Team:Tsinghua/Project2

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== PHA Project ==
== PHA Project ==
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&nbsp;&nbsp;&nbsp;[[Project Description ]]
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&nbsp;&nbsp;&nbsp;[[Designed Charts]]
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DNA Template Resources:
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&nbsp;&nbsp;&nbsp;[[Molecular Cloning]]
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PhbCAB: PBHR68 Plasmid
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Lac I: PET28a
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CRE:
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PpPhaP: Ralstonia eutropha H16 genome
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PrPhaR: Ralstonia eutropha H16 genome
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SRRz:
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EGFP:
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Primers:
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(1) SRRz Primers
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IGEM-ZouYLP-SRRZ-NcoI-for
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5- ATCCATGGATGAAGATGCCAGAAAAACATGACCTGTTG-3
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IGEM-ZouYLP-SRRZ-rev-in
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5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3
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IGEM-ZouYLP-SRRZ-BamHI-rev-out
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5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3
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(2)
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IGEM-ZouYLP-PP-PhaP-NotI-for
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5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3
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IGEM-ZouYLP-PP-PhaP-HindIII-rev
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5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3
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IGEM-ZouYLP-LacILVA-HindIII-for
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5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3
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IGEM-ZouYLP-LacILVA-rev-in
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5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3
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IGEM-ZouYLP-LacILVA-rev-PstI-out
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5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3
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IGEM-ZouYLP-CreT7ter—PstI-for
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5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3
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IGEM-ZouYLP-CreT7ter-rev-in
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5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3
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IGEM-ZouYLP-CreT7ter-EcoRI-rev-out
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5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3
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(3)
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IGEM-ZouYLP-PR-PhaR-NotI-for
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5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3
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IGEM-ZouYLP-PR-PhaR-NdeI-rev
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5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3
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IGEM-ZouYLP-LRLacILP-for-in
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5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3
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IGEM-ZouYLP-LRLacILP-NdeI-for-out
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5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3
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IGEM-ZouYLP-LRLacILP-rev-in
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5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3
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IGEM-ZouYLP-LRLacILP-KpnI-rev-out
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5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3
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(4) EGFP
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IGEMZouYLP-EGFP-for-KoZg-for
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5-ccatgggcagcaagggcgaggagctgttc-3
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IGEMZouYLP-EGFP-rev-in
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5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3
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IGEMZouYLP-EGFP-rev-out
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5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3
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IGEMZouYLP-EGFP-KoZg-rev
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5-TTTTCGAGCTCGAATTCGG-3
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Designed Molecule
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[[Image:SRRz-Duet.jpg]]
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[[Image:EGFP-Duet.jpg]]
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Plasmid constructs:
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1  pUCPhbCAB
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2  pACYC(lacI+) DuetSRRz
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3  pACYC(lacI+) DuetEGFP
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4  pACYC(lacI-) DuetSRRz
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5  pACYC(lacI-) DuetEGFP
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6  pACYC(LacI-)DuetSRRzPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP
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7  pACYC(LacI-)DuetEGFPPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP
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Cloning Strategy:
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(1) Fragment Preparation:
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SRRz: PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;
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    Purify the product and run PCR with primer SRRZ-NcoI-for and primer SRRZ-BamHI-rev-out;
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EGFP: PCR with primer EGFP-for-KoZg-for and EGFP-rev-in;
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    Purify the product and run PCR with primer EGFP-for-KoZg-for and primer EGFP-rev-out;
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PpPhaP: PCR with primer PP-PhaP-NotI-for and PP-PhaP-HindIII-rev;
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LacI(LVA): PCR with primer LacILVA-HindIII-for and LacILVA-rev-in;
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    Purify the product and run PCR with primer LacILVA-HindIII-for and primer LacILVA-rev-PstI-out;
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CRE(T7ter): PCR with primer CreT7ter—PstI-for and CreT7ter-rev-in
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    Purify the product and run PCR with primer CreT7ter—PstI-for and  CreT7ter-EcoRI-rev-out;
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PrPhaR: PCR with primer PR-PhaR-NotI-for and PR-PhaR-NdeI-rev;
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LoxPLacILoxP: PCR with primer LRLacILP-for-in and LRLacILP-rev-in
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    Purify the product and run PCR with primer LRLacILP-NdeI-for-out and primer LRLacILP-KpnI-rev-out.
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(2) Fragment Fusion
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Fuse LacI(LVA) and CRE(T7ter) together and amplify with the primer LacILVA-HindIII-for and CreT7ter-EcoRI-rev-out;
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Fuse PrPhaR and LoxPLacILoxP together and amplify with the Primer PR-PhaR-NotI-for and LRLacILP-KpnI-rev-out.
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We’ve tried to fuse more by PCR, but failed.
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(3) Vector preparation
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We choose pACYCDuet-1 as our second vector while PhbCAB operon is cloned into PUC18.
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Since lacI is one of the elements we also used in our own system, we knocked out the lacI coding region in pACYCDuet-1 successfully.
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(4) Cut and Insert
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The restriction enzymes are chosen as following:
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Fragment Enzyme A Enzyme B
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SRRz/EGFP NcoI BamHI
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PpPhaP NotI HindIII
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LacI(LVA)Cre(T7ter) HindIII SacI
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PrPhaRLoxPLacILoxP NotI KpnI
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== Reference ==
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1
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A sensitive, viable-colony staining method using Nile red
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for direct screening of bacteria that accumulate polyhydroxyalkanoic
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acids and other lipid storage compounds. Patricia Spiekermann · Bernd H. A. Rehm ·
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Rainer Kalscheuer · Dirk Baumeister ·
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Alexander Steinbüchel. Arch Microbiol (1999) 171:73–80
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'''This literature explains how pBHR68 works and how PHA granules are detected.'''
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2  Construction and Selection of  the  Novel  Recombinant Escherichia cob Strain  for Poly(Hydroxybutyrate)  Production.HUIMIN YU,JIN  YIN,HONGQI LI,SHENGLI  YANG,AND  ZHONGYAO. JOURNALOF BIOSCIENCEAND BIOENGWEERING
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Vol. 89,  No. 4, 307-311.  2000
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'''This literature indicates how PHA sysnthesis is realized in Escherichia coli.'''
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3 Spontaneous liberation of intracellular polyhydroxybutyrate granules in Escherichia coli.Il Lae Jung, Ki Heon Phyo, Kug Chan Kim, Hyo Kook Park, In Gyu Kim.Research in Microbiology 156 (2005) 865–873
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'''This literature introduced a method to release PHB granules from Escherichia coli.'''
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4 Simultaneous Expression of Vitreoscilla Globin Gene and Lytic Genes of Phage lambda Novel RecombinantEscherichia Coli Used for Production of PHB.YU Huimin,SHI Yue,YIN Jin,and SHEN Zhongyao.ChineseJ.ofChem.Eug，(4)407一411(2001)
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'''This literature introduced phage lambda lytic gene into Escherichia coli strains.'''
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&nbsp;&nbsp;&nbsp;[[WetLab Results]]
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5 Autoregulator Protein PhaR for Biosynthesis of Polyhydroxybutyrate [P(3HB)] Possibly Has Two Separate Domains That Bind to the Target DNA and P(3HB): Functional Mapping of Amino Acid Residues Responsible for DNA Binding.Miwa Yamada,Koichi Yamashita,Akiko Wakuda,Kazuyoshi Ichimura,Akira Maehara,Michihisa Maeda,and Seiichi Taguchi.JOURNAL  OF  BACTERIOLOGY, Feb. 2007, p. 1118–1127
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&nbsp;&nbsp;&nbsp;[[PHA Project Modeling]]
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'''This literature illustrates how PhaR works in the PHB synthesis regulation.'''
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&nbsp;&nbsp;&nbsp;[[Reference]]
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6 Influence  of  homologous  phasins  (PhaP)  on PHA  accumulation  and  regulation  of  their expression  by  the  transcriptional  repressor PhaR  in  Ralstonia  eutropha  H16.Markus  Po¨tter,  Helena  Muller  and  Alexander  Steinbuchel.Microbiology  (2005),  151,  825–833.
 
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'''This literature illustrates in the interaction of PhaP and PhaR.'''
 
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
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!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
|}
|}
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<!--- The Mission, Experiments --->
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[[image:06.jpeg|center|]]

Latest revision as of 04:30, 30 October 2008

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HOME Team Project 1 Project 2 Parts Modelling Notebook Doodle Board

PHA Project

   Project Description

   Designed Charts

   Molecular Cloning

   WetLab Results

   PHA Project Modeling

   Reference


HOME Team Project 1 Project 2 Parts Modelling Notebook Doodle Board
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