Week 26

Flagella

• Studied inhibition and activation FlhD/C operon.
  
• Found out that PskA is an important inhibitor, which also is part of ribosome synthesis.

Phototaxis

• Ordered proteorhodopsin biobrick [http://partsregistry.org/Part:BBa_I711040 I711040]
  
• Did research on the light-sensitive proteorhodopsin ionpump.

Week 27

Flagella

• Read up on the flagella regulon using following articles;

[http://ncbi.nlm.nih.gov/pmc/articles/PMC179437/pdf/1795602.pdf] Cell Cycle Regulation of Flagellar Genes
Birgit M. Prüß and Philip Matsumura
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60612-7344

[http://ncbi.nlm.nih.gov/pubmed/7961507]
The FlhD/FlhC Complex, a Transcriptional Activator of the Escherichia coli Flagellar Class II Operons
Xiaoying Liu and Philip Matsumura
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60612-7344

• 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.
• Ordered FlhD/C primers

Phototaxis

• Decided to go back to the original idea for controlling phototaxis via a SRII/HtrII/EcTsr fusion-chimeric protein described in the following article;
  
  

[http://pubs.acs.org/doi/abs/10.1021/bi034399q] Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro†
Vishwa D. Trivedi and, John L. Spudich
Biochemistry 2003 42 (47), 13887-13892

which is shown to work in K-12 E. coli in this article;

[http://jb.asm.org/cgi/content/full/183/21/6365] An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli
Jung, Kwang-Hwan, Spudich, Elena N., Trivedi, Vishwa D., Spudich, John L.
J. Bacteriol. 2001 183: 6365-6371

• Contacted the original researchers on the mentioned papers for protein sequences and plasmids
  
  

Retinal Production

• Decided to atempt to make our backteria synthesise retinal endogenously.
• 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].
• Isolated the gene sequence, and suggested biobricks for the coding region (K343001)
• Proposed four biobricks

BBa_K343000 – FlhDCmut coding sequence

BBa_K343001 - Sandboxed coding sequence from ninaB gene.

BBa_K343002 - Sandboxed coding sequence from ninaB gene on one of the weaker Anderson promoters, with rbs and dual terminator.

BBa_K343003 - Sandboxed coding sequence for the SopII-HtrII-Tsr fusion protein.

References:
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.

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.

Modelling

• Read articles about modelling the flagella movement and their influence on the water flow.
• Looked at the equations describing the system from the article to get an idea of how the physics is applied. The following articles was used in this progress;
  
  

[http://docs.google.com/viewer?a=v&pid=wave&srcid=8e-fLRxU2&chrome=true] Synchronization in a carpet of hydrodynamically coupled rotors with random intrinsic frequency N. Uchida 1 and R. Golestanian 2, ELP(Europhysics Letters) volume 89, number 5.

[http://docs.google.com/viewer?a=v&pid=wave&srcid=sz1nqWu52&chrome=true] The physics of flagellar motion of E. coli during chemotaxis M. Siva Kumar and P. Philominathan Biophysical Reviews Volume 2, Number 1 / February, 2010.

• Met with our advisor Associate professor Julian C. Shillcock, PhD (SDU) who helped us plan a model for the system using the article;
  
  

[http://docs.google.com/viewer?a=v&pid=wave&srcid=8e-fLRxU2&chrome=true] Synchronization in a carpet of hydrodynamically coupled rotors with random intrinsic frequency N. Uchida 1 and R. Golestanian 2, ELP(Europhysics Letters) volume 89, number 5.

Week 28

Flagella

• Extracted FlhDC master operon from E. coli strain MG1655
  
• Showed pressence of pst1 site in FlhC
  
• Introduced silent mutation into FlhC
  
  

Retinal

• Transformed pSB1A2-J13002(TetR repressed generator), pSB1A2-K274210 (CrtEBIY under constitutive promoter), pSB1AK3-B0015 into E.coli MG1655

Modelling

• Discussed Stokes and Navier-Stokes equations in relation to the system we would like to model.
• Tied to identify a differential equation describing the system. The following physics books have been used:
  
  

The theory of polymer dynamics, M. DOI, S. F. Edwards, oxford science publications, 4. edition 1992.

Physics of continuous matter, B. Lautrup, IOP publishing, 2005.

• Started implementing our model.

Week 29

Flagella

• Made FlhDCmut
  

Retinal

• pSB3C5-J04450 and pSB3T5-J04450 were transformed into E.coli TOP10 cells
  
• pSB1A2-R0011 was transformed into E.coli TOP10 cells
  
  

Week 30

• Ligated J13002 into pSB3T5
  

Week 31

Flagella

• Showing prescence of pst1 site in native FlhDC and anscence of pst1 site in mutated FlhDC

Week 32

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.

Week 33

* No Progress Report

Week 34

Photosensor

• Transformed B0015 into pSB3T5

Week 35

Photosensor

• NpSopII-NpHtrII-StTar coding sequence was isolated from pKJ606

Week 36

Flagella

• flhD/Cmut was made using new mutaion primers
• flhD/Cmut was ligated into pSB1C3 and plasmid was transformed into E.coli TOP10 cells.

Photosensor

• NpSopII-NpHtrII-StTar coding sequence (K343003) was ligated into pSB1C3 and plasmid was transformed into E.coli Top10 cells.
• NpSopII-NpHtrII-StTar coding sequence (K343003) was assembled with the double terminator (B0015) in pSB1AK3-B0015 and plasmid was transformed into E.coli TOP10 cells

Week 37

Flagella

• FlhD/Cmut coding sequence (K343000) was assembled with the double terminator (B0015) in pSB1AK3-B0015 and plasmid was transformed into E.coli TOP10 cells.

Photosensor

• NpSopII-NpHtrII-StTar coding sequence (K343003) + double terminator (B0015) was assambled with promoter + RBS (J13002)in pSB3T5-J13002 and plasmid was transformed into E.coli TOP10 cells.

Week 38

Retinal

• NinaB coding sequence (K343001) was assembled with Promoter + RBS (J13002) and ligated into pSB1C3. Plasmid was ligated into E.coli TOP10 cells

Photosensor

• 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

Week 39

Retinal

• 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.
• pSB1C3-K343005 and pSB1A2-K274210 (CrtEBIY under constitutive promoter) was transformed into E.coli TOP10 and MG1655 cells

week 40

Retinal

• 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

Week 41

Flagella

• flhD/Cmut composite part (K343004) was ligated into pSB1C3 and pSB3K3 and plasmids were transformed into E.coli TOP10 and MG1655 cells.

Retinal

• NinaB composite part (K343006) was ligated into pSB1C3 and plasmid was transformed into E.coli TOP10 and MG1655 cells

Week 42

Flagella

• Results of sequencing of flhD/Cmut composite part (K343004) in pSB1C3 was OK

Photosensor

• MG1655/pSB3T5-K343007 (NpSopII-NpHtrII-StTar composite part) was sequenced on THOR from [http://www.unisensor.dk/ Unisensor]

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