Team:SDU-Denmark/project-r

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= Results =
= Results =
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== Photosensor ==
== Photosensor ==
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=== Motility assay ===  
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=== Biobrick assembly and sequence ===
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<br>
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We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343007 K343007], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343003 and the terminators B0010 and B0012.<br>
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In this experiment we shone blue light on one half of the plate, while the other half was in the dark. We then placed one colony in each half and observed their motility pattern after 24 hours. We did this for three different strains of E.Coli - DH5alpha, Wildtype Mg1655 and Mg1655 containing a plasmid with our photosensor constitutively on. See the results for yourself: Light shone on the right half of the plates, the left half was in the dark.<br>
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This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
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[[Image:Team-SDU-Denmark-MG1655.JPG|250px|MG1655]]
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<br>
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[[Image:Team-SDU-Denmark-Photosensor.JPG|250px|Photosensor]]
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[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8039| Sequencing results K343007]
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[[Image:Team-SDU-Denmark-DH5alpha.JPG|250px|DH5alpha]]<br>
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<br><br>
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Interpreting these results is not totally obvious since the effect of different motility patterns can be counter intuitive in semisolid agar. An explanation as to how different motility patterns show up on semisolid agar plates is explained by Englert et al. in the book "Microfluidic techniques for the analysis of bacterial chemotaxis":<br><br>
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The sequence obtained from sequencing is identical with the theoretical sequence of the part, except for a silent mutation in the coding sequence, which does not change the amino acid sequence.
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''Polymerized agar consists of chains of extended polymers permeated by water-filled channels. At low agar concentrations (0.25– 0.4% semisolid agar) the channels are sufficiently large that the bacteria can swim through them. In the absence of chemoeffectors, cells conduct a 3D random walk through the agar matrix much as they would in liquid and spread out randomly from the point of inoculation. Since the cells are typically growing, the result is an expanding colony that forms within the agar. This “pseudotaxis” occurs in the absence of any tactic behavior. Mutant
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=== Effect ===
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cells that only swim smoothly get trapped cul de sacs in the agar matrix (16), and their colonies do not expand significantly faster than those of nonmotile cells.'' <br><br>
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Our results from the experiments with semi-solid agar confirms that the bacteria carrying a plasmid with the biobrick have a altered motility, most likely by an alteration in tumbling frequency, when exposed to blue light compared to the wild type MG1655.<br>
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<br>
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The videomicroscopy indicates that bacteria carrying a plasmid with the biobrick travel father and in a more straight path than the wild type MG1655 when exposed to blue light with a wavelength around 500nm, Thereby suggesting a lowered tubling frequency. <br>
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Computeranalysis of videos recorded of swimming baceria containing this part, indicate that these bacteria have a tendency to swim towards the source of light, when exposed to a light gradient. A trackdiagram was constructed from the gathered data, and swimming bacteria tended to orientate their movement in the direction of the light source. This could mean that blue light acts as an attractant stimulus on the SopII-HtrII-Tar complex. <br><br>
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The stability of pSB1C3-K343007 is most likely <20 generations, however the stability of pSB3T5-K343007 was not determined. <br>
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Plasmids expressing K343007 does not seem to hinder the bacterial growth in any way.<br>
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For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343007 characterization of K343007].
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This means that if the photosensor leads to a reduced tumbling rate when exposed to blue light, it should look a lot like the colony of DH5alpha whichw as exposed to blue light. This is exactly the case which proves that the photosensor has an effect on the tumbling rate of the bacteria. The photosensor colony in the dark behaved like the Mg1655 colony in the dark and spread out. This is normal as cells that do not tend to either tumble or run a lot, will spread the most. <br><br>
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==Flagella==
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=== Biobrick assembly and sequence ===
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We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343004 K343004], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343000 and the terminators B0010 and B0012.<br>
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This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
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<br>
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[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8031| Sequencing results for K343004]
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<br><br>
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When comparing the theoretical sequence with the sequence done on the part K343004 the two sequences were identical.
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=== Effect ===
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=== Video microscopy results ===  
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Our results from the motility assay with semi-solid agar confirm that the biobrick does in fact increase the motility of the cells. We believe this is caused by hyperflagellation facilitated by the overexpression of the flhD/C operon. <br>
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The stability of pSB1C3-K343004 is most likely <20 generations, however the stability of pSB3T5-K343004 was not determined.<br>
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Bacteria containing the photosensor will exhibit a lowered tumbling rate when exposed to blue light (wavelengths around 350nm - 450nm). This was analysed with the help of video microscopy and the open source software [http://db.cse.ohio-state.edu/CellTrack/ "CellTrack"]. The individual cells trajectory was tracked and their speed measured. The tracking results are as follows:
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The growth assay showed no significant difference between the wild type and the cells containing either pSB1C3-K343004 or pSB3K3-K343004.<br>
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In a phase contrast microscope we observed that MG1655 containing pSB1C3-K343004 was longer that wild type. This support the theory that the cells are hyperflagellated and therefore stays longer in the G2-phase in the cell cycle.  
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[[Image:Team-SDU-Denmark-PSblue1.png |250px|Photosensor bacteria exposed to blue light]]
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For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343004 characterization of K343004]. <br><br>
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[[Image:PSred sample 1 trajectory - Cell 1.png |250px|Photosensor bacteria exposed to red light (~580nm wavelength)]]
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[[Image:Team-SDU-Denmark-WTblue1.png |250px|Wildtype bacteria exposed to blue light]]<br>
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The phototaxic bacteria move more in a straight line when exposed to bluelight, as can be seen when comparing the trajectories of the thee bacteria given earlier. These were taken from a batch of 10 cells tracked per sample.
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== Retinal ==
== Retinal ==
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=== Biobrick assembly and sequence ===
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=== UV-Vis spectrophotometer determination of beta-carotene production ===  
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We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343006 K343006], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343002 and the terminators B0010 and B0012.<br>
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<br>
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This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
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In this experiment cells were prepared and harvested according to protocol [https://2010.igem.org/Team:SDU-Denmark/protocols#EX1.1]. This experiment was performed with four different strains of ''E. coli'': <br>
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Sequencing results (as .ab1 files):<br>
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Wildtype Top10 <br>
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Wildtype MG1655 <br>
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Top10 containing PSB1A2 with constitutively active K274210 <br>
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MG1655 containing PSB1A2 with  constitutively active K274210 <br> <br>
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The measurements were performed on cells both in the exponential and stationary phase. The resulting graphs are presented below:<br>
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[[Image:Team-SDU-denmarkBetacarotene samples with controles (exponential_phase).png |350px]]
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[[Image:Team-SDU-denmarkBetacarotene samples with controles (stationary phase).png |350px]]
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In order to assess the results, a series of standard dilutions of beta-carotene were made. The concentrations were 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standard dilutions were also measured on the spectrophotometer. The resulting graphs are presented low: <br>
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[[Image:Team-SDU-denmarkStandart betacaroten curves.png |350px]]
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[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8025| Sequencing results for K343006]
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<br><br>
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When combining the graphs of the samples and the standard dilutions, it is clear that the spectres are uniform. This similarity of the spectres indicates that the cells actually do produce beta-carotene. The graphs also give a qualitative indication of the amount of beta-carotene produced by the MG1655 and the Top10 ''E. coli'' cells. The resulting graphs are presented below: <br><br>
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[[Image:Image-Team-SDU-denmarkBetacarotene standarts and stationary samples.png |350px]]
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<br><br>
<br><br>
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When comparing the theoretical sequence with the sequence done on the part K343006 the two sequences was identical.
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=== Effect ===
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UV-Vis spectroskopy and HPLC analysis of organic extractions of the bacteria containing K343006 didn't give conclusive results conserning the Retinal production.<br>
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The stability of pSB1C3-K343006 is most likely <20 generations.<br>
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The growth assay showed no significant difference between the wild type and the cells containing pSB1C3-K343006.<br>
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For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343006 characterization of K343006]
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Latest revision as of 02:22, 28 October 2010

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