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From 2010.igem.org

Phototaxis

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,

[1] 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,

[2] 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)

Flagella

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:

[3] 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

[4] 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)