Team:Tsinghua/Project2

From 2008.igem.org

(Difference between revisions)
(→PHA Project)
 
(13 intermediate revisions not shown)
Line 1: Line 1:
 +
<!--- The Mission, Experiments --->
 +
[[image:331.jpeg|center|]]
{| 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"
!align="center"|[[Team:Tsinghua|HOME]]
!align="center"|[[Team:Tsinghua|HOME]]
Line 10: Line 12:
|}
|}
== PHA Project ==
== PHA Project ==
-
[[Project Description ]]
+
&nbsp;&nbsp;&nbsp;[[Project Description ]]
-
Charts
+
-
Molecular Cloning
+
-
Results
+
 +
&nbsp;&nbsp;&nbsp;[[Designed Charts]]
 +
&nbsp;&nbsp;&nbsp;[[Molecular Cloning]]
 +
&nbsp;&nbsp;&nbsp;[[WetLab Results]]
-
DNA Template Resources:<br>
+
&nbsp;&nbsp;&nbsp;[[PHA Project Modeling]]
-
PhbCAB: PBHR68 Plasmid<br/>
+
-
Lac I: PET28a
+
-
CRE:
+
-
PpPhaP: Ralstonia eutropha H16 genome
+
-
PrPhaR:
+
-
<span style="color: Orange">Ralstonia eutropha H16 genome</span>
+
&nbsp;&nbsp;&nbsp;[[Reference]]
-
SRRz:
+
-
EGFP:
+
-
Primers:
 
-
(1) SRRz Primers
 
-
IGEM-ZouYLP-SRRZ-NcoI-for
 
-
5- ATCCATGGATGAAGATGCCAGAAAAACATGACCTGTTG-3
 
-
 
-
IGEM &nbsp;&nbsp;&nbsp -ZouYLP-SRRZ-rev-in
 
-
5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3
 
-
 
-
IGEM-ZouYLP-SRRZ-BamHI-rev-out
 
-
5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3
 
-
 
-
(2)
 
-
IGEM-ZouYLP-PP-PhaP-NotI-for
 
-
5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3
 
-
 
-
IGEM-ZouYLP-PP-PhaP-HindIII-rev
 
-
5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3
 
-
 
-
IGEM-ZouYLP-LacILVA-HindIII-for
 
-
5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3
 
-
 
-
IGEM-ZouYLP-LacILVA-rev-in
 
-
5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3
 
-
 
-
IGEM-ZouYLP-LacILVA-rev-PstI-out
 
-
5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3
 
-
 
-
IGEM-ZouYLP-CreT7ter—PstI-for
 
-
5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3
 
-
 
-
IGEM-ZouYLP-CreT7ter-rev-in
 
-
5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3
 
-
 
-
IGEM-ZouYLP-CreT7ter-EcoRI-rev-out
 
-
5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3
 
-
 
-
(3)
 
-
IGEM-ZouYLP-PR-PhaR-NotI-for
 
-
5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3
 
-
 
-
IGEM-ZouYLP-PR-PhaR-NdeI-rev
 
-
5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3
 
-
 
-
IGEM-ZouYLP-LRLacILP-for-in
 
-
5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3
 
-
 
-
IGEM-ZouYLP-LRLacILP-NdeI-for-out
 
-
5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3
 
-
 
-
IGEM-ZouYLP-LRLacILP-rev-in
 
-
5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3
 
-
 
-
IGEM-ZouYLP-LRLacILP-KpnI-rev-out
 
-
5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3
 
-
 
-
(4) EGFP
 
-
IGEMZouYLP-EGFP-for-KoZg-for
 
-
5-ccatgggcagcaagggcgaggagctgttc-3
 
-
 
-
IGEMZouYLP-EGFP-rev-in
 
-
5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3
 
-
 
-
IGEMZouYLP-EGFP-rev-out
 
-
5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3
 
-
 
-
IGEMZouYLP-EGFP-KoZg-rev
 
-
5-TTTTCGAGCTCGAATTCGG-3
 
-
 
-
 
-
Designed Molecule
 
-
 
-
[[Image:SRRz-Duet.jpg]]
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
[[Image:EGFP-Duet.jpg]]
 
-
 
-
 
-
 
-
Plasmid constructs:
 
-
1  pUCPhbCAB
 
-
 
 
-
2  pACYC(lacI+) DuetSRRz
 
-
 
 
-
3  pACYC(lacI+) DuetEGFP
 
-
 
 
-
4  pACYC(lacI-) DuetSRRz
 
-
 
 
-
5  pACYC(lacI-) DuetEGFP
 
-
 
-
6  pACYC(LacI-)DuetSRRzPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP
 
-
 
-
7  pACYC(LacI-)DuetEGFPPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP
 
-
 
-
 
-
 
-
Cloning Strategy:
 
-
(1) Fragment Preparation:
 
-
SRRz: PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;
 
-
    Purify the product and run PCR with primer SRRZ-NcoI-for and primer SRRZ-BamHI-rev-out;
 
-
 
-
EGFP: PCR with primer EGFP-for-KoZg-for and EGFP-rev-in;
 
-
    Purify the product and run PCR with primer EGFP-for-KoZg-for and primer EGFP-rev-out;
 
-
 
-
PpPhaP: PCR with primer PP-PhaP-NotI-for and PP-PhaP-HindIII-rev;
 
-
 
 
-
LacI(LVA): PCR with primer LacILVA-HindIII-for and LacILVA-rev-in;
 
-
    Purify the product and run PCR with primer LacILVA-HindIII-for and primer LacILVA-rev-PstI-out;
 
-
 
-
CRE(T7ter): PCR with primer CreT7ter—PstI-for and CreT7ter-rev-in
 
-
    Purify the product and run PCR with primer CreT7ter—PstI-for and  CreT7ter-EcoRI-rev-out;
 
-
 
-
PrPhaR: PCR with primer PR-PhaR-NotI-for and PR-PhaR-NdeI-rev;
 
-
 
-
LoxPLacILoxP: PCR with primer LRLacILP-for-in and LRLacILP-rev-in
 
-
    Purify the product and run PCR with primer LRLacILP-NdeI-for-out and primer LRLacILP-KpnI-rev-out.
 
-
 
-
(2) Fragment Fusion
 
-
Fuse LacI(LVA) and CRE(T7ter) together and amplify with the primer LacILVA-HindIII-for and CreT7ter-EcoRI-rev-out;
 
-
Fuse PrPhaR and LoxPLacILoxP together and amplify with the Primer PR-PhaR-NotI-for and LRLacILP-KpnI-rev-out.
 
-
We’ve tried to fuse more by PCR, but failed.
 
-
(3) Vector preparation
 
-
We choose pACYCDuet-1 as our second vector while PhbCAB operon is cloned into PUC18.
 
-
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.
 
-
(4) Cut and Insert
 
-
The restriction enzymes are chosen as following:
 
-
Fragment Enzyme A Enzyme B
 
-
SRRz/EGFP NcoI BamHI
 
-
PpPhaP NotI HindIII
 
-
LacI(LVA)Cre(T7ter) HindIII SacI
 
-
PrPhaRLoxPLacILoxP NotI KpnI
 
-
 
-
== Reference ==
 
-
 
-
 
-
 
-
 
-
1
 
-
A sensitive, viable-colony staining method using Nile red
 
-
for direct screening of bacteria that accumulate polyhydroxyalkanoic
 
-
acids and other lipid storage compounds. Patricia Spiekermann · Bernd H. A. Rehm ·
 
-
Rainer Kalscheuer · Dirk Baumeister ·
 
-
Alexander Steinbüchel. Arch Microbiol (1999) 171:73–80
 
-
 
-
'''This literature explains how pBHR68 works and how PHA granules are detected.'''
 
-
 
-
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
 
-
Vol. 89,  No. 4, 307-311.  2000
 
-
 
-
'''This literature indicates how PHA sysnthesis is realized in Escherichia coli.'''
 
-
 
-
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
 
-
 
-
'''This literature introduced a method to release PHB granules from Escherichia coli.'''
 
-
 
-
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)
 
-
 
-
'''This literature introduced phage lambda lytic gene into Escherichia coli strains.'''
 
-
 
-
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
 
-
 
-
'''This literature illustrates how PhaR works in the PHB synthesis regulation.'''
 
-
 
-
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.
 
-
 
-
'''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"
Line 218: Line 36:
!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
|}
|}
 +
<!--- The Mission, Experiments --->
 +
[[image:06.jpeg|center|]]

Latest revision as of 04:30, 30 October 2008

331.jpeg
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
06.jpeg
Retrieved from "http://2008.igem.org/Team:Tsinghua/Project2"