Week 26

Flagella

- Studied inhibition and activation FlhD,C operon.
- PskA is an important inhibitor, which also is part of ribosome synthesis.
- It is probably not a good idea to use a constitutive promoter in front of the FlhD,C operon, because we are unsure what will happen if the transcription is active non-stop. (cell death?)

Phototaxis

- Ordered proteorhodopsin biobrick.
- Did research on the light-sensitive proteorhodopsin ionpump. It seems like we will have to use this for the phototaxis functionality, so I (LC) asked Mike to order the already constructed biobrick (BBa_I711040). Sent the relevant article to our instructors to get their feedback.

Week 27

Flagella

July 1st
Progress report:
The Last couple of days I’ve been reading up on the flagella regulon. We want to hyper flagellate our cells to see if this will create more power in our system. Therefore we need to know how the flagellar genes are regulated and if these genes are coupled to other processes than flagella synthesis. For this I’ve used the 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

- The flagellar regulon consists of at least 14 operons which encode more than 40 genes. The operons are divided into three classes with one master operon (FlhDC)in class I which encodes the transcription factors (TF)FlhD and -C. These TF activate the class II genes which entail FliA and Sigma factor-28. The class II gene products are TF's for the class III genes which amongst others code for the FliC protein.
- The FlhDC operon is not only important in expression of flagella but also functions in the cell division.
- The FlhDC operon is activated by environmental factors such as cAMP-CAP and H-NS.
- All three classes of genes can be deactivated by DksA and ppGpp. DksA is also important for ribosomal expression.
Working Hypothesis:
We thought about knocking DskA out and thereby rendering the FlhDC operon active. But since DksA is important for ribosomal expression knocking this gene out might have other severe consequences for the cells.
We know that the FlhDC operon is an important factor in the cell cycle and we don't know if over-activation of this master operon will have important effects on the cell division.

So we are now following the popular "trial-and-error" method and testing what effects it will have to put the FlhDC operon after a constitutive promoter. If this does not have serious effects that will disturb our project, we hope to be able to get hyper flagellated cells.

We hope the hyper flagellation will produce a more powerful flow in our system, but we are aware of the risk that the extra flagellas will only produce chaos. The extra flagellas increase the tumbling rate of the cells and this we don't want. We need a steady laminar flow so we want to reduce the tumbling as much as possible,but this is the focus of the phototaxis group. However, if the hyper flagellation does disrupt the system we have to drop the idea of overexpression and use E. coli with normal flagella expression.

BioBrick Design:
I will work on the biobrick for this project tomorrow.

--Louch07 09:43, 2 July 2010 (UTC)

July 2nd
Biobrick design:
We want to make a constitutive active FlhDC operon. For this we will use a sigma 70 promotor. Sigma 70 is a constitutive active housekeeping gene. We have ordered FlhDC and are waiting for it to arrive. Also we need a terminator and a ribosomal binding sequence (RBS). The promotor (BBa_J23100), the terminator (BBa_B0015) and the RBS (BBa_J61100) are all in the 2010 spring kit.

We have created this as a part on Partsregistry.org

Phototaxis

July 1st
Progress report:
We've gone back to our 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

- It works primarily by inducing autophosphorylation in CheA that in turn phosphorylates CheY into it's active state, that controls the flagellar switch.
- It is activated by blue light.
- CheY's effect on flagellar function is to increase the frequency of tumbling episodes.

Working Hypothesis:
Right now our work with the Fusion-Chimeric photosensitive protein is oriented towards creating a negative feed-back regulation of our flow-generating system. We are working from the idea that inducing more frequent tumbling events in our bacterial colony will increase the amount of turbulence generated in our system, so as to reduce flow. Hereby we can control flow with light.

BioBrick Design:
Since the fusion-chimeric protein is very large we will need to assemble it as a composite part. Luckily parts of it are connected by AA linker chains, we can exploit in our assembly, to reduce the effect of the BioBrick scar. Important considerations concerning staying in-frame and designing good BioBricks for assembly will be met in the coming days.

Note: We need to contact the original researchers on the mentioned papers for protein sequences and possibly plasmids or strains of their bacteria.

--CKurtzhals 19:38, 1 July 2010 (UTC)

July 2nd
Progress report:
Today the decision to atempt to make our backteria synthesise retinal endogenously was made. We will be using old material from the Cambridge 2009 project (Part:BBa_K274210) to make our bacteria synthesise beta-carotene, and we have 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]. An enzyme that catalyses cleavage beta-carotene into two retinal molecules. This protein has been shown to function in E.
coli[http://www.jbc.org/content/275/16/11915.long[1]], along with a synthetic operon closely matching the one from the cambridge project.

We have already isolated the gene sequence, and suggested biobricks for the coding region (BBa_K343001) and a combined part (BBa_K343002) that will work as a protein generator. The relevant cDNA is available for purchase, and should be easy to acquire.

On top of this we deduced the coding sequence for the SopII-HtrII-Tsr fusion, chimera protein from Spudich et al.
[http://jb.asm.org/cgi/content/full/183/21/6365] From the information given in the materials and methods section of the article, we could deduce the exact sequence. The amount of amino acid residues were given and the gene sequences known. From there we could find out where and how the gene sequences were stitched together. By that we could already sandbox the planned biobrick for the bluelight receptor sensory rhodopsin II coupled to the chemotaxis pathway of E.Coli via the HtrII and Tsr fusion. The next part is obtaining the physical DNA, for which we will have to contact Spudich lab and ask for a plasmid containing the chimera-protein.

BioBrick Design:
Today we have 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.

The sequence for the D. melanogaster gene is taken from the cDNA sequence from flybase.org, and matched by length to the b-diox protein. Choice of promoter was made on the consideration that much of this enzyme might not be needed to supply sufficient retinal. Rbs and terminators are entirely standard.

The Cambridge part needed is already on one of our distribution plates: Spring 2010 Distribution 2010 Kit Plate 3, Well 6N, pSB1A2

--CKurtzhals 17:11, 2 July 2010 (UTC), --Lclund 13:34, 3 July 2010 (UTC)

References:
1. 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.

2. 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.

3. "An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli", Jung K-H, Spudich EN, Trivedi VD and Spudich JL ; Journal of Bacteriology, Nov. 2001, p. 6365–6371.

July 3rd
Progress report:
We've not been doing any real project work today. Instead we've done work on the project description on the wiki, and made a couple of graphics.

Other
We've found an interesting project from the 2008 competition. Paris has apparently already worked with the flagellas regulation, although not with intent to produce flagella themselves. Instead they have worked on the regulation, and expression-cascade of flagella proteins, and their work might well help us figure out how we might hyperflagellate our backteria, or how we will model the system.

--CKurtzhals 21:04, 3 July 2010 (UTC)

Modelling

July 1st
Progress report:
The last couple of days we have been reading articles about modelling the flagella movement and their influence on the water flow. Because we need to understand the physics of the system before we are able to model it. Today we have been looking at the equations describing the system from the article to get an idea of how the physics is applied. The following articles have been 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.

Working hypothesise:
Understanding the physics of flagella systems, their movement in a fluid and their effects on the fluids, is important for setting up models describing the system we would like to produce. We haven’t yet found a model describing our system fully and this is therefore our next assignment.

--Toand 15:29, 2 July 2010 (UTC)

July 2nd
Progress report:
Today we continued looking at the equations from the article, then had a meeting with Associate professor Julian C. Shillcock, PhD (SDU) who help us plan a model for the system. Our further work will consist of connecting the physics to the system and device equations that describe it. The article uses was:

[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.

Working hypothesise:
A set of rigid screws with one end attached to a surface with the ability to bend in an angel θi and generate a force Fi. The forces generated by a screw may influence the angel of other screws and the effect the entire system witch in term affects the first screw. When the physics of the system is determined we hope to model variations of θi with the force Fi.

--Toand 08:46, 4 July 2010 (UTC)

Week 29

Flagella

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

Retinal

• Transformed the [http://partsregistry.org/Part:BBa_K274210 Cambridge part] in to Top10 cells
  
• Transformed an [http://partsregistry.org/Part:BBa_I0500 inducible promoter] in to Top10 cells
  
• Transformed [http://partsregistry.org/Part:BBa_E0040 GFP] in to MG1655 cells
  

http://igem.sdu.dk/wp-content/uploads/sponsor-sdu.png http://igem.sdu.dk/wp-content/uploads/sponsor-fermentas.png http://igem.sdu.dk/wp-content/uploads/sponsor-dnatech.png

Week 29

Flagella

• Made FlhDCmut
  

Retinal

• Ligated BBa_J04450 into pSB3C5 and pSB3T5, and transformed the plasmid into Top10 cells
  
• Ligated BBa_R0011 into pSB1A2 and transformed the plasmid into top10 cells
  

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