- | <div class="body" style="white-space:pre-wrap; line-break:strict;"><p class="s1"><span class="c1">四个改造质粒的</span>part<span class="c1">介绍:</span></p><p class="s2 s3"><span class="c2">1.</span><span class="c3">pBlue</span><span class="c3">script II KS(+) ScaI deletion</span><span class="c3"> </span></p><p class="s1"><span class="c3">2</span><span class="c4">、</span><span class="c3">pBlue</span><span class="c3">script II KS(+) EcoRV deletion</span></p><p class="s1"><span class="c3">3</span><span class="c4">、</span><span class="c3">pBlue</span><span class="c3">script II KS(+) 3 copy</span><span class="c3"> TTCGATATCAAGCT</span></p><p class="s1"><span class="c3">4</span><span class="c4">、</span><span class="c3">pBlue</span><span class="c3">script II KS(+) 5 copy</span><span class="c3"> TTCGATATCAAGCT</span></p><p class="s2"> </p><p class="s2 s4"><span class="c5">1.</span><span class="c6">Vector</span></p><p class="s2 s5">We used <span class="c3">pBlue</span><span class="c3">script II KS(+) </span>as our origin vector and then transformed it into the Connector of our desire.</p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:34.583em;"><img src="images/image1.jpg" alt="C:\Users\FlyFreedom\Desktop\pBluescript_II_KS(+)_1x.png" style="width:34.583em;"/></span></span></p><p class="s7 s8"><span class="c8">Figure 1 Vector map of </span><span class="c9">pBluescript II KS(+)</span></p><p class="s7 s9"> </p><p class="s7 s10"><span class="c10">This is the sequence of </span><span class="c3">pBluescript II KS(+):</span></p><p class="s6"><span class="c11">CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAG</span><span class="c12">G^AA</span><span class="c13">T</span><span class="c14">T^C</span><span class="c15">GAT^ATC</span><span class="c16">AAGC</span><span class="c17">T</span><span class="c11">TATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGAT</span><span class="c17">T</span><span class="c18">TTGGTCATGAGA</span><span class="c17">T</span><span class="c11">TATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTC</span><span class="c17">T</span><span class="c18">GTGACTGGTG</span><span class="c19">AG</span><span class="c20">T</span><span class="c21">^ACT</span><span class="c11">CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC</span></p><p class="s6"><span class="c22">(Restriction site for </span><span class="c23">EcoRI </span><span class="c22">is marked with blue, restriction site for </span><span class="c24">EcoRV</span><span class="c23"> </span><span class="c22">is marked with red, restriction site for </span><span class="c25">ScaI </span><span class="c22">is marked with green. The </span><span class="c26">TAL recognition sequences</span><span class="c22"> are highlighted with yellow while the start </span><span class="c27">T</span><span class="c22"> base and end </span><span class="c27">T</span><span class="c22"> base are grey.) </span></p><p class="s11 s12"><span class="c28">●</span><span class="c7">List for possible TAL recognition sequences:</span></p><p class="s6"><span class="c22">'TAAAAAGGCCGCGT', 'TAAAAATGAAGTTT', 'TAAATCAAAAGAAT', 'TAAATCAGCTCATT', 'TAAATCGGAACCCT', 'TAAATTGTAAGCGT', 'TAACGCCAGGGTTT', 'TAACTCACATTAAT', 'TAAGATGCTTTTCT', 'TAAGCGTTAATATT', 'TAAGGGATTTTGGT', 'TAATACGACTCACT', 'TAATCAAGTTTTTT', 'TACCAATGCTTAAT', 'TACCCAGCTTTTGT', 'TACCGGATACCTGT', 'TACCTGTCCGCCTT', 'TACTCTTCCTTTTT', 'TACTGTCATGCCAT', 'TAGAACTAGTGGAT', 'TAGAAGGACAGTAT', 'TAGAGTAAGTAGTT', 'TAGATAACTACGAT', 'TAGCAGAGCGAGGT', 'TAGCGGTGGTTTTT', 'TAGCTGTTTCCTGT', 'TAGGGTTGAGTGTT', 'TAGGTATCTCAGTT', 'TAGTGAGGGTTAAT', 'TATATGAGTAAACT', 'TATCACTCATGGTT', 'TATCCGCTCACAAT', 'TATCCGGTAACTAT', 'TATCTCAGCGATCT', 'TATCTCAGTTCGGT', 'TATTATTGAAGCAT', 'TATTGGGCGCTCTT', 'TATTTGAATGTATT', 'TATTTTGTTAAAAT', 'TCAAAAAGGATCTT', 'TCAAAGGCGGTAAT', 'TCAACCAAGTCATT', 'TCAAGAAGATCCTT', 'TCAAGCTTATCGAT', 'TCAATCTAAAGTAT', 'TCACCAGCGTTTCT', 'TCACCTAGATCCTT', 'TCACGCTCGTCGTT', 'TCACGTTAAGGGAT', 'TCACTCATGGTTAT', 'TCACTGCCCGCTTT', 'TCAGCCCGACCGCT', 'TCAGCGATCTGTCT', 'TCAGCTCATTTTTT', 'TCAGTGAGGCACCT', 'TCATACTCTTCCTT', 'TCATAGCTCACGCT', 'TCATGGTCATAGCT', 'TCATTGGAAAACGT', 'TCCAGTCTATTAAT', 'TCCATCCAGTCTAT', 'TCCCATTCGCCATT', 'TCCCCCTGGAAGCT', 'TCCGCTTCCTCGCT', 'TCCGTAAGATGCTT', 'TCCTGCAACTTTAT', 'TCCTGTGTGAAATT', 'TCCTTTGATCTTTT', 'TCCTTTTTCAATAT', 'TCGATATCAAGCTT', 'TCGCCATTCAGGCT', 'TCGCGTTAAATTTT', 'TCGCTGCGCTCGGT', 'TCGGAAAAAGAGTT', 'TCGGCAAAATCCCT', 'TCGGGGCGAAAACT', 'TCGGTCGTTCGGCT', 'TCGGTGTAGGTCGT', 'TCGTCGTTTGGTAT', 'TCGTGCACCCAACT', 'TCGTGCGCTCTCCT', 'TCGTGTAGATAACT', 'TCGTTGTCAGAAGT', 'TCTAAAGTATATAT', 'TCTATCAGGGCGAT', 'TCTATTTCGTTCAT', 'TCTCAGCGATCTGT', 'TCTCAGTTCGGTGT', 'TCTCATGAGCGGAT', 'TCTCTTACTGTCAT', 'TCTGAGAATAGTGT', 'TCTGTCTATTTCGT', 'TCTTCAGCATCTTT', 'TGAACCATCACCCT', 'TGAAGTGGTGGCCT', 'TGAATACTCATACT', 'TGACAGTTACCAAT', 'TGACTCCCCGTCGT', 'TGACTCGCTGCGCT', 'TGAGAATAGTGTAT', 'TGAGCGCGCGTAAT', 'TGAGGCACCTATCT', 'TGAGTAAACTTGGT', 'TGAGTGAGCTAACT', 'TGATCCCCCATGTT', 'TGCAAAAAAGCGGT', 'TGCAAGCAGCAGAT', 'TGCACCCAACTGAT', 'TGCAGGAATTCGAT', 'TGCATAATTCTCTT', 'TGCCCGGCGTCAAT', 'TGCCGTAAAGCACT', 'TGCGCAACGTTGTT', 'TGCGCGCTTGGCGT', 'TGCGCTCGGTCGTT', 'TGCGCTCTCCTGTT', 'TGCGGCGACCGAGT', 'TGCTACAGAGTTCT', 'TGCTGAAGCCAGTT', 'TGCTGCAAGGCGAT', 'TGGAAAACGTTCTT', 'TGGCAGCACTGCAT', 'TGGCAGCAGCCACT', 'TGGCGCTTTCTCAT', 'TGGCGTTTTTCCAT', 'TGGTAGCGGTGGTT', 'TGGTAGCTCTTGAT', 'TGGTATGGCTTCAT', 'TGGTCATAGCTGTT', 'TGGTCCTGCAACTT', 'TGGTTTTTTTGTTT', 'TGTAACCCACTCGT', 'TGTCAGAAGTAAGT', 'TGTGAAATTGTTAT', 'TGTGACTGGTGAGT', 'TGTGTGAAATTGTT', 'TGTTGAATACTCAT', 'TGTTGAGATCCAGT', 'TGTTGTTCCAGTTT', 'TTAAAAATGAAGTT', 'TTAAAAGTGCTCAT', 'TTAAAATTCGCGTT', 'TTAAATCAGCTCAT', 'TTAATTGCGCGCTT', 'TTAGCTCCTTCGGT', 'TTATCAAAAAGGAT', 'TTATCACTCATGGT', 'TTATCAGGGTTATT', 'TTATCCGCCTCCAT', 'TTATTGAAGCATTT', 'TTCACCTAGATCCT', 'TTCCGCGCACATTT', 'TTCCTGTGTGAAAT', 'TTCGATATCAAGCT', 'TTCGCGTTAAATTT', 'TTCGGAAAAAGAGT', 'TTCGGTCCTCCGAT', 'TTGCGCAACGTTGT', 'TTGCTGGCGTTTTT', 'TTGGAAAACGTTCT', 'TTGGCCGCAGTGTT', 'TTGGGAAGGGCGAT', 'TTGGTATCTGCGCT', 'TTGGTCATGAGATT', 'TTGGTCTGACAGTT', 'TTGTAAGCGTTAAT', 'TTGTTAAATCAGCT', 'TTGTTCCCTTTAGT', 'TTGTTGCCATTGCT', 'TTTAAATTAAAAAT', 'TTTATCAGGGTTAT', 'TTTCACCAGCGTTT', 'TTTCCCCGAAAAGT', 'TTTCGTTCATCCAT', 'TTTCTACGGGGTCT', 'TTTCTGTGACTGGT', 'TTTGGAACAAGAGT', 'TTTGGGGTCGAGGT', 'TTTGGTCATGAGAT', 'TTTTAAATCAATCT', 'TTTTCAATATTATT', 'TTTTTCAATATTAT', 'TTTTTCCATAGGCT'</span></p><p class="s7 s13"> </p><p class="s11 s14"><span class="c29">2.</span><span class="c30">Connector</span></p><p class="s15"><span class="c7">The so-called Connector is plasmid designed to bind with several different Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000). Connectees are </span></p><p class="s7 s10"><span class="c7">fusion proteins who carry out functional enzymes and have TAL(Transactivator-like effectors) at one end that can bind to certain DNA sequences and you can design your own Connectees with different enzymes. For </span><span class="c3">pBluescript II KS(+)</span><span class="c7">, we have selected three 14-nucleotide-long sequences called RS</span><span class="c7">Ⅰ</span><span class="c7">, RS</span><span class="c7">Ⅱ</span><span class="c7"> and RS</span><span class="c7">Ⅲ</span><span class="c7"> as the recognition sequences(RS).</span></p><p class="s7 s10"><span class="c7">The recognition sequences must start with a T and end with a T.</span></p><p class="s7 s16"><span class="c22">RS </span><span class="c22">Ⅰ</span><span class="c22">: </span><span class="c23">TTC</span><span class="c31">GATATC</span><span class="c22">AAGCT </span></p><p class="s7 s16"><span class="c22">RS </span><span class="c22">Ⅱ</span><span class="c22">: TGTGACTGGTG</span><span class="c25">AGT</span></p><p class="s7 s16"><span class="c22">RS </span><span class="c22">Ⅲ</span><span class="c22">: TTTGGTCATGAGAT</span></p><p class="s7 s10"><span class="c22">( RS </span><span class="c22">Ⅰ </span><span class="c22">contains partial restriction enzyme cutting site of </span><span class="c23">EcoRI </span><span class="c22">and </span><span class="c24">EcoRV </span><span class="c22">)</span></p><p class="s7 s10"><span class="c22">( RS </span><span class="c22">Ⅱ </span><span class="c22">contains partial restriction enzyme cutting site of </span><span class="c25">ScaI </span><span class="c22">)</span></p><p class="s7 s17"><span class="c7"> <span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:26.812em;"><img src="images/image2.jpg" alt="Macintosh HD:Users:chenming:Desktop:2)S$H4H`C{]S)M29%C6UJ}3.jpg" style="width:26.812em;"/></span></span></p><p class="s7 s18"><span class="c8">Figure 2 A Connector binds with three different Connectees</span></p><p class="s7 s19"> </p><p class="s7 s19"> </p><p class="s7 s19"> </p><p class="s11 s14"><span class="c29">3.</span><span class="c30">Combination</span></p><p class="s7 s10"><span class="c7">In order to prove that Connectors are able to bind with three kinds of Connectees. We designed the combination experiment. We selected a pathway: substrate becomes intermediate product through enzyme</span><span class="c7">Ⅰ</span><span class="c7">,then the intermediate product can either go to production A through enzyme</span><span class="c7">Ⅱ</span><span class="c7"> or production B through enzyme </span><span class="c7">Ⅲ</span><span class="c7">. </span></p><p class="s7 s20"><span class="c7"> <span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:34.566em;"><img src="images/image3.jpg" alt="Macintosh HD:Users:chenming:Desktop:6~2I1}LP1{6UJJW~100NIIL.jpg" style="width:34.566em;"/></span></span></p><p class="s7 s18"><span class="c8">Figure 3 Diagrams of our pathway design</span></p><p class="s7 s21"> </p><p class="s6"><span class="c7"> To achieve this, we need three kinds of Connectees fused with enzyme </span><span class="c7">Ⅰ</span><span class="c7">, </span><span class="c7">Ⅱ</span><span class="c7">and</span><span class="c7">Ⅲ</span><span class="c7"> independently and two kinds of Connectors. One has RS</span><span class="c7">Ⅰ</span><span class="c7">and RS</span><span class="c7">Ⅱ</span><span class="c7"> while the other has RS</span><span class="c7">Ⅰ</span><span class="c7">and RS</span><span class="c7">Ⅲ</span><span class="c7">. So in the end, Connectors with RS</span><span class="c7">Ⅰ</span><span class="c7"> and RS</span><span class="c7">Ⅱ</span><span class="c7"> get production A while Connectors with RS</span><span class="c7">Ⅰ</span><span class="c7"> and RS</span><span class="c7">Ⅲ</span><span class="c7"> production B.</span></p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.228em;"><img src="images/image4.png" alt="image4.png" style="width:36.228em;"/></span></span></p><p class="s6"><span class="c8">Figure 4 Connector is originally designed with three recognition sequence(RS). Then we transformed it into two different Connector, one with RS </span><span class="c8">Ⅰ</span><span class="c8"> and RS </span><span class="c8">Ⅱ</span><span class="c8"> while the other one with RS </span><span class="c8">Ⅰ</span><span class="c8"> and RS </span><span class="c8">Ⅲ</span><span class="c8">. Connector with RS</span><span class="c8">Ⅰ</span><span class="c8">and RS</span><span class="c8">Ⅱ</span><span class="c8"> can bind with Connectee-enzyme </span><span class="c8">Ⅰ</span><span class="c8"> and Connectee-enzyme </span><span class="c8">Ⅱ</span><span class="c8"> and get production A in the end, while Connector with RS </span><span class="c8">Ⅰ</span><span class="c8"> and RS </span><span class="c8">Ⅲ</span><span class="c8"> can bind with Connectee-enzyme </span><span class="c8">Ⅰ</span><span class="c8"> and Connectee-enzyme </span><span class="c8">Ⅲ</span><span class="c8"> and get production B.</span></p><p class="s7 s19"> </p><p class="s6"><span class="c7"> The corresponding Connectors are</span><span class="c32"> </span><span class="c3">pBluescript II KS(+) ScaI deletion</span><span class="c7"> and </span><span class="c3">pBluescript II KS(+) EcoRV deletion.</span></p><p class="s11 s22"><span class="c33">●</span><span class="c3">pBluescript II KS(+) ScaI deletion</span><span class="c4">:</span></p><p class="s7 s10"><span class="c7">We delete the RS</span><span class="c7">Ⅱ</span><span class="c7"> site so this transformed Connector can only bind to Connectees with two kinds of enzyme. </span></p><p class="s7 s10"><span class="c7">In order to delete this site and surrounding sequence, we adopted the Inverse PCR to delete the following sequence: </span></p><p class="s7 s10"><span class="c7">CTGTGACTGGTGAGTACTCAACCAAGTCATTCTG</span></p><p class="s7 s21"> </p><p class="s7 s10"><span class="c7">Primers for Sac</span><span class="c7">Ⅰ</span><span class="c7">:</span></p><p class="s7 s10"><span class="c7">Forward: AGAATAGTGTATGCGGCGCGAC Tm:57</span><span class="c7">℃</span></p><p class="s7 s10"><span class="c7">Reverse: AAAAGCATCTTACGGATGGCA Tm:58</span><span class="c7">℃</span></p><p class="s11 s23"> </p><p class="s11 s24"><span class="c3">pBluescript II KS(+) ScaI deletion </span><span class="c7">can be used along with</span><span class="c3"> pBluescript II</span></p><p class="s11 s24"><span class="c3">KS(+) EcoRV deletion </span><span class="c7">and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s11 s23"> </p><p class="s11 s22"><span class="c33">●</span><span class="c3">pBluescript II KS(+) EcoRV deletion</span><span class="c4">:</span></p><p class="s7 s10"><span class="c7">We delete the RS</span><span class="c7">Ⅲ</span><span class="c7"> site so this transformed Connector can only bind to Connectees with two kinds of enzyme. </span></p><p class="s7 s10"><span class="c7">In order to delete this site and surrounding sequence, we adopted the Inverse PCR to delete the following sequence: </span></p><p class="s7 s10"><span class="c7">GGCTGCAGGAATTCGATATCAAGC</span></p><p class="s7 s19"> </p><p class="s7 s10"><span class="c7">Primers for EcoRV:</span></p><p class="s7 s10"><span class="c7">Forward: TTATCGATACCGTCGACCTCG Tm:56</span><span class="c7">℃</span></p><p class="s15"><span class="c7">Reverse: CGGGGGATCCACTAGTTCTA Tm:53</span><span class="c7">℃</span></p><p class="s7 s25"> </p><p class="s11 s24"><span class="c3">pBluescript II KS(+) EcoRV deletion </span><span class="c7">can be used along with</span><span class="c3"> pBluescript II</span></p><p class="s11 s24"><span class="c3">KS(+) ScaI deletion </span><span class="c7">and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s7 s25"> </p><p class="s7 s26"> </p><p class="s11 s14"><span class="c29">4.</span><span class="c30">Maximization</span></p><p class="s6"><span class="c7"> We are trying to combine as many as enzymes as possible because more enzymes</span><span class="c7">’ </span><span class="c7">combination can produce more complicated reaction chains. So our purpose is to figure out the maximum number of Connectees that binding to a Connector.</span></p><p class="s6"><span class="c7"> So we intended to add more RS on the Connectors. On the one hand, more RSs mean we can bind more kinds of enzymes on one Connector. On the other hand ,we doubt the binding efficiency between Connectors and Connectees so more RSs can improve the possibility of Connectees binding to Connectors.</span></p><p class="s1"> The corresponding Connectors are<span class="c3"> pBlue</span><span class="c3">script II KS(+)_3_copy</span><span class="c3"> </span>and<span class="c3"> pBlue</span><span class="c3">script II KS(+)_5_copy</span>.</p><p class="s7 s19"> </p><p class="s11 s12"><span class="c28">●</span><span class="c3">pBluescript II KS(+)_3_copy </span></p><p class="s7 s10"><span class="c7">In order to add 3 copies of RS on Connectors, we firstly use restriction enzyme BstXI and BamHI to make a nick and replace the original sequence with one RS. Secondly, in the same way, we use restriction enzyme SalI and KpnI to add another RS.</span></p><p class="s7 s10"><span class="c7">The RS sequence is TTCGATATCAAGCT.</span></p><p class="s7 s10"><span class="c7">Here we have designed the new short sequence:</span></p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.208em;"><img src="images/image5.png" alt="image5.png" style="width:36.208em;"/></span></span></p><p class="s11 s24"><span class="c7">and</span></p><p class="s6"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.235em;"><img src="images/image6.png" alt="Macintosh HD:Users:chenming:Desktop:2.png" style="width:36.235em;"/></span></p><p class="s7 s10"><span class="c7">This is the what we get in the end:</span></p><p class="s6"><span class="c7">CT...CT</span><span class="c34">CCACCGCGGTGG</span><span class="c35">TTCGATATCAAGCT</span><span class="c36">GGATCC</span><span class="c7">CCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACC</span><span class="c37">GTCGAC</span><span class="c7">TTCGATATCAAGCT</span><span class="c38">GGTACC</span><span class="c7">CA</span><span class="c7">…</span><span class="c7">.AC</span></p><p class="s27"><span class="c3">pBluescript II KS(+)_3_copy</span><span class="c7"> can be used along with our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s2 s28"> </p><p class="s2 s29"><span class="c39">●</span><span class="c40">pBlue</span><span class="c40">script II KS(+)_5_copy</span></p><p class="s7 s10"><span class="c7">In order to add 4 more copies of RS on Connectors, we firstly use restriction enzyme BstXI and BamHI to make a nick and replace the original sequence with 4 consecutive repeats of RS. </span></p><p class="s7 s10"><span class="c7">The RS sequence is TTCGATATCAAGCT.</span></p><p class="s7 s10"><span class="c7">Here we have designed the new short sequence:</span></p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.167em;"><img src="images/image7.png" alt="Macintosh HD:Users:chenming:Desktop:3.png" style="width:36.167em;"/></span></span></p><p class="s7 s10"><span class="c7">This is the what we get in the end:</span></p><p class="s6"><span class="c7">CT</span><span class="c7">…</span><span class="c7">CT</span><span class="c37">CCACCGCGGTGG<span id="b1"/></span><span class="c35">TTCGATATCAAGCT</span><span class="c41">TTCGATATCAAGCT</span><span class="c35">TTCGATATCAAGCT</span><span class="c41">TTCGATATCAAGCT</span><span class="c38">GGATCC</span><span class="c7">CC</span><span class="c7">…</span><span class="c7">AC</span></p><p class="s27"><span class="c3">pBluescript II KS(+)_5_copy</span><span class="c7"> can be used along with our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s11 s30"> </p><p class="s11 s14"><span class="c29">5.</span><span class="c42">Inverse PCR</span></p><p class="s2 s5"><span class="c43">We used Inverse PCR to transform our plasmid into two kinds of Connectors:</span><span class="c40"> pBlue</span><span class="c40">script II KS(+) ScaI deletion</span><span class="c40"> </span><span class="c43">and </span><span class="c40">pBlue</span><span class="c40">script II KS(+) EcoRV deletion</span><span class="c43">.</span></p><p class="s1"><span class="c43">Inverse PCR method is using cyclic DNA (such as a plasmid) as the template, with two primers designed in a reverse direction to achieve completed PCR. In this way, by designing primers,we can introduce a mutation, insertion or deletion.</span></p><p class="s7 s10"><span class="c7">We used KOD-Plus-Mutagenesis Kit by TOYOBO.CO.LTD. </span></p><p class="s7 s31 c7">More details please click http://www.bio-toyobo.cn.</p></div>
| + | <div class="body" style="white-space:pre-wrap; line-break:strict;"><p class="s1"><span class="c1">四个改造质粒的</span>part<span class="c1">介绍:</span></p><p class="s2 s3"><span class="c2">1.</span><span class="c3">pBlue</span><span class="c3">script II KS(+) ScaI deletion</span><span class="c3"> </span></p><p class="s1"><span class="c3">2</span><span class="c4">、</span><span class="c3">pBlue</span><span class="c3">script II KS(+) EcoRV deletion</span></p><p class="s1"><span class="c3">3</span><span class="c4">、</span><span class="c3">pBlue</span><span class="c3">script II KS(+) 3 copy</span><span class="c3"> TTCGATATCAAGCT</span></p><p class="s1"><span class="c3">4</span><span class="c4">、</span><span class="c3">pBlue</span><span class="c3">script II KS(+) 5 copy</span><span class="c3"> TTCGATATCAAGCT</span></p><p class="s2"> </p><p class="s2 s4"><span class="c5">1.</span><span class="c6">Vector</span></p><p class="s2 s5">We used <span class="c3">pBlue</span><span class="c3">script II KS(+) </span>as our origin vector and then transformed it into the Connector of our desire.</p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:34.583em;"><img src="images/image1.jpg" alt="C:\Users\FlyFreedom\Desktop\pBluescript_II_KS(+)_1x.png" style="width:34.583em;align=center"/></span></span></p><p class="s7 s8"><span class="c8">Figure 1 Vector map of </span><span class="c9">pBluescript II KS(+)</span></p><p class="s7 s9"> </p><p class="s7 s10"><span class="c10">This is the sequence of </span><span class="c3">pBluescript II KS(+):</span></p><p class="s6"><span class="c11">CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAG</span><span class="c12">G^AA</span><span class="c13">T</span><span class="c14">T^C</span><span class="c15">GAT^ATC</span><span class="c16">AAGC</span><span class="c17">T</span><span class="c11">TATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGAT</span><span class="c17">T</span><span class="c18">TTGGTCATGAGA</span><span class="c17">T</span><span class="c11">TATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTC</span><span class="c17">T</span><span class="c18">GTGACTGGTG</span><span class="c19">AG</span><span class="c20">T</span><span class="c21">^ACT</span><span class="c11">CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC</span></p><p class="s6"><span class="c22">(Restriction site for </span><span class="c23">EcoRI </span><span class="c22">is marked with blue, restriction site for </span><span class="c24">EcoRV</span><span class="c23"> </span><span class="c22">is marked with red, restriction site for </span><span class="c25">ScaI </span><span class="c22">is marked with green. The </span><span class="c26">TAL recognition sequences</span><span class="c22"> are highlighted with yellow while the start </span><span class="c27">T</span><span class="c22"> base and end </span><span class="c27">T</span><span class="c22"> base are grey.) </span></p><p class="s11 s12"><span class="c28">●</span><span class="c7">List for possible TAL recognition sequences:</span></p><p class="s6"><span class="c22">'TAAAAAGGCCGCGT', 'TAAAAATGAAGTTT', 'TAAATCAAAAGAAT', 'TAAATCAGCTCATT', 'TAAATCGGAACCCT', 'TAAATTGTAAGCGT', 'TAACGCCAGGGTTT', 'TAACTCACATTAAT', 'TAAGATGCTTTTCT', 'TAAGCGTTAATATT', 'TAAGGGATTTTGGT', 'TAATACGACTCACT', 'TAATCAAGTTTTTT', 'TACCAATGCTTAAT', 'TACCCAGCTTTTGT', 'TACCGGATACCTGT', 'TACCTGTCCGCCTT', 'TACTCTTCCTTTTT', 'TACTGTCATGCCAT', 'TAGAACTAGTGGAT', 'TAGAAGGACAGTAT', 'TAGAGTAAGTAGTT', 'TAGATAACTACGAT', 'TAGCAGAGCGAGGT', 'TAGCGGTGGTTTTT', 'TAGCTGTTTCCTGT', 'TAGGGTTGAGTGTT', 'TAGGTATCTCAGTT', 'TAGTGAGGGTTAAT', 'TATATGAGTAAACT', 'TATCACTCATGGTT', 'TATCCGCTCACAAT', 'TATCCGGTAACTAT', 'TATCTCAGCGATCT', 'TATCTCAGTTCGGT', 'TATTATTGAAGCAT', 'TATTGGGCGCTCTT', 'TATTTGAATGTATT', 'TATTTTGTTAAAAT', 'TCAAAAAGGATCTT', 'TCAAAGGCGGTAAT', 'TCAACCAAGTCATT', 'TCAAGAAGATCCTT', 'TCAAGCTTATCGAT', 'TCAATCTAAAGTAT', 'TCACCAGCGTTTCT', 'TCACCTAGATCCTT', 'TCACGCTCGTCGTT', 'TCACGTTAAGGGAT', 'TCACTCATGGTTAT', 'TCACTGCCCGCTTT', 'TCAGCCCGACCGCT', 'TCAGCGATCTGTCT', 'TCAGCTCATTTTTT', 'TCAGTGAGGCACCT', 'TCATACTCTTCCTT', 'TCATAGCTCACGCT', 'TCATGGTCATAGCT', 'TCATTGGAAAACGT', 'TCCAGTCTATTAAT', 'TCCATCCAGTCTAT', 'TCCCATTCGCCATT', 'TCCCCCTGGAAGCT', 'TCCGCTTCCTCGCT', 'TCCGTAAGATGCTT', 'TCCTGCAACTTTAT', 'TCCTGTGTGAAATT', 'TCCTTTGATCTTTT', 'TCCTTTTTCAATAT', 'TCGATATCAAGCTT', 'TCGCCATTCAGGCT', 'TCGCGTTAAATTTT', 'TCGCTGCGCTCGGT', 'TCGGAAAAAGAGTT', 'TCGGCAAAATCCCT', 'TCGGGGCGAAAACT', 'TCGGTCGTTCGGCT', 'TCGGTGTAGGTCGT', 'TCGTCGTTTGGTAT', 'TCGTGCACCCAACT', 'TCGTGCGCTCTCCT', 'TCGTGTAGATAACT', 'TCGTTGTCAGAAGT', 'TCTAAAGTATATAT', 'TCTATCAGGGCGAT', 'TCTATTTCGTTCAT', 'TCTCAGCGATCTGT', 'TCTCAGTTCGGTGT', 'TCTCATGAGCGGAT', 'TCTCTTACTGTCAT', 'TCTGAGAATAGTGT', 'TCTGTCTATTTCGT', 'TCTTCAGCATCTTT', 'TGAACCATCACCCT', 'TGAAGTGGTGGCCT', 'TGAATACTCATACT', 'TGACAGTTACCAAT', 'TGACTCCCCGTCGT', 'TGACTCGCTGCGCT', 'TGAGAATAGTGTAT', 'TGAGCGCGCGTAAT', 'TGAGGCACCTATCT', 'TGAGTAAACTTGGT', 'TGAGTGAGCTAACT', 'TGATCCCCCATGTT', 'TGCAAAAAAGCGGT', 'TGCAAGCAGCAGAT', 'TGCACCCAACTGAT', 'TGCAGGAATTCGAT', 'TGCATAATTCTCTT', 'TGCCCGGCGTCAAT', 'TGCCGTAAAGCACT', 'TGCGCAACGTTGTT', 'TGCGCGCTTGGCGT', 'TGCGCTCGGTCGTT', 'TGCGCTCTCCTGTT', 'TGCGGCGACCGAGT', 'TGCTACAGAGTTCT', 'TGCTGAAGCCAGTT', 'TGCTGCAAGGCGAT', 'TGGAAAACGTTCTT', 'TGGCAGCACTGCAT', 'TGGCAGCAGCCACT', 'TGGCGCTTTCTCAT', 'TGGCGTTTTTCCAT', 'TGGTAGCGGTGGTT', 'TGGTAGCTCTTGAT', 'TGGTATGGCTTCAT', 'TGGTCATAGCTGTT', 'TGGTCCTGCAACTT', 'TGGTTTTTTTGTTT', 'TGTAACCCACTCGT', 'TGTCAGAAGTAAGT', 'TGTGAAATTGTTAT', 'TGTGACTGGTGAGT', 'TGTGTGAAATTGTT', 'TGTTGAATACTCAT', 'TGTTGAGATCCAGT', 'TGTTGTTCCAGTTT', 'TTAAAAATGAAGTT', 'TTAAAAGTGCTCAT', 'TTAAAATTCGCGTT', 'TTAAATCAGCTCAT', 'TTAATTGCGCGCTT', 'TTAGCTCCTTCGGT', 'TTATCAAAAAGGAT', 'TTATCACTCATGGT', 'TTATCAGGGTTATT', 'TTATCCGCCTCCAT', 'TTATTGAAGCATTT', 'TTCACCTAGATCCT', 'TTCCGCGCACATTT', 'TTCCTGTGTGAAAT', 'TTCGATATCAAGCT', 'TTCGCGTTAAATTT', 'TTCGGAAAAAGAGT', 'TTCGGTCCTCCGAT', 'TTGCGCAACGTTGT', 'TTGCTGGCGTTTTT', 'TTGGAAAACGTTCT', 'TTGGCCGCAGTGTT', 'TTGGGAAGGGCGAT', 'TTGGTATCTGCGCT', 'TTGGTCATGAGATT', 'TTGGTCTGACAGTT', 'TTGTAAGCGTTAAT', 'TTGTTAAATCAGCT', 'TTGTTCCCTTTAGT', 'TTGTTGCCATTGCT', 'TTTAAATTAAAAAT', 'TTTATCAGGGTTAT', 'TTTCACCAGCGTTT', 'TTTCCCCGAAAAGT', 'TTTCGTTCATCCAT', 'TTTCTACGGGGTCT', 'TTTCTGTGACTGGT', 'TTTGGAACAAGAGT', 'TTTGGGGTCGAGGT', 'TTTGGTCATGAGAT', 'TTTTAAATCAATCT', 'TTTTCAATATTATT', 'TTTTTCAATATTAT', 'TTTTTCCATAGGCT'</span></p><p class="s7 s13"> </p><p class="s11 s14"><span class="c29">2.</span><span class="c30">Connector</span></p><p class="s15"><span class="c7">The so-called Connector is plasmid designed to bind with several different Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000). Connectees are </span></p><p class="s7 s10"><span class="c7">fusion proteins who carry out functional enzymes and have TAL(Transactivator-like effectors) at one end that can bind to certain DNA sequences and you can design your own Connectees with different enzymes. For </span><span class="c3">pBluescript II KS(+)</span><span class="c7">, we have selected three 14-nucleotide-long sequences called RS</span><span class="c7">Ⅰ</span><span class="c7">, RS</span><span class="c7">Ⅱ</span><span class="c7"> and RS</span><span class="c7">Ⅲ</span><span class="c7"> as the recognition sequences(RS).</span></p><p class="s7 s10"><span class="c7">The recognition sequences must start with a T and end with a T.</span></p><p class="s7 s16"><span class="c22">RS </span><span class="c22">Ⅰ</span><span class="c22">: </span><span class="c23">TTC</span><span class="c31">GATATC</span><span class="c22">AAGCT </span></p><p class="s7 s16"><span class="c22">RS </span><span class="c22">Ⅱ</span><span class="c22">: TGTGACTGGTG</span><span class="c25">AGT</span></p><p class="s7 s16"><span class="c22">RS </span><span class="c22">Ⅲ</span><span class="c22">: TTTGGTCATGAGAT</span></p><p class="s7 s10"><span class="c22">( RS </span><span class="c22">Ⅰ </span><span class="c22">contains partial restriction enzyme cutting site of </span><span class="c23">EcoRI </span><span class="c22">and </span><span class="c24">EcoRV </span><span class="c22">)</span></p><p class="s7 s10"><span class="c22">( RS </span><span class="c22">Ⅱ </span><span class="c22">contains partial restriction enzyme cutting site of </span><span class="c25">ScaI </span><span class="c22">)</span></p><p class="s7 s17"><span class="c7"> <span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:26.812em;"><img src="images/image2.jpg" alt="Macintosh HD:Users:chenming:Desktop:2)S$H4H`C{]S)M29%C6UJ}3.jpg" style="width:26.812em;"/></span></span></p><p class="s7 s18"><span class="c8">Figure 2 A Connector binds with three different Connectees</span></p><p class="s7 s19"> </p><p class="s7 s19"> </p><p class="s7 s19"> </p><p class="s11 s14"><span class="c29">3.</span><span class="c30">Combination</span></p><p class="s7 s10"><span class="c7">In order to prove that Connectors are able to bind with three kinds of Connectees. We designed the combination experiment. We selected a pathway: substrate becomes intermediate product through enzyme</span><span class="c7">Ⅰ</span><span class="c7">,then the intermediate product can either go to production A through enzyme</span><span class="c7">Ⅱ</span><span class="c7"> or production B through enzyme </span><span class="c7">Ⅲ</span><span class="c7">. </span></p><p class="s7 s20"><span class="c7"> <span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:34.566em;"><img src="images/image3.jpg" alt="Macintosh HD:Users:chenming:Desktop:6~2I1}LP1{6UJJW~100NIIL.jpg" style="width:34.566em;"/></span></span></p><p class="s7 s18"><span class="c8">Figure 3 Diagrams of our pathway design</span></p><p class="s7 s21"> </p><p class="s6"><span class="c7"> To achieve this, we need three kinds of Connectees fused with enzyme </span><span class="c7">Ⅰ</span><span class="c7">, </span><span class="c7">Ⅱ</span><span class="c7">and</span><span class="c7">Ⅲ</span><span class="c7"> independently and two kinds of Connectors. One has RS</span><span class="c7">Ⅰ</span><span class="c7">and RS</span><span class="c7">Ⅱ</span><span class="c7"> while the other has RS</span><span class="c7">Ⅰ</span><span class="c7">and RS</span><span class="c7">Ⅲ</span><span class="c7">. So in the end, Connectors with RS</span><span class="c7">Ⅰ</span><span class="c7"> and RS</span><span class="c7">Ⅱ</span><span class="c7"> get production A while Connectors with RS</span><span class="c7">Ⅰ</span><span class="c7"> and RS</span><span class="c7">Ⅲ</span><span class="c7"> production B.</span></p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.228em;"><img src="images/image4.png" alt="image4.png" style="width:36.228em;"/></span></span></p><p class="s6"><span class="c8">Figure 4 Connector is originally designed with three recognition sequence(RS). Then we transformed it into two different Connector, one with RS </span><span class="c8">Ⅰ</span><span class="c8"> and RS </span><span class="c8">Ⅱ</span><span class="c8"> while the other one with RS </span><span class="c8">Ⅰ</span><span class="c8"> and RS </span><span class="c8">Ⅲ</span><span class="c8">. Connector with RS</span><span class="c8">Ⅰ</span><span class="c8">and RS</span><span class="c8">Ⅱ</span><span class="c8"> can bind with Connectee-enzyme </span><span class="c8">Ⅰ</span><span class="c8"> and Connectee-enzyme </span><span class="c8">Ⅱ</span><span class="c8"> and get production A in the end, while Connector with RS </span><span class="c8">Ⅰ</span><span class="c8"> and RS </span><span class="c8">Ⅲ</span><span class="c8"> can bind with Connectee-enzyme </span><span class="c8">Ⅰ</span><span class="c8"> and Connectee-enzyme </span><span class="c8">Ⅲ</span><span class="c8"> and get production B.</span></p><p class="s7 s19"> </p><p class="s6"><span class="c7"> The corresponding Connectors are</span><span class="c32"> </span><span class="c3">pBluescript II KS(+) ScaI deletion</span><span class="c7"> and </span><span class="c3">pBluescript II KS(+) EcoRV deletion.</span></p><p class="s11 s22"><span class="c33">●</span><span class="c3">pBluescript II KS(+) ScaI deletion</span><span class="c4">:</span></p><p class="s7 s10"><span class="c7">We delete the RS</span><span class="c7">Ⅱ</span><span class="c7"> site so this transformed Connector can only bind to Connectees with two kinds of enzyme. </span></p><p class="s7 s10"><span class="c7">In order to delete this site and surrounding sequence, we adopted the Inverse PCR to delete the following sequence: </span></p><p class="s7 s10"><span class="c7">CTGTGACTGGTGAGTACTCAACCAAGTCATTCTG</span></p><p class="s7 s21"> </p><p class="s7 s10"><span class="c7">Primers for Sac</span><span class="c7">Ⅰ</span><span class="c7">:</span></p><p class="s7 s10"><span class="c7">Forward: AGAATAGTGTATGCGGCGCGAC Tm:57</span><span class="c7">℃</span></p><p class="s7 s10"><span class="c7">Reverse: AAAAGCATCTTACGGATGGCA Tm:58</span><span class="c7">℃</span></p><p class="s11 s23"> </p><p class="s11 s24"><span class="c3">pBluescript II KS(+) ScaI deletion </span><span class="c7">can be used along with</span><span class="c3"> pBluescript II</span></p><p class="s11 s24"><span class="c3">KS(+) EcoRV deletion </span><span class="c7">and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s11 s23"> </p><p class="s11 s22"><span class="c33">●</span><span class="c3">pBluescript II KS(+) EcoRV deletion</span><span class="c4">:</span></p><p class="s7 s10"><span class="c7">We delete the RS</span><span class="c7">Ⅲ</span><span class="c7"> site so this transformed Connector can only bind to Connectees with two kinds of enzyme. </span></p><p class="s7 s10"><span class="c7">In order to delete this site and surrounding sequence, we adopted the Inverse PCR to delete the following sequence: </span></p><p class="s7 s10"><span class="c7">GGCTGCAGGAATTCGATATCAAGC</span></p><p class="s7 s19"> </p><p class="s7 s10"><span class="c7">Primers for EcoRV:</span></p><p class="s7 s10"><span class="c7">Forward: TTATCGATACCGTCGACCTCG Tm:56</span><span class="c7">℃</span></p><p class="s15"><span class="c7">Reverse: CGGGGGATCCACTAGTTCTA Tm:53</span><span class="c7">℃</span></p><p class="s7 s25"> </p><p class="s11 s24"><span class="c3">pBluescript II KS(+) EcoRV deletion </span><span class="c7">can be used along with</span><span class="c3"> pBluescript II</span></p><p class="s11 s24"><span class="c3">KS(+) ScaI deletion </span><span class="c7">and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s7 s25"> </p><p class="s7 s26"> </p><p class="s11 s14"><span class="c29">4.</span><span class="c30">Maximization</span></p><p class="s6"><span class="c7"> We are trying to combine as many as enzymes as possible because more enzymes</span><span class="c7">’ </span><span class="c7">combination can produce more complicated reaction chains. So our purpose is to figure out the maximum number of Connectees that binding to a Connector.</span></p><p class="s6"><span class="c7"> So we intended to add more RS on the Connectors. On the one hand, more RSs mean we can bind more kinds of enzymes on one Connector. On the other hand ,we doubt the binding efficiency between Connectors and Connectees so more RSs can improve the possibility of Connectees binding to Connectors.</span></p><p class="s1"> The corresponding Connectors are<span class="c3"> pBlue</span><span class="c3">script II KS(+)_3_copy</span><span class="c3"> </span>and<span class="c3"> pBlue</span><span class="c3">script II KS(+)_5_copy</span>.</p><p class="s7 s19"> </p><p class="s11 s12"><span class="c28">●</span><span class="c3">pBluescript II KS(+)_3_copy </span></p><p class="s7 s10"><span class="c7">In order to add 3 copies of RS on Connectors, we firstly use restriction enzyme BstXI and BamHI to make a nick and replace the original sequence with one RS. Secondly, in the same way, we use restriction enzyme SalI and KpnI to add another RS.</span></p><p class="s7 s10"><span class="c7">The RS sequence is TTCGATATCAAGCT.</span></p><p class="s7 s10"><span class="c7">Here we have designed the new short sequence:</span></p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.208em;"><img src="images/image5.png" alt="image5.png" style="width:36.208em;"/></span></span></p><p class="s11 s24"><span class="c7">and</span></p><p class="s6"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.235em;"><img src="images/image6.png" alt="Macintosh HD:Users:chenming:Desktop:2.png" style="width:36.235em;"/></span></p><p class="s7 s10"><span class="c7">This is the what we get in the end:</span></p><p class="s6"><span class="c7">CT...CT</span><span class="c34">CCACCGCGGTGG</span><span class="c35">TTCGATATCAAGCT</span><span class="c36">GGATCC</span><span class="c7">CCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACC</span><span class="c37">GTCGAC</span><span class="c7">TTCGATATCAAGCT</span><span class="c38">GGTACC</span><span class="c7">CA</span><span class="c7">…</span><span class="c7">.AC</span></p><p class="s27"><span class="c3">pBluescript II KS(+)_3_copy</span><span class="c7"> can be used along with our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s2 s28"> </p><p class="s2 s29"><span class="c39">●</span><span class="c40">pBlue</span><span class="c40">script II KS(+)_5_copy</span></p><p class="s7 s10"><span class="c7">In order to add 4 more copies of RS on Connectors, we firstly use restriction enzyme BstXI and BamHI to make a nick and replace the original sequence with 4 consecutive repeats of RS. </span></p><p class="s7 s10"><span class="c7">The RS sequence is TTCGATATCAAGCT.</span></p><p class="s7 s10"><span class="c7">Here we have designed the new short sequence:</span></p><p class="s6"><span class="c7"><span style="display:inline-block;text-indent:0px;vertical-align:baseline;width:36.167em;"><img src="images/image7.png" alt="Macintosh HD:Users:chenming:Desktop:3.png" style="width:36.167em;"/></span></span></p><p class="s7 s10"><span class="c7">This is the what we get in the end:</span></p><p class="s6"><span class="c7">CT</span><span class="c7">…</span><span class="c7">CT</span><span class="c37">CCACCGCGGTGG<span id="b1"/></span><span class="c35">TTCGATATCAAGCT</span><span class="c41">TTCGATATCAAGCT</span><span class="c35">TTCGATATCAAGCT</span><span class="c41">TTCGATATCAAGCT</span><span class="c38">GGATCC</span><span class="c7">CC</span><span class="c7">…</span><span class="c7">AC</span></p><p class="s27"><span class="c3">pBluescript II KS(+)_5_copy</span><span class="c7"> can be used along with our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.</span></p><p class="s11 s30"> </p><p class="s11 s14"><span class="c29">5.</span><span class="c42">Inverse PCR</span></p><p class="s2 s5"><span class="c43">We used Inverse PCR to transform our plasmid into two kinds of Connectors:</span><span class="c40"> pBlue</span><span class="c40">script II KS(+) ScaI deletion</span><span class="c40"> </span><span class="c43">and </span><span class="c40">pBlue</span><span class="c40">script II KS(+) EcoRV deletion</span><span class="c43">.</span></p><p class="s1"><span class="c43">Inverse PCR method is using cyclic DNA (such as a plasmid) as the template, with two primers designed in a reverse direction to achieve completed PCR. In this way, by designing primers,we can introduce a mutation, insertion or deletion.</span></p><p class="s7 s10"><span class="c7">We used KOD-Plus-Mutagenesis Kit by TOYOBO.CO.LTD. </span></p><p class="s7 s31 c7">More details please click http://www.bio-toyobo.cn.</p></div> |