Team:SDU-Denmark/notebook

From 2010.igem.org

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== Flagella ==
== Flagella ==
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* Studied inhibition and activation FlhD/C operon. <br>
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* Studied inhibition and activation of the FlhD/C operon. <br>
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* Found out that PskA is an important inhibitor, which also is part of ribosome synthesis.
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* We found out that PskA is an important inhibitor, which also is part of ribosome synthesis.
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<br>
<br>
== Flagella ==  
== Flagella ==  
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* Read up on the flagella regulon using following articles;
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* Read up on the flagella regulon using the following articles;
<br><br>
<br><br>
[http://ncbi.nlm.nih.gov/pmc/articles/PMC179437/pdf/1795602.pdf]  
[http://ncbi.nlm.nih.gov/pmc/articles/PMC179437/pdf/1795602.pdf]  
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Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60612-7344 <br><br>
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60612-7344 <br><br>
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<br>
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* Decided to follow the popular "trial-and-error" method and test what effects it will have to put the FlhDC operon after a constitutive promoter.
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* Decided to follow the popular "trial-and-error" method and tested what effects it will have to put the FlhD/C operon after a constitutive active promoter.
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* Ordered FlhD/C primers  
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* Ordered the FlhD/C primers.
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* Designed the biobrick ''K343000'' – ''FlhDCmut'' coding sequence <br><br>
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Jung, Kwang-Hwan, Spudich, Elena N., Trivedi, Vishwa D., Spudich, John L. <br>
Jung, Kwang-Hwan, Spudich, Elena N., Trivedi, Vishwa D., Spudich, John L. <br>
J. Bacteriol. 2001 183: 6365-6371 <br><br>
J. Bacteriol. 2001 183: 6365-6371 <br><br>
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* Contacted the original researchers on the mentioned papers for protein sequences and plasmids <br><br>
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* Contacted the original researchers on the mentioned papers for the protein sequences and plasmids <br><br>
== Retinal Production ==
== Retinal Production ==
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* Decided to atempt to make our bacteria synthesize retinal endogenously.  
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* Decided that we would try to make our bacteria synthesize retinal endogenously.  
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* Designed a new BioBrick around the ''ninaB'' gene from ''D. melanogaster'', that has been shown to produce a beta-carotene 15,15'-monooxygenase [http://www.jbc.org/content/275/16/11915.long][http://www.ncbi.nlm.nih.gov/pmc/articles/PMC14720/?tool=pubmed].  
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* Designed a new BioBrick around the ''ninaB'' gene from ''D. melanogaster'', which has shown to produce a beta-carotene 15,15'-monooxygenase [http://www.jbc.org/content/275/16/11915.long][http://www.ncbi.nlm.nih.gov/pmc/articles/PMC14720/?tool=pubmed].  
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* Isolated the gene sequence, and suggested biobricks for the coding region (K343001)
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* Isolated the gene sequence and suggested biobricks for the coding region (K343001)
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* Proposed four biobricks
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* Proposed three new biobricks
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''K343000'' – ''FlhDCmut'' coding sequence <br><br>
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''K343001'' - Sandboxed coding sequence from ''ninaB'' gene. <br><br>
''K343001'' - Sandboxed coding sequence from ''ninaB'' gene. <br><br>
''K343002'' - Sandboxed coding sequence from ''ninaB'' gene on one of the weaker Anderson promoters, with rbs and dual terminator. <br><br>
''K343002'' - Sandboxed coding sequence from ''ninaB'' gene on one of the weaker Anderson promoters, with rbs and dual terminator. <br><br>
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<br>  
<br>  
== Flagella ==
== Flagella ==
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* Extracted FlhDC master operon from ''E. coli'' strain MG1655 <br>
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* Extracted the FlhDC master operon from ''E. coli'' strain MG1655 <br>
* Showed pressence of pst1 site in FlhC <br>
* Showed pressence of pst1 site in FlhC <br>
* Introduced silent mutation into FlhC <br><br>
* Introduced silent mutation into FlhC <br><br>
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<br>
== Flagella ==
== Flagella ==
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* Made FlhDCmut <br>
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* Made the mutation the silent in the FlhDC operon <br>
== Retinal ==
== Retinal ==
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= Week 31 =
= Week 31 =
== Flagella ==
== Flagella ==
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* Showing prescence of pst1 site in native FlhDC and anscence of pst1 site in mutated FlhDC
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* Showing prescence of pst1 site in native FlhDC and absence of pst1 site in mutated FlhDC
= Week 32 =
= Week 32 =
== Retinal==
== Retinal==
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== Flagella ==
== Flagella ==
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* flhD/Cmut was made using new mutaion primers
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* FlhD/Cmut was made using new mutaion primers
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* flhD/Cmut was ligated into pSB1C3 and plasmid was transformed into ''E.coli'' TOP10 cells.
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* FlhD/Cmut was ligated into pSB1C3 and the plasmid was transformed into ''E.coli'' TOP10 cells.
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== Photosensor ==
== Photosensor ==
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<br>
== Flagella ==
== Flagella ==
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* FlhD/Cmut coding sequence (K343000)  was assembled with the double terminator (B0015) in pSB1AK3-B0015 and plasmid was transformed into ''E.coli'' TOP10 cells.
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* FlhD/Cmut coding sequence (K343000)  was assembled with the double terminator (B0015) in pSB1AK3 and the plasmid was transformed into ''E.coli'' TOP10 cells.
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== Photosensor ==
== Photosensor ==
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== Photosensor ==
== Photosensor ==
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* Results from sequencing of NpSopII-NpHtrII-StTar coding sequence (K343003) in pSB1C3 and NpSopII-NpHtrII-StTar coding sequence (K343003) + Promoter + RBS (J13002) in pSB1AK3 was OK
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* Results from sequencing of NpSopII-NpHtrII-StTar coding sequence (K343003) in pSB1C3 and NpSopII-NpHtrII-StTar coding sequence (K343003) + Promoter + RBS (J13002) in pSB1AK3 was OK.
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= Week 39 =
= Week 39 =
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<br>
== Retinal ==
== Retinal ==
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* NinaB coding sequence + Promoter + RBS (K343005) was assembled with the double terminator (B0015) in pSB1AK3-B0015 and plasmid was transformed into ''E.coli'' TOP10 cells.
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* NinaB coding sequence + Promoter + RBS (K343005) was assembled with the double terminator (B0015) in pSB1AK3 and the plasmid was transformed into ''E.coli'' TOP10 cells.
* pSB1C3-K343005 and pSB1A2-K274210 (CrtEBIY under constitutive promoter) was transformed into ''E.coli'' TOP10 and MG1655 cells
* pSB1C3-K343005 and pSB1A2-K274210 (CrtEBIY under constitutive promoter) was transformed into ''E.coli'' TOP10 and MG1655 cells
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== Retinal ==
== Retinal ==
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* NinaB coding sequence + Promoter + RBS (K343005) was assembled with the double terminator (B0015) in pSB1AK3-B0015 and transformed into ''E.coli'' TOP10 and MG1655 cells
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* NinaB coding sequence + Promoter + RBS (K343005) was assembled with the double terminator (B0015) in pSB1AK3 and transformed into ''E.coli'' TOP10 and MG1655 cells
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<br><br>  
= Week 41 =
= Week 41 =
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<br>
== Flagella ==
== Flagella ==
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* flhD/Cmut composite part (K343004) was ligated into pSB1C3 and pSB3K3 and plasmids were transformed into ''E.coli'' TOP10 and MG1655 cells.
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* FlhD/Cmut composite part (K343004) was ligated into pSB1C3 and pSB3K3 and plasmids were transformed into ''E.coli'' TOP10 and MG1655 cells.
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== Retinal ==
== Retinal ==
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== Flagella ==
== Flagella ==
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* Results of sequencing of flhD/Cmut composite part (K343004) in pSB1C3 was OK
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* Results of sequencing of flhD/Cmut composite part (K343004) in pSB1C3 was OK. Motility plates, flagella staining and phase microscopy was made.
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== Photosensor ==
== Photosensor ==
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* MG1655/pSB3T5-K343007 (NpSopII-NpHtrII-StTar composite part) was sequenced on THOR from [http://www.unisensor.dk/ Unisensor]
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* MG1655/pSB3T5-K343007 (NpSopII-NpHtrII-StTar composite part) was examined on THOR from [http://www.unisensor.dk/ Unisensor]
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Latest revision as of 02:11, 28 October 2010