From 2009.igem.org

Latest revision as of 19:50, 21 October 2009

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.

sialic acid alpha2,6 organism

• 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

• 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)

• The differences of biosynthesis of sialic acid between the bacteria and mammals.[6]

Circuit Design

sialic acid synthesis

pTet R0040	RBS B0034	neu genes operon	B0015

sialic acid synthesis

pTet R0040	RBS B0034	sialyltransferase	B0015

• 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

sialic acid support-involved in transport of polysialic acid in E.coli K1(7953bp)

pTet R0040	RBS B0034	kps genes cluster	B0015

sialic acid support-involved in transport of polysialic acid in E.coli K1(1432bp)

pTet R0040	RBS B0034	kpsM、kpsT	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

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. 2009/09/11

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. 2009/09/16

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. 2009/09/18

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. 2009/10/01

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

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