Team:SDU-Denmark/project-t

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= Theory =
= Theory =
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In this section we will review the theory behind our approach to establish a flow through a microtube.  
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In this section we will review the theory behind our biological work.  
== Phototaxis ==
== Phototaxis ==
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====The mechanism of bacterial motility====
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====Bacterial flagellar motility====
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Phototaxis is naturally not present in E.Coli, so that the wildtype does not change it's motility in response to light. The bacteria do move in response to different chemical gradients, so called chemotaxis. By this ability the cell can choose whether it wants to "run" or "tumble", which are the 2 known modes of bacterial propulsion. When running the bacteria just go in a straight line. When the bacteria tumble they will randomly reorientate themselves, until the mode is switched back to run and they run off into another direction. The cells use these mechanisms so that they will increase the rate of tumbling in an unfavorable environment, so that they tumble a lot and thereby get away from the unfavorable environment. In a favorable environment the bacteria will reduce their tumbling frequency so as they won't leave the good environment they are in at the moment.
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Bacteria have evolved many modes of propulsion for the microscale environments they inhabit. At these scales materials behave very different than at macroscale, and of particular interest to us is the way liquids seem more viscous [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 1]]. One of the many ways bacteria move around in liquids, is by means of flagella. A single flagellum is a thin filament around 100-150 Å thick, that extends many cell lengths out from the cell [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 2]]. It consists mainly of flagellin subunits that assemble into a helical structure forming a long hollow cylindrical filament [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 3]]. In ''E. coli'' the mean number of flagella per cell is 4 [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], but there is a wide variance between strains, and even between individual cells of each strain. The environment around the cell also has a large influence on how many flagellae are present, or if they are present at all [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. Flagella rotate to generate force that allows bacterial cells to swim through fluids in characteristic patterns, more on which later, in search of better conditions for proliferation or survival [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 5]]. Normally flagellated strains of ''E. coli'' can achieve speeds up to 20µm/sec [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], and considering a cell length of only 1-2µm, this is an impressive feat indeed.<br><br>
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The bacterial propulsion works with the help of flagellae, small bacterial motors that rotate. The 6 - 8 flagella of E. Coli bacteria will rotate counter clockwise when being in "run-mode" and three of them will bundle up to create one big flagellum. This results in a smooth-straight line pattern of movement. When the bacteria tumble the flagellae rotate clockwise, which breaks the bigger bundle up and makes the flagella whip around randomly instead of their normal rotation.
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A flagellum  is anchored to the cell body by a large, wheel-like protein complex spanning both inner and outer membranes [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. Through this complex subunits are secreted to the tip of the flagellar tube [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 3]], thus elongating the filament (This mechanism is slightly different in archaea, where the filament is assembled from the base of the flagellum). The membrane anchor also functions as a rotary engine, driven by the proton motive force [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. In ''E. coli'' the flagellar motor rotates at up to 300hz and can turn either clockwise or counter-clockwise [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], both resulting in a distinct movement pattern for the cell.<br><br>
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Spinning in the counter-clockwise direction, the flagella will twist into a bundle in the shape of a corkscrew, and create a linear driving force [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], propelling the cell in a straight line through the liquid. This form of movement is termed run. Spun in the clockwise direction one might then expect the cell to reverse, but this is not the case. Instead the flagellar bundle will unwind, thereby creating chaotic movement [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. This movement reorients the cell randomly and is termed tumbling. A cell will typically run for some time, then change it’s orientation by tumbling, and then run again [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 5]]. The direction of flagella rotation is controlled by the binding of a cytosolic protein CheY, more on which later [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]].<br><br>
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We want to be able to control the amount of flow in the tube through a remote signal. The signal we have chosen is light, since light does not have any effect on the composition of the fluid. This means that the probability of unwanted chemical interactions is reduced. Having looked at previous iGEM work on light sensitive systems, which have all been focused on transcriptional regulation, we realised that we would need a different approach for the fast response times our system requires. We have therefore focused our work on photorhodopsins that integrate into the chemotaxis pathway, giving us very fast response to light stimulation. <br><br>
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The type of light that we will use is blue light, which functions as a repellent in our case. This will make the bacteria want to get away from the light source which in turn results in an increased tumbling frequency, why will be explained a little further down this text. Since we chose ''E. coli'' as our model organism and wanted to use a light signal, we would have to increase it's sensitivity to bluelight, which naturally is very, very small. Through research we found out that the ''Halobacterium salinarum'' has a very well researched phototaxis mechanism, where the individual membrane domains role in the process had been solved ''and'' transferred to ''E. coli''. Which means that we would have to pick up on that research and create this mechanism as biobricks. <br><br>
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Different taxis pathways that steer cells towards favorable conditions and away from danger work by regulating the frequency of tumbling events [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 5]]. An example is when a bacterium gets close to the source of a lethal toxin, then intracellular pathways will increase the frequency of tumbling events, in effect preventing the cell from dying. Since the frequency of tumbling events will decrease if the cell is going in a direction away from the toxin, it will “encourage” the cell to continue in that direction. In the case of an attractant such as an increase in nutrient concentration, the pattern will be opposite, so that the cell is encouraged to continue towards the source of the attractant. This form of movement, combining tumbling and running, with regulation of the tumbling frequency is termed a biased random walk [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 5]]. <br><br>
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[[Image:Team-SDU-Denmark-Biased_random_walk.png | 300px | thumb |right | '''Figure 1:''' A biased random walk motion pattern.]] <br>
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The following model shows the way we want to couple the phototaxis pathway to ''E. coli'''s natural chemotaxis pathway. This is almost identical to the phototaxis pathway in ''H. salinarum'' except that the HtrII is directly coupled to CheA, so that there is no Tsr involved.<br><br><html>
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To understand how this can work we need a simplified understanding of the chemotaxis pathway at a molecular level. Chemotactic receptors can both increase and decrease tumbling frequencies to generate a biased random walk behavior [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 6]]. Increased tumbling is achieved through a phosphorylation cascade beginning with the binding of a repellant to a transmembrane receptor [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. The receptor is linked to two proteins, CheW and CheA [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. CheA is a histidine-kinase that will autophosphorylate when the repellant binds [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. The phosphoryl group is then transferred to CheY [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. The flagellar motor complex has high affinity for phosphorylated CheY (CheY-p), and binding reverses the mode of movement from run to tumble [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 7]]. CheY-p is continuously dephosphorylated back to CheY by CheZ [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 8]]. A receptor sensing an attractant might instead switch from the default active CheA state to an inactive state when it’s ligand is bound, thus decreasing CheY phosphorylation [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 6]].<br><br>
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<img width="600px" height="364px" src="https://static.igem.org/mediawiki/2010/a/a8/Team-SDU-Denmark-Phototaxis_mechanism.png" </img></html><br><br>
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The way the halobacterial pathway works is that the photonreceptor is a protein called sensory rhodopsin II, which absorbs the blue light and in response changes it's conformation. HtrII is just a transducer and signals this to CheA, which in turn gets phosphorylated and afterwards passes the phosphate group on to CheB. Phosphorylated CheB binds to the flagellar motor switch, so that the flagella starts rotating clockwise, which induces the tumbling motility pattern. The more CheY gets phosphorylated the higher the tumbling frequency will be.
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====Photosensor====
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Our focus is to get this working in ''E. coli'', which craves a few extra steps. First we will have to link the SopII and HtrII domains together with a 9 amino acid residue linker, so that the signal transducing happens succesfully in ''E. coli''. We also have to fuse HtrII and Tsr in their cytoplasmic domains, which is the HAMP domain, that both proteins contain. Fusion in this HAMP domain effectively couples the phototaxic receptors to the chemotaxis pathway, so that a phototactic effect is possible in ''E. coli''. These construction informations were obtained from the article: ''An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli'' by Spudich et Al. [http://jb.asm.org/cgi/content/full/183/21/6365]. That this system is functional in vitro in ''E. coli'' has also been shown by Spudich et Al in the article ''Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro† '' [http://www.ncbi.nlm.nih.gov/pubmed/14636056].
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<br> We will only have to add retinal to the system, which is needed for proper function of the fusion,chimera-protein. Therefore we want ''E. coli'' to produce retinal on its own, by transferring the gene for the enzyme that cleaves beta-carotene to retinal from flies (drosophila melanogaster). For the accumulation of beta-carotene we will use the biobrick BBa_K274210, which was constructed by the Cambridge team in 2009 [https://2009.igem.org/Team:Cambridge]. We will expand this brick's functionality by coupling it with the enzyme that cleaves beta-carotene to retinal. In that way we will be able to construct a retinal generator with the help of Cambridge's and our part. Here is a model of the retinal generator:<br><br><html><img width="600px" height="400px" src="https://static.igem.org/mediawiki/2010/c/cb/Team-SDU-Denmark-Retinal_generator.png"></img></html><br><br>
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In the end we want to split the whole fusion, chimer into two biobricks that can be fused as a composite part. By doing this we hopefully introduce biobricks that give ''E. coli'' phototaxic abilities and also introduce modularity into the complex, so that its signalling function can be coupled to other pathways than chemotaxis.<br><br>
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</p>
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=== BioBrick design ===
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In our system we want to be able to control the amount of flow in the channel, through a remote signal. The signal we have chosen is light since we want to avoid altering the chemical composition of the fluid running through the channel. Having looked at previous iGEM work on light sensitive systems which have all been focused on transcriptional regulation ([http://partsregistry.org/wiki/index.php/Part:BBa_I15010 e.g. I15010]), we realized that we would need a different approach for the fast response times our system requires. We have therefore focused our work on proteorhodopsins that integrate into the chemotaxis pathway, giving us very fast responses to light stimulation. <br><br>
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SopII-HtrII-Tsr fusion,chimer coding sequence: [http://partsregistry.org/Part:BBa_K343003 BBa_K343003]<br>
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Our construct centers around a synthetic protein created by Spudich ''et al.'' [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 9]]. It is a fusion chimeric protein that consists of an archaeal proteorhodopsin SopII (Sensory Rhodopsin II) and its transducer protein HtrII, both from ''Natronomonas pharaonis''. These are coupled to a Tar domain from a transmembrane receptor from ''Salmonella enterica''. The Tar domain is the part of the receptor that couples with CheA and CheW, and although it is taken from a different species, it has been shown to work in ''E. coli'' as well [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 10]]. In the construct we are working with light acting as an attractant, reducing the tumbling rate upon illumination (see picture). This might help us to control our pumping power, by decreasing the fraction of bacteria tumbling in the channel by increasing light stimulus, thus promoting linear drive. The photosensor should be most active in light with a wavelength of about 500nm, according to the original article. We have used DNA sent to us from the original authors to isolate the coding sequence for the protein generator.<br><br>
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Photosensor generator: [http://partsregistry.org/Part:BBa_K343007 BBa_K343007]<br><br>
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Note that although the bacteria will be stationary in our system, since they are glued to the inner surface of the flowchannel, our construct in reality confers a phototactic ability to ''E. coli''.
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== Retinal production ==
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[[image:Team-sdu-denmark-Phototaxis_mechanism.png | 600px | thumb |'''Figure 2:''' '''A.''' The SopII proteorhodopsin has not been activated. Note that CheA is active by default, continuosly autophosphorylating itself, and cycling back to it's unphosphorylated state by transfering the phosphoryl group to CheY. High levels of CheY-p will induce tumbling motion in the flagella. Also, note that CheZ continuously dephosphorylates CheY.
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'''B.''' SopII is hit with a photon, causing conformational change of the entire complex. The inactivation of CheA halts production of CheY-p, and CheZ rapidly dephosphorylates the remaining CheY-p, resulting in a reduced frequency of tumbling.]]
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=== Background ===
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====BioBrick design====
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[[Image:Team-Sdu-denmark-PS_CS.png]]<br>
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Photosensor coding biobrick: [http://partsregistry.org/Part:BBa_K343003 K343003] <br>
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[[Image:Team-Sdu-denmark-PS_BB.png]]<br>
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Photosensor composite part: [http://partsregistry.org/Part:BBa_K343007 K343007]
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<br>
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== Retinal Generator ==
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====Retinal requirements of light-sensing proteins====
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Proteorhodopsins belong to a larger group of proteins called retinylidene proteins consisting of proteins called an opsins with a retinoid cromophore such as retinal attached as a prosthetic group[[https://2010.igem.org/Team:SDU-Denmark/project-t#References 11]]. In our system retinal is responsible for the initial light-activation, as it undergoes photoizomerization when it is struck by a photon[[https://2010.igem.org/Team:SDU-Denmark/project-t#References 12]]. It is this change in conformation of the retinal molecule that is relayed through the entire protein complex to regulate chemotaxis [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 12]]. Thus either an external supply of retinal or an internal supply, generated by means of genes coding for enzymes in the retinal biosynthesis pathway are required, if we wish to see phototactic behaviour in our cells [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 10]].
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Light-sensitive pigments can be found in a large variety of organisms from archaea and bacteria to both uni- and multicellular eukaryotes [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 11]], [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 13]], [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 14]]. Many plants and microbes have complete retinal biosynthesis pathways in their genomes [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 15]]. In these organisms rhodopsins play an essential role, not only for photosensing but also directly in energy production [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 11]],[[https://2010.igem.org/Team:SDU-Denmark/project-t#References 13]],[[https://2010.igem.org/Team:SDU-Denmark/project-t#References 14]]. In fact in some organisms rhodopsins are used to create proton motive force directly by pumping protons out into the extracellular space using light energy to drive the process [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 14]]. Humans and other animals on the other hand often only have enzymes coding for the final steps of the pathway, more on which later. They rely on a supply of retinal precursors or vitamin A (a group of molecules consisting of retinal and it's metabolites) in their diet [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 16]]. This is why vitamin A deficiency causes night-blindness as an early symptom in humans [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 17]].
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=== Retinal biosynthesis ===
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==== Retinal biosynthesis ====
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<p style="text-align: justify;">
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Retinal can be synthesized from the enzymatic cleavage of some carotenes. In our system we focus on cleavage of beta-carotene, partly because it yields 2 ''all-trans'' retinal molecules which are the molecules we desire, and partly because the beta-carotene biosynthesis pathway has already been introduced to ''E. coli'' by the [http://partsregistry.org/Part:BBa_K274210 Cambridge 2009 ] iGEM team.
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Retinal is produced naturally in many species. Our construct uses genes from the plant pathogen ''Pantoea ananatis'' and from the fruit fly, ''Drosophila melanogaster''.  <br></p>
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The Cambridge construct uses genes from the plant pathogen ''Pantoea ananatis'' and our construct completes the pathway to retinal with a gene from the common fruit fly, ''Drosophila melanogaster''.  <br>
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In our construct, part of the synthesis is carried out by the BioBrick BBs_K274210, created by the Cambridge team in 2009. It consists of four genes ''crtE'', ''crtB'', ''crtI'' and ''crtY'' from Pantoea ananatis that together make up the pathway that converts farnesyl pyrophosphate to beta-carotene, which is a precursor for retinal. <br>
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The Cambridge 2009 construct consists of four genes ''crtE'', ''crtB'', ''crtI'' and ''crtY'' from ''P. ananatis'' that together make up the pathway that converts farnesyl pyrophosphate to beta-carotene, which is a precursor for retinal. Farnesyl pyrophosphate is naturally present in ''E. coli''. <br>
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• ''crtE'' encodes the protein geranyl-geranyl pyrophosphate synthase that converts farnesyl pyrophosphate to geranyl-geranyl pyrophosphate by elongating it by one unit of isopentenyl. <br>
• ''crtE'' encodes the protein geranyl-geranyl pyrophosphate synthase that converts farnesyl pyrophosphate to geranyl-geranyl pyrophosphate by elongating it by one unit of isopentenyl. <br>
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The pathway (including the step that generates retinal) is summed up below: <br><br>
The pathway (including the step that generates retinal) is summed up below: <br><br>
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<html><img width="630px" height="420px" src="https://static.igem.org/mediawiki/2010/c/cb/Team-SDU-Denmark-Retinal_generator.png"></img></html>
 
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To introduce the final step from beta-carotene to retinal, we use the gene ''ninaB'' from ''Drosophila melanogaster''. This gene encodes the protein beta-carotene 15,15’-monooxygenase, which cleaves beta-carotene to produce two molecules of retinal under the consumption of oxygene. <br>
 
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We have inserted the part BBa_343006 into a different plasmid from the BBa_K274210 part because both parts are very long and thus wouldn’t have fitted in one plasmid.
 
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=== The effect of retinal on the system ===
 
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=== Characterizing the retinal BioBrick ===
 
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We characterized our BioBrick using two methods: Photospectrometry and high performance liquid chromatography (HPLC). While characterizing our own construct, we simultaneously characterized the Cambridge part, BBa_K274210 seeing as we wish to find out if the two parts work in concert.<br>
 
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Seeing as both beta-carotene and retinal have unique and characteristic spectres when studied by UV-vis spectrometry, this is a good place to start. The spectra can be obtained by harvesting beta-carotene- and retinal-producing cells, resuspending them in acetone and lysing them, which we chose to do by sonication.  Afterwards, the cell debris can be pelleted and the supernatant can be examinated. <br>
 
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The acetone suspension of beta-carotene and retinal can then be subjected to photospectrometry, and the obtained values and spectra can be compared to those of pure beta-carotene or retinal in known concentrations. This method provides both a qualitative answer to whether or not the desired compound is present and a qualitative indication of the concentration in the cells. <br>
 
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This is the method by which the Cambridge team characterized their beta-carotene-generating BioBrick in 2009. <br>
 
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Apart from photospectrometry, the resuspension of beta-carotene or retinal in acetone can be subjected to analysis by HPLC. HPLC can be used to separate retinal from beta-carotene, to get a better indication of whether or not our retinal-generating part actually produces retinal from beta-carotene. For this particular purpose, we use a C-18 column, and the eluents used are as follows: <br>
 
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A-buffer: 100% methanol with 0,1% trifluoroacetic acid. <br>
 
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B-buffer: A mixture consisting of 60% methanol and 40% acetone with 0,1% trifluoroacetic acid. <br>
 
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Due to the chemical properties of beta-carotene and retinal, respectively, retinal will come through the column before beta-carotene when a gradient is run from 100% A-buffer to 100% B-buffer. <br>
 
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Afterwards, the solutions of purified retinal or beta-carotene are studied using UV-vis photospectrometry and the values and spectra are compared to those of the same compounds of known concentrations. Again, this gives both qualitative and quantitative indications of whether the compound in question is present and, if it is, in what concentration. <br>
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[[Image:Team-SDU-Denmark-Retinal_generator.png |400px|thumb|Figure 3: The retinal Biosynthesis pathway.]]
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See our results [https://2010.igem.org/Team:SDU-Denmark/project-r#UV-Vis_spectrophotometer_determination_of_beta-carotene_production here].
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To introduce the final step from beta-carotene to retinal, we use the gene ninaB from D. melanogaster. This gene encodes the protein beta-carotene 15,15’-monooxygenase, which cleaves beta-carotene to produce two molecules of trans-retinal under the consumption of oxygen [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 18]]. We have inserted the part K343006 into a different plasmid from the K274210 part since both parts are very long, so a plasmid containing both wouldn't have been viable for transformations.
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</p>
 
=== BioBrick design ===
=== BioBrick design ===
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Retinal coding biobrick: [http://partsregistry.org/Part:BBa_K343001 BBa_K343001 ]<br>
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[[Image:Team-sdu-denmark-NinaB_CS.png]]<br>
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Retinal composite part: [http://partsregistry.org/Part:BBa_K343002 BBa_K343002]
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Retinal coding biobrick: [http://partsregistry.org/Part:BBa_K343001 K343001 ]<br>
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[[Image:Team-sdu-denmark-NinaB_BB.png]]<br>
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Retinal composite part: [http://partsregistry.org/Part:BBa_K343006 K343006]
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=== Further use of the retinal BioBrick ===
=== Further use of the retinal BioBrick ===
==== Role in light-based signal transduction ====
==== Role in light-based signal transduction ====
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Retinal plays an essential role in photosensation in both eukaryotes as well as bacteria and acrhaea. All work with rhodopsins and proteorhodopsins will need a retinal supply to function. This supply might come from the external environment, but it is an appealing thought that we might be able to supply the retinal from an internal source. Our project centers around phototaxis, but other constructs combining photorhodopsins with other membrane associated tyrosine kinases may also be imagined, opening vast posibilities for regulation of phopsphorylation cascades using light as input. In such systems retinal biosynthesis could play a very valuable role.<br>
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Since retinal plays such an essential role in photosensing in both eukaryotes as well as bacteria and archaea, all work with rhodopsins and proteorhodopsins will need a retinal supply to function. This supply might come from the external environment, but it is an appealing thought that we might be able to supply the retinal internally. Our project centers around phototaxis, but constructs combining photorhodopsins with other membrane associated tyrosine kinases may also be imagined, opening vast posibilities for regulation of phopsphorylation cascades using light as input. In such systems, retinal biosynthesis could play a very valuable role.<br>
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==== Physiological functions functions in higher animals ====
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Vitamin A (retinal and related metabolites such as retinol) are important nutrients in humans and animals. Vitamin A is important in many physiological processes including regulation of gene transcription, skin and teeth maintenance, prenatal development and vision. <br>
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== Hyperflagellation ==
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=== Background ===
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Motility can be a very beneficial quality for a microorganism. Evolution has therefore provided bacteria with two general means of transportation; the flagella and the pili. These qualities allow the bacteria to move away from a hostile environment and towards more favorable conditions. Flagella and pili are however viewed as a virulence factor as they also serve as an advantage in colonizing a host organism and yet they can cause a strong immune response [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 19]]. <br><br>
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Many organisms are able to synthesize a flagellum, if the external environment calls for it. The synthesis of a flagellum is a huge and energy consuming process and is therefore tightly regulated by the bacteria’s external environment. One of the most well characterized flagellation systems is the one found in ''E. coli''. Here at least 50 genes are involved in the hierarchical synthesis and operation of the flagella. These genes are sorted into 15 operons which are expressed in a transcriptional cascade separated into three classes. Class I consists of the master operon ''flhDC''. The active FlhDC protein is a hexamer organized into an FlhD<sub>4</sub>C<sub>2</sub> complex with a computed value of 96,4kDa [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 20]]. The homodimeric FlhC protein is able to bind DNA, while the FlhD homodimers are not. The formation of the FlhDC complex however, stabilizes and increases the DNA binding ability [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 21]]. The transcription of ''flhDC'' is heavily regulated by nutritional and environmental conditions. Flagellum synthesis is inhibited at high temperatures, at high salt concentrations, at extreme pH or in the presence of carbohydrates, low molecular alcohols or DNA gyrase inhibitors, as these conditions stimulate growth as opposed to motility [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 22]]. Because the flagellum synthesis is so energy consuming, the process is not started unless the environment calls for motility rather than growth. In fact, in situations where nutrition is plenty over a long period, the bacteria will focus on growth and over time lose the ability to synthesize the flagellum, as seen with the ''E. coli'' strain MG1655 localized in mouse intestines [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 23]].
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==== Global health issues ====
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Vitamin A deficiency is a serious global problem, estimated to affect one in three children under the age of five around the world according to WHO()
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[[Image:Team-SDU-Denmark-flagella-overview-1.png|600px|thumb|center|Figure 4: Overviews cascade of the flagellum synthesis.]]
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[[Image:Team SDU-Denmark FlhD4C2 structure.JPG|thumb|right|210px|'''Figure 5:'''3D structure of the FlhD<sub>4</sub>C<sub>2</sub> hexamer.[[https://2010.igem.org/Team:SDU-Denmark/project-t#References 23]][[https://2010.igem.org/Team:SDU-Denmark/project-t#References 24]]]] <br>
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The FlhD<sub>4</sub>C<sub>2</sub> hexamer acts as a transcription factor for the Class II genes, which encodes the basal body, that is embedded in the cell membrane as well as hook proteins, which are transported to the cell exterior through the basal body. Another Class II gene is the σ<sup>28</sup> transcription factor, which is responsible for the transcription of the Class III genes. This includes ''fliC'', which encodes the flagellin subunit that composes the flagella “tail”. To ensure that the Class III genes are not transcribed before the assembly of the basal body and the hook is complete another Class II protein FliM acts as an anti-sigma factor and bind σ<sup>28</sup>, thereby preventing the transcription of ''fliC''.<br><br>
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Several studies regarding the motility of ''E. coli'' has shown the expression of the ''flhDC'' operon to be crucial [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 23]][[https://2010.igem.org/Team:SDU-Denmark/project-t#References 25]]. These focused on insertion sequence (IS) elements upstream of the ''flhDC'' regulon. IS are sequences that can be inserted randomly within the DNA and therefore serve as an important factor in the plasticity of the ''E. coli'' genome as well as in many other organisms. Generally they do not encode any genes apart from those responsible for their movement within the genome, however, they can also serve as activators of neighboring genes, by disrupting repression or by the formation of hybrid promoters [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 25]]. In the beforementioned studies, bacteria containing an activating IS upstrem of the ''flhDC'' operon showed an increased motility compared to bacteria without this IS. It is therefore resonable to asume that by placing a constitutive active promoter in front of the ''flhDC'' operon, hyperflagellation will be induced.
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== Hyperflagellation ==
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=== Our system ===
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=== Background ===
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The flagella regulon in ''E. coli'' is composed of at least 50 genes organized in no less than 14 operons that all contribute to the synthesis and operation of flagella. The operons are synthesized in a three-level transcriptional cascade where the ''FlhDC'' operon is the master regulator at the top of the cascade. The flagella regulon is tightly controlled by nutritional and environmental conditions, ''E. coli'' starved of amino acids showed temporarily decrease of the flagella regulon transcripts which are needed for the synthesis and operation of the flagellum.[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06939.x/full (1)]
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As we are trying to create a microflow in our system using flagella, we want to increase the flow created by a single bacteria by increasing the number of flagella on the bacteria. However, inducing flagellation in a wildtype ''E. coli'' strain requires too much of the bacterial environment. In order to avoid the tight control of the ''flhDC'' operon, we insert it into a plasmid backbone containing a constitutive active promoter. As we bypass the original regulations and continuously express the master regulon of the flagellum cascade we hope to see a significant difference in motility of the cells containing our composite part.<br>
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The synthesis and assembly of flagella are regulated by the transcriptional cascade composed of three levels of gene products (class I, -II and –III). Class I genes consist of a single operon encoding the proteins ''FlhD'' and ''FlhC'' that form a multimeric (''FlhD4C2'') transcriptional activation complex. This ‘master regulator’ stimulates transcription by binding upstream of Class II promoters. Class II genes encode proteins that assemble to form the basal body and hook of the flagellum, as well as the ''fliA'' gene that encodes the alternative σ factor σ28, also called σF. σ28 binds to RNA polymerase (RNAP) core enzyme and directs it to Class III promoters. Class III genes encode the rest of the structural genes of the flagellum, including ''fliC'' encoding flagellin, as well as the chemotaxis apparatus. [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06939.x/full (1)]
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As ''E. coli'' already express the ''flhDC'' operon, we isolate this coding sequence by purifying genomic DNA from the ''E. coli'' MG1655 strain and then amplifying the operon using PCR with specially designed [https://2010.igem.org/Team:SDU-Denmark/primers primers], before assembly and the insertion into the plasmid backbone <br>
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Furthermore, it will be interesting to see whether the overexpression of the flhDC operon will have an effect on the bacterial growth.
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It has been shown that overexpression of the ''FlhDC'' operon restores motility in mutants that have been made immotile [http://jb.asm.org/cgi/content/short/181/24/7500 (2)]. Also, overexpression of ''FlhDC'' in the ''E. coli'' K12 strain MG1655 made the cells hypermotile.[http://iai.asm.org/cgi/content/abstract/75/7/3315 (3)]  
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'''Our system'''
 
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To maximize the microflow system's effectivity, we want to increase the force each single bacterium can generate. Since the main motor for the flow is the flagellum we will have to modify this factor for increasing the force. Flagella in ''E. coli'' rotate at a maximum speed around 6000 rpm, which can not easily be exceeded. So if we wanted to increse the generated force we would have to opt for more flagella on the surface of our bacteria, instead of faster flagella. This is called hyperflagellation, which is a process that is not all too well studied in normally-flagellated ''E. coli'', so we will have to test it.<br>
 
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The way we are hoping to achieve the hyperflagellation is by upregulating the ''FlhDC'' operon. Since ''FlhDC'' is sitting on top of the regulating cascade we want to overexpress it, so that we will get an increase in flagellar count.<br>
 
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The way we are going to achieve that is by isolating the operon from an ''E. coli'' and inserting the coding sequence into a BioBrick where we can control both the ribosome binding site and the type of promoter. We are going to use a constitutive promoter, so that flagella will be constantly expressed. The effects of this on other areas of the organism like the cell cycle are not entirely clear, but we will first try to overxpress the operon and then analyse the effect on the cells behavior.<br><br>
 
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=== Biobrick design ===  
=== Biobrick design ===  
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FlhD,C mutated coding sequence: [http://partsregistry.org/Part:BBa_K343000 BBa_K343000 ]<br>
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[[Image:Team-sdu-denmark-FlhDC_CS.png]]<br>
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FlhDC composite part: [http://partsregistry.org/Part:BBa_K343004 BBa_K343004]
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FlhD,C mutated coding sequence: [http://partsregistry.org/Part:BBa_K343000 K343000 ]<br>
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[[Image:Team-sdu-denmark-FlhDC_BB.png]]<br>
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FlhDC composite part: [http://partsregistry.org/Part:BBa_K343004 K343004]
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# Claret, L. and Hughes, C. (2000) [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-45M7T8W-51-1&_cdi=6899&_user=644074&_pii=S0022283600941494&_origin=search&_coverDate=11%2F03%2F2000&_sk=996969995&view=c&wchp=dGLbVzW-zSkzV&md5=25cecb82828b382819b79c207eaaf63b&ie=/sdarticle.pdf Functions of the Subunits in the FlhD<sub>2</sub>C<sub>2</sub> Transcriptional Master Regulator of Bacterial Flagellum Biogenesis and Swarming.] J. Mol. Biol. 303, 467-478. <br>
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And now for all of the truly brainy stuff!
And now for all of the truly brainy stuff!
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Latest revision as of 00:00, 28 October 2010