Team:SDU-Denmark/notebook

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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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== 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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* 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 backteria synthesise 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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''BBa_K343000'' – FlhDCmut coding sequence <br><br>
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''BBa_K343001'' - Sandboxed coding sequence from ninaB gene. <br><br>
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''K343001'' - Sandboxed coding sequence from ''ninaB'' gene. <br><br>
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''BBa_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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''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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''BBa_K343003'' - Sandboxed coding sequence for the SopII-HtrII-Tsr fusion protein. <br><br>
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''K343003'' - Sandboxed coding sequence for the ''SopII-HtrII-Tsr'' fusion protein. <br><br>
''References:'' <br>
''References:'' <br>
5. ''Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal.'',von Lintig J, Vogt K., J Biol Chem. 2000 Apr 21;275(16):11915-20. <br><br>
5. ''Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal.'',von Lintig J, Vogt K., J Biol Chem. 2000 Apr 21;275(16):11915-20. <br><br>
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6. ''Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo'', Johannes von Lintig,* Armin Dreher, Cornelia Kiefer, Mathias F. Wernet, and Klaus Vogt, Proc Natl Acad Sci U S A. 2001 January 30; 98(3): 1130–1135. <br><br>
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6. ''Analysis of the blind ''Drosophila mutant'' ''ninaB'' identifies the gene encoding the key enzyme for vitamin A formation in vivo'', Johannes von Lintig,* Armin Dreher, Cornelia Kiefer, Mathias F. Wernet, and Klaus Vogt, Proc Natl Acad Sci U S A. 2001 January 30; 98(3): 1130–1135. <br><br>
== Modelling ==  
== Modelling ==  
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== 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==
* Showing, by uV-VIS absorbance spectre, that TOP10- and MG1655-cells containing the K274210 biobrick(camebridge part)show grater absorbance than the wild types.
* Showing, by uV-VIS absorbance spectre, that TOP10- and MG1655-cells containing the K274210 biobrick(camebridge part)show grater absorbance than the wild types.
= Week 33 =
= Week 33 =
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* No Progress Report
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No Progress Report
= Week 34 =
= Week 34 =
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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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== 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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== 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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= Week 41 =
= Week 41 =
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== 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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[[Image:Team SDU-Denmark-pearl logo.JPG |150px]]
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The iGEM team had a fun afternoon playing kindergarteners with the Students' Association and Louise made this lovely iGEM DENMARK logo out of plastic pearls. She didn't realize she had written 21010 instead of 2010 until the pearls were melted together though... rookie mistake. </p><br>
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[[Image:Team SDU-Denmark-pearl logo.JPG |150px]]
 
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The iGEM team had a fun afternoon with many other students when it was "kindergarden day" at the students assiciation on the BMB institute. There were cake, popcorn and juice. You could play different games, like Mario Cart on Nintendo 64 and Kalaha, some painted their own coffee mug and Louise made a plastic pearl iGEM DENMARK logo. She didn't realize she had written 21010 instead of 2010 untill she had melted the pearls together though...rookie mistake <br>
 
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Latest revision as of 02:11, 28 October 2010