP, is expressed: cells turn green indicating that the oil is ''extra virgin''. When PHO80 is transcribed by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p inhibiting the trascription of PHO8. As PHO80 is integrated with RFP, when it is expressed cells turn red, indicating that the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence: yellow-orange.<br />
<br />
The input of the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, oil will be classified as:<br />
*''extra virgin'', if the oleic acid conentration is less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration is less than 2 gr<br />
*''not edible'', if the oleic acid concentration is greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-24T17:01:52Z<p>Giov1984: /* '''System Model''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
[[images from Naples]]<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== <br />
[[image:logo2.jpg|center|160px]]<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast. This system will detect the quality of olive oil, one of the wordly famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some brainstorming we had this idea!!!<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
The whole circuit is based on the reaction of the transcription factor for PHO4p which is activated when there is a low oleic acid concentration, <br />
i.e. ''extra virgin olive oil'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. ''not edible oil''. <br />
When PHO4p is activated it transcribes PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, oil will be classified as:<br />
*''extra virgin'', if the oleic acid conentration is less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration is less than 2 gr<br />
*''not edible'', if the oleic acid concentration is greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-24T16:58:02Z<p>Giov1984: /* '''System Model''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
[[images from Naples]]<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== <br />
[[image:logo2.jpg|center|160px]]<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast. This system will detect the quality of olive oil, one of the wordly famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had this idea!!!<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
The whole idea is based on the reaction of the transcription factor for PHO4p which is activated when there is a low oleic acid concentration, i.e. ''extra virgin olive oil'', while the gene PHO80 is activated when the oleic acid concentration is high, i.e. ''not edible oil''. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, oil will be classified as:<br />
*''extra virgin'', if the oleic acid conentration is less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration is less than 2 gr<br />
*''not edible'', if the oleic acid concentration is greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-24T16:52:09Z<p>Giov1984: /* '''Our Project - YeSOil: A Yeast Sensor for real Extra Virgin Olive oil''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
[[images from Naples]]<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== <br />
[[image:logo2.jpg|center|160px]]<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast. This system will detect the quality of olive oil, one of the wordly famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had an the idea!!!<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, oil will be classified as:<br />
*''extra virgin'', if the oleic acid conentration is less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration is less than 2 gr<br />
*''not edible'', if the oleic acid concentration is greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-24T16:44:17Z<p>Giov1984: /* '''University of Naples "Federico II"''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
[[images from Naples]]<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== <br />
[[image:logo2.jpg|center|160px]]<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast able to detect the quality of olive of oil, one of the most famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had the idea represented in the picture.<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, oil will be classified as:<br />
*''extra virgin'', if the oleic acid conentration is less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration is less than 2 gr<br />
*''not edible'', if the oleic acid concentration is greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-24T16:41:05Z<p>Giov1984: /* '''Tigem''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
[[images from Naples]]<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was established by the King of "Sacro Romano Impero" Federico II of Svevia. It's considered the most ancient public school of the world. It consists of 13 departments divided in three areas: Sciences and technologies, humanistic and social, medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== <br />
[[image:logo2.jpg|center|160px]]<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast able to detect the quality of olive of oil, one of the most famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had the idea represented in the picture.<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, oil will be classified as:<br />
*''extra virgin'', if the oleic acid conentration is less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration is less than 2 gr<br />
*''not edible'', if the oleic acid concentration is greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-24T15:36:36Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|220px|center]][[image:Undici.JPG|220px|center]] [[image:Tre.JPG|200px|center]] <br />
[[image:Otto.JPG|center|200px]] [[image:Quattro.JPG|180px|center]] <br />
[[image:Dieci.JPG|140px|center]] [[image:Cinque.JPG|210px|center]]<br />
[[image:Belle.jpg|240px|center]]<br />
[[image:P1000186.JPG|240px|center]]<br />
[[image:New.JPG|180px|center]]<br />
[[image:New2.JPG|180px|center]]<br />
[[image:Belle2.jpg|180px|center]]<br />
[[image:Belle77.jpg|220px|center]]<br />
[[image:Belle4.jpg|240px|center]]</div>Giov1984http://2007.igem.org/wiki/index.php/File:P1000186.JPGFile:P1000186.JPG2007-10-24T15:32:33Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/File:New2.JPGFile:New2.JPG2007-10-24T15:31:46Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/File:New.JPGFile:New.JPG2007-10-24T15:30:57Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-24T10:24:28Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|200px|center]][[image:Undici.JPG|200px|center]] [[image:Tre.JPG|180px|center]] <br />
[[image:Otto.JPG|center|200px]] [[image:Quattro.JPG|180px|center]] <br />
[[image:Dieci.JPG|140px|center]] [[image:Cinque.JPG|180px|center]]<br />
[[image:Belle.jpg|240px|center]]<br />
[[image:Belle2.jpg|180px|center]]<br />
[[image:Belle77.jpg|200px|center]]<br />
[[image:Belle4.jpg|230px|center]]</div>Giov1984http://2007.igem.org/wiki/index.php/File:Belle77.jpgFile:Belle77.jpg2007-10-24T10:19:47Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-19T17:09:43Z<p>Giov1984: </p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM mission is the understanding of the pathogenic mechanisms of genetic diseases with the aim of developing preventive and therapeutic strategies.TIGEM currently hosts 17 research groups, and a total of more than 120 persons, including students, postdoctoral fellows, staff scientists, technicians, and administrators and offers training programs in medical human genetics and Synthetic Biology in cooperation with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was established by the King of "Sacro Romano Impero" Federico II of Svevia. It's considered the most ancient public school of the world. It consists of 13 departments divided in three areas: Sciences and technologies, humanistic and social, medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== [[image:yesoil.jpg|center|160px]]<br />
<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast able to detect the quality of olive of oil, one of the most famouse product of Italy [http://en.wikipedia.org/wiki/Italy], now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had the idea represented in the picture.<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, it will be:<br />
*''extra virgin'', if the oleic acid conentration will be less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration will be less than 2 gr<br />
*''not edible'', if the oleic acid concentration will be greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-19T17:09:22Z<p>Giov1984: </p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|420px]]<br />
[[Naples]] by night<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM mission is the understanding of the pathogenic mechanisms of genetic diseases with the aim of developing preventive and therapeutic strategies.TIGEM currently hosts 17 research groups, and a total of more than 120 persons, including students, postdoctoral fellows, staff scientists, technicians, and administrators and offers training programs in medical human genetics and Synthetic Biology in cooperation with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was established by the King of "Sacro Romano Impero" Federico II of Svevia. It's considered the most ancient public school of the world. It consists of 13 departments divided in three areas: Sciences and technologies, humanistic and social, medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== [[image:yesoil.jpg|center|160px]]<br />
<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast able to detect the quality of olive of oil, one of the most famouse product of Italy [http://en.wikipedia.org/wiki/Italy], now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had the idea represented in the picture.<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, it will be:<br />
*''extra virgin'', if the oleic acid conentration will be less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration will be less than 2 gr<br />
*''not edible'', if the oleic acid concentration will be greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/File:NAPOLI_ariprova.jpgFile:NAPOLI ariprova.jpg2007-10-19T17:02:37Z<p>Giov1984: </p>
<hr />
<div>[[Image:NAPOLI_ariprova.jpg|500px]]</div>Giov1984http://2007.igem.org/wiki/index.php/Lucia_MarucciLucia Marucci2007-10-19T16:55:51Z<p>Giov1984: </p>
<hr />
<div>[[Image:Lucia.jpg|450px]] [[Image:Lucia3.jpg|370px]]<br />
[[Image:Lucia2.jpg|390px|380px]] [[Image:Lucia1.jpg|350px]]</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-19T09:57:05Z<p>Giov1984: /* '''Mathematical Model''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center|900px]]<br />
<br />
[[Naples]] by night<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM mission is the understanding of the pathogenic mechanisms of genetic diseases with the aim of developing preventive and therapeutic strategies.TIGEM currently hosts 17 research groups, and a total of more than 120 persons, including students, postdoctoral fellows, staff scientists, technicians, and administrators and offers training programs in medical human genetics and Synthetic Biology in cooperation with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was established by the King of "Sacro Romano Impero" Federico II of Svevia. It's considered the most ancient public school of the world. It consists of 13 departments divided in three areas: Sciences and technologies, humanistic and social, medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== [[image:yesoil.jpg|center|160px]]<br />
<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast able to detect the quality of olive of oil, one of the most famouse product of Italy [http://en.wikipedia.org/wiki/Italy], now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had the idea represented in the picture.<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, it will be:<br />
*''extra virgin'', if the oleic acid conentration will be less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration will be less than 2 gr<br />
*''not edible'', if the oleic acid concentration will be greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/NaplesNaples2007-10-17T21:57:34Z<p>Giov1984: /* '''System Model''' */</p>
<hr />
<div> [[Image:NAPOLI_ariprova.jpg|center]]<br />
<br />
<br />
<br />
<br />
== '''About Us''' == <br />
<br />
[[Image:TUTTI3.jpg]][[Image:capi2.jpg]] <br />
<br />
'''Students''':<br />
<br />
*[[Giovanni Russo]] <br />
*[[Lucia Marucci]]<br />
*[[Velia Siciliano]]<br />
*[[Irene Cantone]] <br />
*[[Roberta Bergamasco]][https://2007.igem.org/User_talk:Robertina] <br />
*[[Maria Aurelia Ricci]] <br />
*[[Mafalda Graziano]] <br />
<br />
'''Instructors''' <br />
<br />
*Diego di Bernardo <br />
*Maria Pia Cosma <br />
*Mario di Bernardo <br />
<br />
'''Advisor'''<br />
<br />
*[[Giulia Cuccato]]<br />
<br />
<br />
[[more pictures]] <br />
<br />
<br />
----<br />
<br />
== '''Tigem'''==<br />
<br />
[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] <br />
was created by the Italian Telethon Foundation in 1994. TIGEM mission is the understanding of the pathogenic mechanisms of genetic diseases with the aim of developing preventive and therapeutic strategies.TIGEM currently hosts 17 research groups, and a total of more than 120 persons, including students, postdoctoral fellows, staff scientists, technicians, and administrators and offers training programs in medical human genetics and Synthetic Biology in cooperation with the University of Naples Federico II.<br />
<br />
<br />
----<br />
<br />
== '''University of Naples "Federico II"'''== <br />
[[Image:Uni.jpg|left|110px]] <br />
<br />
<br />
<br />
University of Naples "Federico II"[http://www.international.unina.it/] was established by the King of "Sacro Romano Impero" Federico II of Svevia. It's considered the most ancient public school of the world. It consists of 13 departments divided in three areas: Sciences and technologies, humanistic and social, medicine.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''== [[image:yesoil.jpg|center|160px]]<br />
<br />
<br />
The aim of our project is to engineer a synthetic biological network in yeast able to detect the quality of olive of oil, one of the most famouse product of Italy [http://en.wikipedia.org/wiki/Italy], now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.<br />
<br />
<br />
<br />
<br />
==='''System Model''' === <br />
<br />
After some ''brainstorming'' we had the idea represented in the picture.<br />
<br />
The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.<br />
<br />
The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.<br />
<br />
[[image:Circuito3.jpg|left|thumb|580px|YeSOil circuit]]<br />
<br />
<br />
<br />
<br />
<br />
We recall that, for 100 gr of the oil, it will be:<br />
*''extra virgin'', if the oleic acid conentration will be less than 0.8 gr<br />
*''virgin'', if oleic the acid concentration will be less than 2 gr<br />
*''not edible'', if the oleic acid concentration will be greater than 3-4 gr<br />
<br />
<br />
Now, we need to convert gr in mol and we found that:<br />
<br />
*the oil is ''extra virgin'' if the oleic acid concentration is less than 2.8 mM<br />
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM<br />
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM<br />
<br />
<br />
<br />
<br />
[[Image:tfOre.jpg|right|420px]]<br />
<br />
<br />
<br />
Oleate is the principal olive oil element and acidity indicator.<br />
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,<br />
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]<br />
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the<br />
proliferation of these organelles in Saccharomyces cerevisiae. <br />
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. <br />
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
==='''Mathematical Model'''=== <br />
<br />
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.<br />
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.<br />
<br />
<br />
[[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]]<br />
<br />
<br />
<br />
----<br />
<br />
==='''Yeast Strain''' ===<br />
Yeast strain used is [[W303]].<br />
<br />
All DNA manipulations and subcloning were done in [[Escherichia coli]]<br />
<br />
----<br />
<br />
=== '''Materials & Methods''' ===<br />
We have adopted a strategy of parallel cloning.<br />
<br />
A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 gene.<br />
<br />
'''Background'''<br />
<br />
[[References]]<br />
<br />
'''Cloning Strategies in E.coli'''<br />
*[[Biobrick Vector choice]]<br />
*[[Biobrick Restriction Enzyme]] <br />
*[[Biobrick Primers Design]]<br />
<br />
<br />
'''Cloning Strategies in S.cerevisiae'''<br />
*[[Vector choice]]<br />
*[[Restriction Enzyme]] <br />
*[[Primers Design]]<br />
*[[Primers Design For Yeast Integration]]<br />
<br />
'''Cloning Process'''<br />
*[[Yeast DNA extraction]]<br />
*[[PCR]]<br />
*[[Agarose Gel Electrophoresis]]<br />
*[[PCR Purification]]<br />
*[[Digestion]]<br />
*[[Extraction from Gel]]<br />
*[[Ligation]]<br />
*[[E.coli Transformation]] <br />
*[[Mini and Midi prep]]<br />
*[[Transformation]]<br />
<br />
<br />
----<br />
<br />
=== '''Experimental Results'''===<br />
*[[Luciferase assay]]<br />
*[[Cloning in BioBrick vectors]]<br />
*[[Cloning in yeast vector]]<br />
<br />
<br />
----<br />
<br />
== '''Thanks to...''' ==<br />
<br />
[[Image:Synbiocomm.jpg]] [[Image:Bandiera_comunita_europea.jpg]]<br />
<br />
'''We are partly funded by the European Union SYNBIOCOMM project'''</div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-17T21:56:58Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|200px|center]][[image:Undici.JPG|200px|center]] [[image:Tre.JPG|180px|center]] <br />
[[image:Otto.JPG|center|200px]] [[image:Quattro.JPG|180px|center]] <br />
[[image:Dieci.JPG|140px|center]] [[image:Cinque.JPG|180px|center]]<br />
[[image:Belle.jpg|240px|center]]<br />
[[image:Belle2.jpg|180px|center]]<br />
[[image:Belle3.jpg|200px|center]]<br />
[[image:Belle4.jpg|230px|center]]</div>Giov1984http://2007.igem.org/wiki/index.php/File:Belle4.jpgFile:Belle4.jpg2007-10-17T21:54:59Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/File:IMGP2750.jpgFile:IMGP2750.jpg2007-10-17T21:52:13Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-17T21:51:38Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|180px|center]][[image:Undici.JPG|180px|center]] [[image:Tre.JPG|180px|center]] <br />
[[image:Otto.JPG|center|200px]] [[image:Quattro.JPG|180px|center]] <br />
[[image:Dieci.JPG|180px|center]] [[image:Cinque.JPG|180px|center]]<br />
[[image:Belle.jpg|200px|center]]<br />
[[image:Belle2.jpg|180px|center]]<br />
[[image:Belle3.jpg|200px|center]]</div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-17T21:46:50Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|160px]][[image:Undici.JPG|160px]] [[image:Tre.JPG|160px]] <br />
[[image:Otto.JPG|center|160px]] [[image:Quattro.JPG|160px]] <br />
[[image:Dieci.JPG|160px]] [[image:Cinque.JPG|160px]]<br />
[image:Belle.jpg|160px]]<br />
[image:Belle2.jpg|160px]]<br />
[image:Belle3.jpg|160px]]</div>Giov1984http://2007.igem.org/wiki/index.php/File:Belle3.jpgFile:Belle3.jpg2007-10-17T21:44:23Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/File:Belle2.jpgFile:Belle2.jpg2007-10-17T21:43:42Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/File:Belle.jpgFile:Belle.jpg2007-10-17T21:42:56Z<p>Giov1984: </p>
<hr />
<div></div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-17T11:49:35Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.JPG|160px]][[image:Undici.JPG|160px]] [[image:Tre.JPG|160px]] <br />
[[image:Otto.JPG|center|160px]] [[image:Quattro.JPG|160px]] [[image:Dodici.JPG|160px]]<br />
[[image:Dieci.JPG|160px]] [[image:Cinque.JPG|160px]]</div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-17T11:41:00Z<p>Giov1984: </p>
<hr />
<div>[[image:Sei.jpg|center|160px]]</div>Giov1984http://2007.igem.org/wiki/index.php/More_picturesMore pictures2007-10-17T11:39:24Z<p>Giov1984: </p>
<hr />
<div>[[image:Uno.jpg|center|160px]]</div>Giov1984