iGEM 2010 — Team SDU-Denmark

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# Team:SDU-Denmark/notebook

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# Week 26

## Flagella

* Studied inhibition and activation of the FlhD/C operon.
* We found out that PskA is an important inhibitor, which also is part of ribosome synthesis.

## Phototaxis

* Ordered proteorhodopsin biobrick [I711040](http://partsregistry.org/Part%3ABBa_I711040)
* Did research on the light-sensitive proteorhodopsin ionpump.

# Week 27

## Flagella

* Read up on the flagella regulon using the following articles;

[[1]](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

[[2]](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 tested what effects it will have to put the FlhD/C operon after a constitutive active promoter.
* Ordered the FlhD/C primers.
* Designed the biobrick *K343000* – *FlhDCmut* coding sequence

## 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;

[[3]](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;

[[4]](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 the protein sequences and plasmids

## Retinal Production

* Decided that we would try to make our bacteria synthesize retinal endogenously.
* Designed a new BioBrick around the *ninaB* gene from *D. melanogaster*, which has shown to produce a beta-carotene 15,15'-monooxygenase [[5]](http://www.jbc.org/content/275/16/11915.long)[[6]](http://www.ncbi.nlm.nih.gov/pmc/articles/PMC14720/?tool=pubmed).
* Isolated the gene sequence and suggested biobricks for the coding region (K343001)
* Proposed three new biobricks

*K343001* - Sandboxed coding sequence from *ninaB* gene.

*K343002* - Sandboxed coding sequence from *ninaB* gene on one of the weaker Anderson promoters, with rbs and dual terminator.

*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 mutantninaB *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;

[[7]](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.

[[8]](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;

[[9]](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 the 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 the mutation the silent in the FlhDC operon

## 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 absence 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 the 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 and the 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 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

# week 40

## Retinal

* NinaB coding sequence + Promoter + RBS (K343005) was assembled with the double terminator (B0015) in pSB1AK3 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. Motility plates, flagella staining and phase microscopy was made.

## Photosensor

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

[![Team SDU-Denmark-pearl logo.JPG](/wiki/images/d/d1/Team_SDU-Denmark-pearl_logo.JPG)](/File%3ATeam_SDU-Denmark-pearl_logo.JPG)

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.

|  |
| --- |
| Contents   * [1 Week 26](#Week_26)   + [1.1 Flagella](#Flagella)   + [1.2 Phototaxis](#Phototaxis) * [2 Week 27](#Week_27)   + [2.1 Flagella](#Flagella_2)   + [2.2 Phototaxis](#Phototaxis_2)   + [2.3 Retinal Production](#Retinal_Production)   + [2.4 Modelling](#Modelling) * [3 Week 28](#Week_28)   + [3.1 Flagella](#Flagella_3)   + [3.2 Retinal](#Retinal)   + [3.3 Modelling](#Modelling_2) * [4 Week 29](#Week_29)   + [4.1 Flagella](#Flagella_4)   + [4.2 Retinal](#Retinal_2) * [5 Week 30](#Week_30) * [6 Week 31](#Week_31)   + [6.1 Flagella](#Flagella_5) * [7 Week 32](#Week_32)   + [7.1 Retinal](#Retinal_3) * [8 Week 33](#Week_33) * [9 Week 34](#Week_34)   + [9.1 Photosensor](#Photosensor) * [10 Week 35](#Week_35)   + [10.1 Photosensor](#Photosensor_2) * [11 Week 36](#Week_36)   + [11.1 Flagella](#Flagella_6)   + [11.2 Photosensor](#Photosensor_3) * [12 Week 37](#Week_37)   + [12.1 Flagella](#Flagella_7)   + [12.2 Photosensor](#Photosensor_4) * [13 Week 38](#Week_38)   + [13.1 Retinal](#Retinal_4)   + [13.2 Photosensor](#Photosensor_5) * [14 Week 39](#Week_39)   + [14.1 Retinal](#Retinal_5) * [15 week 40](#week_40)   + [15.1 Retinal](#Retinal_6) * [16 Week 41](#Week_41)   + [16.1 Flagella](#Flagella_8)   + [16.2 Retinal](#Retinal_7) * [17 Week 42](#Week_42)   + [17.1 Flagella](#Flagella_9)   + [17.2 Photosensor](#Photosensor_6) |

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