IGEM:Harvard/2010/Team Allergy

Description
Allergies to fruits and vegetables are (increasingly) common, affecting millions of people around the world with symptoms ranging from mild itchiness to life-threatening anaphylaxis. Allergy is caused by an inappropriate immune response to harmless proteins present in the environment. Several common food allergens are structurally similar to pollens that cause seasonal allergies and are present in a wide range of fruits and vegetables. Many of these proteins have been knocked down in plants using RNA interference, leading to plants with reduced allergenicity. As part of our iGarden project we are designing modular BioBrick intron-containing self-complementary hairpin forming RNA (ihpRNA) constructs for the targeted knockdown of proteins with homology to allergens in arabidopsis and strawberry, as well as designing ihpRNA constructs against allergens in a range of other plants common in home gardens, including lettuce, carrots, celery, tomato, and several herbs. Our goal is to use genetic engineering to make food safer, and to specially tailor gardens to the needs of each person with a different set of allergies.

Plant shRNA

 * 1) 1 pmid=11576441
 * 2) 2 pmid=16531494

Allergen silencing

 * 1) 3 pmid=17784907
 * 2) 4 pmid=18467156
 * 3) 5 pmid=17088146
 * 4) 6 pmid=19356919
 * 5) 7 pmid=16950292
 * 6) 8 pmid=12746509
 * 7) 9 pmid=15696096
 * 8) 10 pmid=14962769

Allergen proteins

 * 1) 11 pmid=12811016
 * 2) 12 pmid=12757453
 * 3) 13 pmid=15347364
 * 4) 14 pmid=8946858
 * 5) 15 pmid=16650053
 * 6) 16 pmid=15507096
 * 7) 17 pmid=16945416
 * 8) 18 pmid=10887301
 * 9) 19 pmid=12169176

ihpRNA
 #seqs > div {width:800px; word-wrap:break-word;} .bbheader_seq, .bbfooter_seq {color:red;}

CCTTGAATTCGCGGCCGCATCTAGA CCAATTGGTA AGGAAATAAT TATTTTCTTT TTTCCTTTTA GTATAAAATA GTTAAGTGAT GTTAATTAGT ATGATTATAA TAATATAGTT GTTATAATTG TGAAAAAATA ATTTATAAAT ATATTGTTTA CATAAACAAC ATAGTAATGT AAAAAAATAT GACAAGTGAT GTGTAAGACG AAGAAGATAA AAGTTGAGAG TAAGTATATT ATTTTTAATG AATTTGATCG AACATGTAAG ATGATATACT AGCATTAATA TTTGTTTTAA TCATAATAGT AATTCTAGCT GGTTTGATGA ATTAAATATC AATGATAAAA TACTATAGTA AAAATAAGAA TAAATAAATT AAAATAATAT TTTTTTATGA TTAATAGTTT ATTATATAAT TAAATATCTA TACCATTACT AAATATTTTA GTTTAAAAGT TAATAAATAT TTTGTTAGAA ATTCCAATCT GCTTGTAATT TATCAATAAA CAAAATATTA AATAACAAGC TAAAGTAACA AATAATATCA AACTAATAGA AACAGTAATC TAATGTAACA AAACATAATC TAATGCTAAT ATAACAAAGC GCAAGATCTA TCATTTTATA TAGTATTATT TTCAATCAAC ATTCTTATTA ATTTCTAAAT AATACTTGTA GTTTTATTAA CTTCTAAATG GATTGACTAT TAATTAAATG AATTAGTCGA ACATGAATAA ACAAGGTAAC ATGATAGATC ATGTCATTGT GTTATCATTG ATCTTACATT TGGATTGATT ACAGTTGGGA AATTGGGTTC GAA ACTAGTAGCGGCCGCTGCAGCCTT

CCTTGAATTCGCGGCCGCATCTAGA CTCCGCCACC GTTGGTAAGC ATTTTAATTA CTTAACTTTT TGACAAAAGA TAAAAGCAAT TTTGCACGAT TGTGGTCCTT TATTTGGTGG AAAAATCTTT ACTCTTTTCA TGCACATTAA ATATACACAT GTAGATTCTT GTCTTAGTAC CCCCACTACA ACATATATTC GCGCACATGT AGCCTAGTAG TAACGAGATA TAATTAAATG TAAATTTGAA TTTAGGAAAT TGAACAAAAA ACTTAGTTAA GATCTGAGTC ATGTGAGTGT CTCTATCCAT TATTTTCTTG GCTTTATTCA TATGCTTTTC CGAGTCACGT TATGTGGATT GGTTTTATGG CTCATCCACT CGTGAAGAGA GCCGTTCGTT TTAGGGATGG AGTACCTTTT TTCTTTCTTA TTTTGTTTGT TTCATTGTTG TTAACTTTTC TATCAAAATT TTGTCCATTA ATTAAATTTA TTACATGTGA ACAAAAAATA GAAATATGTT TTTTATCATT TCATTTACAA CTTCGGTATA ATTCGCATTT CTTTTTCCCC TTTATAACAC CAAAATATTA ACATTTTAGT TTTAAAATTT TCGTAAAAGT AAAGGTGATA TTCTTGCAAA AGAAGTCTTG TTTTAATTAG TCTTTTAGCT AATGATGCCG TTAATTAACC GTTGTTTCTT TTTGTGGGGC CCAGCTGCTG TCGGCGAGAG ATTCTTAGCT CGTGACATCA CTTTCCAAAA CACGGCGGGT CCGTCG ACTAGTAGCGGCCGCTGCAGCCTT

CCTTGAATTCGCGGCCGCATCTAGA ACAGAACTCA TTAGGTAATT AATTAAATCC TATACCTTAT ATTATATAAC TAATTAATAA TTAGAGAACT CATTAGGAAT GTGATCAGAG AAATAAATAA ATTAGTGGCT TAAGTGGATA ACGCATACTC ACGAGTATAC AATTTGCATG GGAGATATAC CGGGTCGGTG ATACCGGTCG GGTCGGGTCT ATTTTAATTA ATTAAAGTTT ATTTCCGATT TCAGATTTTT GAACGCCGGA ACTAGTAGCGGCCGCTGCAGCCTT

CCTTGAATTCGCGGCCGCATCTAGA ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC CCATCCTGGT CGAGCTGGAC GGCGACGTAA ACGGCCACAA GTTCAGCGTG TCCGGCGAGG GCGAGGGCGA TGCCACCTAC GGCAAGCTGA CCCTGAAGTT CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTCGTGACCA CCCTGACCTA CGGCGTGCAG TGCTTCAGCC GCTACCCCGA CCACATGAAG CAGCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG CACCATCTTC ACTAGTAGCGGCCGCTGCAGCCTT

 Sense: CCTTGAATTCGCGGCCGCATCTAGA ATGGCTGGAG TGATGAAGTT GGCATGCTTG CTCTTGGCCT GCATGATTGT GGCCGGTCCA ATCACATCGA ACGCTGCGCT AAGCTGTGGC TCAGTTAACA GCAACTTGGC AGCGTGCATT GGCTACGTGC TCCAAGGTGG TGTCATTCCC CCAGCGTGTT GCTCCGGCGT TAAAAACCTC AACAGCATAG CCAAGACGAC CCCAGACCGT CAGCAAGCTT GCAATTGCAT TCAAGGTGCC GCTAGAGCCT TAGGCTCTGG TCTCAACGCT GGCCGTGCAG CTG ACTAGTAGCGGCCGCTGCAGCCTT Antisense: <span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA CAGCTGCACG GCCAGCGTTG AGACCAGAGC CTAAGGCTCT AGCGGCACCT TGAATGCAAT TGCAAGCTTG CTGACGGTCT GGGGTCGTCT TGGCTATGCT GTTGAGGTTT TTAACGCCGG AGCAACACGC TGGGGGAATG ACACCACCTT GGAGCACGTA GCCAATGCAC GCTGCCAAGT TGCTGTTAAC TGAGCCACAG CTTAGCGCAG CGTTCGATGT GATTGGACCG GCCACAATCA TGCAGGCCAA GAGCAAGCAT GCCAACTTCA TCACTCCAGC CAT <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT

<div id="rs300_seq"><span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA CCCACAAACA CACGCTCGGA CGCATATTAC ACATGTTCAT ACACTTAATA CTCGCTGTTT TGAATTGATG TTTTAGGAAT ATATATGTAG A <span style="color:blue" title="Customization Sequence 1 (doesn't match observed RS300 background)">ACAGGTCCCCTTCTGTCCATT TCACAGGT CGTGATATGA TTCAATTAGC TTCCGACTCA TTCATCCAAA TACCGAGTCG CCAAAATTCA AACTAGACTC GTTAAATGAA TGAATGATGC GGTAGACAAA TTGGATCATT GATTCTCTTT GA <span style="color:green" title="Customization Sequence 2 (doesn't match observed RS300 background)">TATGGACTGAAGGGGACCCGT TCTCTCT TTTGTATTCC AATTTTCTTG ATTAATCTTT CCTGCACAAA AACATGCTTG ATCCACTAAG TGACATATAT GCTGCCTTCG TATATATAGT TCTGGTAAAA TTAACATTTT GGGTTTATCT TTATTTAAGG CATCGCCATG GGG <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT

<div id="ger3_seq"> Sense: <span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA ATGAAGATGA TAATCCAAAT TTTCTTCATC ATCTCCCTCA TTTCAACCAT CTCTTTTGCC TCTGTTCAAG ACTTTTGTGT AGCTGACCCA AAGGGTCCTC AAAGCCCATC AGGTTACTCT TGCAAGAACC CTGACCAAGT CACCGAAAAT GACTTTGCAT TTACCGGCCT CGGCACAGCT GGAAACACTT CCAACATCAT TAAAGCCGCA GTGACTCCAG CCTTTGCTCC TGCTTACGCA GGCATCAATG GCCTTGGCGT ATCTCTGGCT CGTCTTGACT TAGCCGGTGG TGGTGTCATC <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT Antisense: <span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA GATGACACCAC CACCGGCTAAG TCAAGACGAGC CAGAGATACGC CAAGGCCATTG ATGCCTGCGTA AGCAGGAGCAA AGGCTGGAGTC ACTGCGGCTTT AATGATGTTGG AAGTGTTTCCA GCTGTGCCGAG GCCGGTAAATG CAAAGTCATTT TCGGTGACTTG GTCAGGGTTCT TGCAAGAGTAA CCTGATGGGCT TTGAGGACCCT TTGGGTCAGCT ACACAAAAGTC TTGAACAGAGG CAAAAGAGATG GTTGAAATGAG GGAGATGATGA AGAAAATTTGG ATTATCATCTT CAT <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT

<div id="bet1_seq"> Sense: <span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA ATGATAGAAGA GGAGATTGAGG TAGACGTCGAT ATTAAAGCCAG GGCTGACAAGT TCCACAAGTTC ATCAGAAGATC ACAACATGTGC CCAAAGCCACT CACTACATTAA GGGATGTGATC TGCTCGAAGGC GAGTGGGGAAA AGTTGGAAGCA TACTCTTGTGG AAATTAGTTTT TGGTAATGGTT ATATATATCGC ACCTTAATATA TTAGATCATGG TAACGTATTTT TTAATATGGTA <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT Antisense: <span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA TACCATATTA AAAAATACGT TACCATGATC TAATATATTA AGGTGCGATA TATATAACCA TTACCAAAAA CTAATTTCCA CAAGAGTATG CTTCCAACTT TTCCCCACTC GCCTTCGAGC AGATCACATC CCTTAATGTA GTGAGTGGCT TTGGGCACAT GTTGTGATCT TCTGATGAAC TTGTGGAACT TGTCAGCCCT GGCTTTAATA TCGACGTCTA CCTCAATCTC CTCTTCTATC AT <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT

<div id="bet2_seq"> Sense: <span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA AAATTCATTTA TAAACGCATGT TGGTGTAGATG GAGAGCCACGA GTGTCAAAGGA TATGATCGAGG TAATAGATGAG GAGAAGAATGT GATTCAGTTGA GGGTGTTAGAG GGACCTCTGAA GAAAGAATACA AGAGCTTCCTG AAAACGATGAA AGTGATGAGCC CTAAGCATGGA GGGCCCGGAAG TGTGGTGAAGT GGAACATGAAA TATGAGAGGAT TGATCAGAACG TGGATCACCCA AACAGGCTTCT CCAGTTCTTCG TCGAAGTGACT AAAGAGATTGA CCAATACCTCT TGTCTAAGGAT TAA <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT Antisense:<span class="bbheader_seq">CCTTGAATTCGCGGCCGCATCTAGA TTAATCCTTA GACAAGAGGT ATTGGTCAAT CTCTTTAGTC ACTTCGACGA AGAACTGGAG AAGCCTGTTT GGGTGATCCA CGTTCTGATC AATCCTCTCA TATTTCATGT TCCACTTCAC CACACTTCCG GGCCCTCCAT GCTTAGGGCT CATCACTTTC ATCGTTTTCA GGAAGCTCTT GTATTCTTTC TTCAGAGGTC CCTCTAACAC CCTCAACTGA ATCACATTCT TCTCCTCATC TATTACCTCG ATCATATCCT TTGACACTCG TGGCTCTCCA TCTACACCAA CATGCGTTTA TAAATGAATT T <span class="bbfooter_seq">ACTAGTAGCGGCCGCTGCAGCCTT

Isolating Genes From Plants

 * Qiagen RNeasy Plant Mini Kit
 * iScript cDNA Synthesis

Strawberry Transformation

 * 1) 1 pmid=17147614
 * 2) 2 pmid=16320068
 * 3) 3 pmid=19956955
 * 4) 4 pmid=7581659

amiRNA

 * amiRNA PCR primer design
 * Web MicroRNA Designer
 * amiRNA results analysis

Plasmids

 * Spreadsheet

Introns
ihpRNA production requires the selection of an intron. We are using PCR to make hairpin parts out of two introns:
 * PME3, 746bp
 * PAL2, 240bp

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