"

 

From 2009.igem.org

You do not have permission to edit this page, for the following reason:

You can view and copy the source of this page:

[[Image:NYMU Wt-rece.png|NYMU Wt-rece.png]] {{:Team:NYMU-Taipei/Header}} = Motivation & Design Background = Influenza has been a pathogen infecting the human body since centuries ago. Recently, H1N1 became prevalent around the world, so we hope our design could make some contribution towards fighting against it. Among so many mechanisms which play important role in the infection process of Influenza virus, sialic acid is the most important one that gets involved in this infection process. There are many different types of sialic acid exist in different species[1]. For example, on the human cell membranes are alpha-2,6-sialic acid that mostly play an important role in the infection of virus. Others such as alpha-2,8-sialic acid is essential for the pathogen of some other kind of bacteria like ''E.coli K1''. The main difference is the enzyme that is used in the process of producing the sialic acid. Sialyltransferase is the enzyme that changes alpha-2,8-sialic acid to alpha-2,6-sialic acid[3][4][5]. Which is aimed at transferring NeuNAc, the substrate that is made from other two enzymes--UDP-GlcNAc-2-Epimerase and NeuNAc Synthase onto cell membrane’s proteins or lipid in the position of alpha- 2,6 or alpha-2,8 sialic acid. Several animal viruses, including the influenza virus, attach to their host cells through interactions with oligosaccharides displayed on the host cell surface. The HA (hemagglutinin) protein is essential for influenza viruses to enter and infect healthy body cells. The residues of Neu5Ac (a type of sialic acid) is situated at the ends of the oligosaccharide chains of many plasma glycoproteins. The oligosaccharide ligands that bind to influenza viruses' HA is Neu5Ac(alpha2-6)Gal(beta1-4)Glc. We try to modify the lipopolysaccharide on the membrane of bacteria because there is already alpha-2,8-sialic acid at the ends of the oligosaccharide chains of plasma glycoproteins. The technique we use is the synthesis of glycoprotein.[2] =Materials and Methods= The neuS gene in ''E.coli K1'' is coding for alpha-2,8-sialyltransferase, and there is also some kinds of photobacteria that have genes coding for alpha-2,6-sialyltransferase. We performed PCR to clone the neuD, neuB, neuA, neuC, neuE genes, from one operon in ''E.coli K1'', and are coding for enzymes that are in charge of sialic acid synthesis. On the other hand, we do PCR for cloning sialyltransferase gene from photobacteria. After performing these PCR, and make sure all the PCR products' length are correct, doing restriction enzyme digestion and then ligation, finally transformation are the next work. We use colony PCR to make sure the colony is what we want. We find the appropriate promoter(which can promote quantitative expression of genes)and ribosome binding site to ligate with the coding sequences-neu genes. =====<br>sialic acid alpha2,6 organism<br>===== *''Photobacterium damselae'' =====sialic acid alpha2,8 organism===== *''Escherichia coli O1:K1'' / APEC with [Complete proteome] in NCBI and ENSEMBL =The comparison of the process of sialic acid synthesis between bacteria and mammals= [[File:NYMU Sialic acid synthesis among bacteria.jpg]] *This picture shows the catabolism of sialic acid among bacteria.[7] *Sialic acid biosynthesis genes(neuA,neuB,neuC)among the bacteria encoding the Nan cluster(the genes encoding NanA, NanB, NanE are clustered together) [[File:NYMU Sialic acid-1.jpg]] *The differences of biosynthesis of sialic acid between the bacteria and mammals.[6] =Circuit Design= {{:Team:NYMU-Taipei/Part4|'''sialic acid synthesis'''|= |P|R0040|pTet|= |R|B0034|RBS|= |C|neu genes operon||= |T|B0015||=}} {{:Team:NYMU-Taipei/Part4|'''sialic acid synthesis'''|= |P|R0040|pTet|= |R|B0034|RBS|= |C|sialyltransferase||= |T|B0015||=}} '''neu genes cluster descriptions:''' *'''neuD'''(623bp)--sugar O-acyltransferase, sialic acid O-acetyltransferase NeuD family *'''neuB'''(1040bp)--N-acetylneuraminate synthase *'''neuA'''(1256bp)--CMP-N-acetylneuraminic acid synthetase *'''neuC'''(1175bp)--UDP-N-acetyl-D-glucosamine 2-epimerase,UDP-hydrolysing *'''neuE'''(1175bp)--polysialic acid biosynthesis protein *'''sialyltransferase 0160'''(2743bp)--Photobacterium damsela gene for sialyltransferase 0160 <br>{{:Team:NYMU-Taipei/Part4|'''sialic acid support-involved in transport of polysialic acid in E.coli K1'''(7953bp)|=<br> |P|R0040|pTet|= |R|B0034|RBS|= |C|kps genes cluster||= |T|B0015||=}} <br>{{:Team:NYMU-Taipei/Part4|'''sialic acid support-involved in transport of polysialic acid in E.coli K1'''(1432bp)|=<br> |P|R0040|pTet|= |R|B0034|RBS|= |C|kpsM、kpsT||= |T|B0015||=}} =Gene Sequences= === [http://www.ncbi.nlm.nih.gov/nuccore/454079?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Escherichia coli polysialic acid gene cluster region 2 (neuD and neuB) genes, complete cds] === atgagtaa aaagttaata 121 atatttggtg cgggtggttt ttcaaaatct ataattgaca gcttaaatca taaacattac 181 gagttaatag gatttatcga taaatataaa agtggttatc atcaatcata tccaatatta 241 ggtaatgata ttgcagacat cgagaataag gataattatt attattttat tgggataggc 301 aaaccatcaa ctaggaagca ctatttaaac atcataagaa aacataatct acgcttaatt 361 aacattatag ataaaactgc tattctatca ccaaatatta tactgggtga tggaattttt 421 attggtaaaa tgtgtatact taaccgtgat actagaatac atgatgccgt tgtaataaat 481 actaggagtt taattgaaca tggtaatgaa ataggctgct gtagcaatat ctctactaat 541 gttgtactta atggtgatgt ttctgttgga gaagaaactt ttgttggtag cgtgactgtt 601 gtaaatggcc agttgaagct aggctcaaag agtattattg gttctgggtc ggttgtaatt 661 agaaatatac caagtaatgt tgtagttgct gggactccaa caagattaat tagggggaat 721 gaatgagtaa tatatatatc gttgctgaaa ttggttgcaa ccataatggt agtgttgata 781 ttgcaagaga aatgatatta aaagccaaag aggccggtgt taatgcagta aaattccaaa 841 catttaaagc tgataaatta atttcagcta ttgcacctaa ggcagagtat caaataaaaa 901 acacaggaga attagaatct cagttagaaa tgacaaaaaa gcttgaaatg aagtatgacg 961 attatctcca tctaatggaa tatgcagtca gtttaaattt agatgttttt tctacccctt 1021 ttgacgaaga ctctattgat tttttagcat ctttgaaaca aaaaatatgg aaaatccctt 1081 caggtgagtt attgaattta ccgtatcttg aaaaaatagc caagcttccg atccctgata 1141 agaaaataat catatcaaca ggaatggcta ctattgatga gataaaacag tctgtttcta 1201 tttttataaa taataaagtt ccggttggta atattacaat attacattgc aatactgaat 1261 atccaacgcc ctttgaggat gtaaacctta atgctattaa tgatttgaaa aaacacttcc 1321 ctaagaataa cataggcttc tctgatcatt ctagcgggtt ttatgcagct attgcggcgg 1381 tgccttatgg aataactttt attgaaaaac atttcacttt agataaatct atgtctggcc 1441 cagatcattt ggcctcaata gaacctgatg aactgaaaca tctttgtatt ggggtcaggt 1501 gtgttgaaaa atctttaggt tcaaatagta aagtggttac agcttcagaa aggaagaata 1561 aaatcgtagc aagaaagtct attatagcta aaacagagat aaaaaaaggt gaggtttttt 1621 cagaaaaaaa tataacaaca aaaagacctg gtaatggtat cagtccgatg gagtggtata 1681 atttattggg taaaattgca gagcaagact ttattccaga tgaattaata attcatagcg 1741 aattcaaaaa tcagggggaa taa === [http://www.ncbi.nlm.nih.gov/nuccore/146943?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum E.coli CMP-N-acetylneuraminic acid synthetase (neuA) gene, complete cds] === atgagaa caaaaattat tgcgataatt ccagcccgta 61 gtggatctaa agggttgaga aataaaaatg ctttgatgct gatagataaa cctcttcttg 121 cttatacaat tgaagctgcc ttgcagtcag aaatgtttga gaaagtaatt gtgacaactg 181 actccgaaca gtatggagca atagcagagt catatggtgc tgattttttg ctgagaccgg 241 aagaactagc aactgataaa gcatcatcat ttgaatttat aaaacatgcg ttaagtatat 301 atactgatta tgagagcttt gctttattac aaccaacttc accctttaga gattcgaccc 361 atattattga ggctgtaaag ttatatcaaa ctttagaaaa ataccaatgt gttgtttctg 421 ttactagaag caataagcca tcacaaataa ttagaccatt agatgattac tcgacactgt 481 ctttttttga ccttgattat agtaaatata atcgaaactc aatagtagaa tatcatccga 541 atggagctat atttatagct aataagcagc attatcttca tacaaagcat ttttttggtc 601 gctattcact agcttatatt atggataagg aaagctcttt agatatagat gatagaatgg 661 atttcgaact tgcaattacc attcagcaaa aaaaaaatag acaaaaaatt gacctttatc 721 aaaacataca taatagaatc aatgagaaac gaaatgaatt tgatagtgta agtgatataa 781 ctttaattgg acactcgctg tttgattatt gggacgtaaa aaaaataaat gatatagaag 841 ttaataactt aggtatcgct ggtataaact cgaaggagta ctatgaatat attattgaga 901 aagagctgat tgttaatttc ggagagtttg ttttcatctt ttttggaact aatgatatag 961 ttgttagtga ttggaaaaaa gaagacacat tgtggtattt gaagaaaaca tgccagtata 1021 taaagaagaa aaatgctgca tcaaaaattt atttattgtc ggttcctcct gtttttgggc 1081 gtattgatcg agataataga ataattaatg atttaaattc ttatcttcga gagaatgtag 1141 attttgcgaa gtttattagc ttggatcacg ttttaaaaga ctcttatggc aatctaaata 1201 aaatgtatac ttatgatggc ttacatttta atagtaatgg gtatacagta ttagaaaacg 1261 aaatagcgga gattgttaaa tga === [http://www.ncbi.nlm.nih.gov/nuccore/146945?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum E.coli protein p7 (neu C) gene, complete cds] === atgaaaaaaa tattatacgt aactggatct 181 agagctgaat atggaatagt tcggagactt ttgacaatgc taagagaaac tccagaaata 241 cagcttgatt tggcagttac aggaatgcat tgtgataatg cgtatggaaa tacaatacat 301 attatagaac aagataattt taatattatc aaggttgtgg atataaatat caatacaact 361 tcacatactc acattctcca ttcaatgagt gtttgcctca attcgtttgg tgattttttt 421 tcaaataaca catatgatgc ggttatggtt ttaggcgata gatatgaaat attttcagtc 481 gctatcgcag catcaatgca taatattcca ttaattcata ttcatggtgg tgaaaagaca 541 ttagctaatt atgatgagtt tattaggcat tcaattacta aaatgagtaa actccatctt 601 acttctacag aagagtataa aaaacgagta attcaactag gtgaaaagcc tggtagtgtg 661 tttaatattg gttctcttgg tgcagaaaat gctctttcat tgcatttacc aaataagcag 721 gagttggaac taaaatatgg ttcactgtta aaacggtact ttgttgtagt attccatcct 781 gaaacacttt ccacgcagtc ggttaatgat caaatagatg agttattgtc agcgatttct 841 ttttttaaaa atactcacga ctttattttt attggcagta acgctgacac tggttctgat 901 ataattcaga gaaaagtaaa atatttttgc aaagagtata agttcagata tttgatttct 961 attcgttcag aagattattt ggcaatgatt aaatactctt gtgggctaat tgggaactcc 1021 tcctctggtt taattgaggt tccatcttta aaagttgcaa caattaacat tggtgatagg 1081 cagaaaggcc gtgttcgtgg agccagtgta atagatgtac ccgttgaaaa aaatgcaatc 1141 gtcagaggga taaatatatc tcaagatgaa aaatttatta gtgttgtaca gtcatctagt 1201 aatccttatt ttaaagaaaa tgctttaatt aatgctgtta gaattattaa ggattttatt 1261 aaatcaaaaa ataaagatta caaagatttt tatgacatcc cggaatgtac caccagttat 1321 gactag === [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&RID=6E5CM00T01R&log%24=nucltop&blast_rank=8&list_uids=62866977 Escherichia coli polysialic acid biosynthesis protein (neuE) gene, complete cds] === 61 atgacatccc ggaatgtacc accagttatg actagaaaaa aagtgctttg ttttgtcttt 121 cgttatgatt ctcatttttt agctttgaaa aatatttttg agcagataga tgttgattca 181 tatgatttat ttttttgctg cttggataat tctctacaag agtttgtaaa aaaaaattta 241 gatgaaaaga tagttgtatt ctatcctgat gactttgttt gttttttcac ttttattaat 301 attgagttta ttttttgttc aacaggaggg aaggaccttc atgaaattgt taatactgta 361 agaacaaaag ataccataat tatatcttgt tttcctggca ttgtccttac ttctcagata 421 gaagctttta tttcaaaatc taatagtcac tatttactta ttaactcccc aaaagatatt 481 aaaacgtata aaaaaatttg taaaataata ggggttcctt ttaatggaat tctttttggt 541 ccaccatgga ttaaaaatgt caatatcaat gcaaaaagtg agaattcttg tcttatcgtt 601 gatcaagtta atgaaccctt gacgccaata aagaggatag aatatgcacg ttttttgatt 661 agagtaattc agaaacatcc acatatgaat tttattttta aaactcgaaa tcctcttata 721 tcaccagact caattgtttt tgatattaag gaatacattg aacgcttcga tttgaaaaat 781 ataacattta gcgatgataa tattgattct ttaatttcta aagttgaata ttgtattaca 841 atatcttctt cggtcgcaat atattgtctg gctaataaaa ttaaggttta tttaataaat 901 ggatttaatc atacttgcaa tggacaatgt tatttttcaa gatctggact tattgttgac 961 tataataagt ttaattttaa acacattcca cgtattaaaa aaaaatggat ggaggagaac 1021 ttttattact ctagggatat tcaacataag attttgaatg atattttaaa aatgccgtca 1081 aatgttaatg ttaggacttt tggaattaaa agatctacat tgattatatt gtttttgatc 1141 ttttttaatt tctttttctc attaggacca aaaaaaataa aaacattgaa aaaaatccat 1201 aaagttttat taaggtataa gaaagatgat atttga primer design: gaattcgcggccgcttctag atgagtaaaaagttaataatatttggtg Length=20+28, Tm=51, GC%=21% ctgcagcggccgctactagta tcaaatatcatctttcttataccttaat Length=21+28, Tm=51, GC%=21% === [http://www.ncbi.nlm.nih.gov/nuccore/2988378 Photobacterium damsela gene for sialyltransferase 0160, complete cds] === atgaa gaaaatactg acagttctat 421 ctatttttat tctttcagcg tgtaatagtg acaataccag cttgaaagaa acggtaagct 481 ctaattctgc agatgtagta gaaacagaaa cttaccaact gacaccgatt gatgctccta 541 gctctttttt atctcattct tgggagcaaa catgtggcac acctatcttg aatgaaagtg 601 acaagcaagc gatatctttt gattttgttg ctccagagtt aaagcaagat gaaaagtatt 661 gttttacttt taaaggtatt acaggcgatc ataggtatat cacaaataca acattaactg 721 ttgttgcacc tacgctagaa gtttacatcg atcatgcatc cttaccatcg ctacagcagc 781 ttatccacat tattcaagca aaagatgaat acccaagtaa tcaacgtttt gtctcttgga 841 agcgtgtaac tgttgatgct gataatgcca ataagttaaa cattcatact tatccattaa 901 aaggcaataa tacctcacca gaaatggtgg cagcgattga tgagtatgct cagagcaaaa 961 atcgattgaa tatagagttc tatacaaata cagctcatgt ttttaataat ttaccaccta 1021 ttattcaacc tttatataat aacgagaagg tgaaaatttc tcatattagt ttgtatgatg 1081 atggttcttc tgaatatgta agtttatatc aatggaaaga tacaccaaat aagatagaaa 1141 cattagaagg tgaagtatcg cttcttgcta attatttagc aggaacatct ccggatgcac 1201 caaaaggaat gggaaatcgt tataactggc ataaattata tgacactgat tattactttt 1261 tgcgcgaaga ttaccttgac gttgaagcaa acctacatga tttacgtgat tatttaggct 1321 cttccgcaaa gcaaatgcca tgggatgaat ttgctaaatt atctgattct cagcaaacac 1381 tatttttaga tattgtgggt tttgataaag agcaattgca acaacaatat tcacaatccc 1441 cactaccaaa ctttattttt accggcacaa caacttgggc tgggggggaa acgaaagagt 1501 attatgctca gcaacaagta aatgtgatta ataatgcgat caatgaaact agcccttatt 1561 atttaggtaa agactacgat ctatttttca aggggcatcc tgctggtggc gttattaacg 1621 acatcattct tggaagcttc cctgatatga tcaatattcc agccaagatt tcatttgagg 1681 tcttgatgat gacggatatg ttgcctgata cagtagctgg tattgcgagc tctctgtact 1741 tcacaattcc tgccgataaa gttaatttta ttgtatttac ttcatctgac actattactg 1801 atcgtgaaga ggctcttaaa tcaccattag tacaagtgat gctaacgttg ggtattgtta 1861 aagaaaaaga tgttctgttc tgggctgatc ataaagtaaa ctcgatggaa gttgccattg 1921 atgaagcctg tactcggatc attgcaaagc gacaaccaac cgcgagtgat ttacgcttgg 1981 ttattgctat tatcaaaaca attactgatc ttgagcgtat tggcgatgtg gcagaaagta 2041 ttgctaaagt cgcattagag agctttagta ataagcaata taacctattg gtttctttag 2101 aatctcttgg ccagcatacg gttcgaatgc tgcatgaggt gttagatgcg tttgctcgta 2161 tggatgttaa agccgcaata gaagtgtacc aagaagatga tcgaattgat caagagtatg 2221 agtcgatagt cagacagcta atggcccata tgatggaaga tccaagctca attcctaatg 2281 taatgaaagt gatgtgggcg gcacgttcta ttgagcgagt gggtgatcgc tgtcaaaaca 2341 tttgtgagta cattatctac tttgtgaagg gtaaagacgt tcgccatacc aaaccagatg 2401 attttggtac tatgctcgat taa =Experiment results= {| border="1" | [[image:NYMU UVP02637.PNG|640px]] ||'''Figure1.''' The first time we tried to clone neu's gene operon using Taq. Except markers, lane1 should be the PCR product of neu genes operon in E.coli K1 using Taq, but it seems to be failed. Only lane2, the positive control-hflB genes in E.coli K1(1934bp)was successful. Lane3 is the negative control, and there is no contamination. <br>2009/09/11<br> |} {| border="1" | [[image:NYMU UVP02655.PNG|640px]]||'''Figure2.''' It was failed to clone the neuD,neuB,neuA,neuC,neuE gene operon in ''E.coli K1'' even using gradient PCR. The reason may be that the sequences are too complicated to do cloning correctly. The strange bands in gel may be contamination because six of the PCR products using different temperatures protocol are neu gene operon,but the bands are not in the same position. We thought that only one lane of the PCR product of neu gene operon was successful, but we cannot make sure. Maybe this gel is not good. <br>2009/09/16<br> |} {| border="1" |[[image:NYMU Gel-2.png|640px]]||'''Figure3.''' It was the third time we tried to clone neu genes operon using both Taq(lane1,2,3) and Pfu enzymes(lane5,6,7). Unfortunately, we still failed because only positive control appeared. Lane4 and lane8 are the negative control. <br>2009/09/18<br> |} {| border="1" | [[image:NYMU UVP02663.PNG|640px]] ||'''Figure4.''' We still failed to clone these neu genes operon by using Pfu. The positive control-hflB genes was also not cloned successfully. We can see many primers under the gel. Maybe it was because the enzyme-Pfu was not good. <br>2009/10/01<br> |} =References= *[1]Tatsunori Iwataa, Kaori Fukuzawab, Katsuhisa Nakajimac, Sachiko Aida-Hyugaji d,Yuji Mochizukie,f, HirofumiWatanabea,f, Shigenori Tanakaa,f,:'''Theoretical analysis of binding specificity of influenza viral hemagglutinin to avian and human receptors based on the fragment molecular orbital method.'''Computational Biology and Chemistry 32 (2008) 198–211 *[2]Hai Yu, Harshal Chokhawala, Shengshu Huang, and Xi Chen:'''One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities.'''Nat Protoc. 2006 ; 1(5): 2485–2492. doi:10.1038/nprot.2006.401. *[3]Hai Yu, Shengshu Huang, Harshal Chokhawala,Mingchi Sun, Haojie Zheng, and Xi Chen:'''Highly Efficient Chemoenzymatic Synthesis of Naturally Occurring and Non-Natural a-2,6-Linked Sialosides: A P. damsela a-2,6-Sialyltransferase with Extremely Flexible Donor–Substrate Specificity.'''DOI: 10.1002/anie.200600572 *[4]Chin-Fen Teo,a, c Tzann-Shun Hwang,a, b, c Pei-Heng Chen,a Chih-Hung Hung,a Heau-Shan Gao,a, d Lee-Shang Chang,a, d Chun-Hung Lina, b:'''Synthesis of Sialyl TN Glycopeptides -- Enzymatic Sialylation by a2,6-Sialyltransferase from Photobacterium damsela.'''Adv. Synth. Catal. 2005, 347, 967 –972 *[5]Anne Harduin-Lepers1,2,4, Rosella Mollicone3,4,Philippe Delannoy2,4, and Rafael Oriol3,4:'''The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.'''Glycobiology 2005 15(8):805-817; doi:10.1093/glycob/cwi063 *[6]Bioorganic Chemistry 33 (2005) 216–228 *[7]Salvador Almagro-Moreno1,2 and E Fidelma Boyd1:'''Insights into the evolution of sialic acid catabolism among bacteria.'''BMC Evolutionary Biology 2009, 9:118doi:10.1186/1471-2148-9-118

Return to Team:NYMU-Taipei/Project/Receptor/Sialic Acid.

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Privacy policy
• Disclaimers