Team:SDU-Denmark/project-r

From 2010.igem.org

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(Effect)
(Biobrick assembly and sequence)
 
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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>
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>
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
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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Sequencing results (as .ab1 files):<br>
 
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[http://2010.igem.org/wiki/images/a/ab/Sequencing_K343007.ab1| Sequencing results K343007]
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[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8039| Sequencing results K343007]
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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.
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.
=== Effect ===
=== Effect ===
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Our results from the experiments with semi-solid agar confirm that the biobrick does indeed couple itself to the bacterial chemotaxis pathway and modify the bacterial motility pattern by modifying the tumbling frequency. For further information on this conclusion see [http://2010.igem.org/Team:SDU-Denmark/project-p#Growth_of_bacterial_culture_on_semi-solid_agar_plates growth of bacteria on semi-solid agar plates]<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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The videomicroscopy indicates that blue light with a wavelength around 500nm leads to CheA's autophosphorylation being downregulated. This means that the bacterial tumbling frequency gets reduced and the bacteria will spend an increased time in the "run" mode of propulsion. This means that the bacteria will travel further in a certain amount of time, compared to wildtype bacteria. More details can be found here [http://2010.igem.org/Team:SDU-Denmark/project-p#Videomicroscopy_and_computer_analysis_of_bacterial_motility videomicroscopy]<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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The stability of pSB1C3-K343007 is most likely <20 generations, however the stability of pSB3T5-K343007 was not determined (see [http://2010.igem.org/Team:SDU-Denmark/project-p#Stability_assay stability assay])<br><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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Plasmids expressing K343007 does not seem to hinder the bacterial growth in any way (see [http://2010.igem.org/Team:SDU-Denmark/project-p#Growth_assay growth assay])<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 [http://2010.igem.org/Team:SDU-Denmark/K343007 characterization of K343007].
==Flagella==
==Flagella==
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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>
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>
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
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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Sequencing results (as .ab1 files):<br>
 
<br>
<br>
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[http://2010.igem.org/wiki/images/a/ab/Sequencing_K343004.ab1| Sequencing results for K343004]
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[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8031| Sequencing results for K343004]
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When comparing the theoretical sequence with the sequence done on the part K343004 the two sequences was identical.
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When comparing the theoretical sequence with the sequence done on the part K343004 the two sequences were identical.
=== Effect ===
=== Effect ===
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Our results from the motility assay with semi-solid agar confirm that the biobrick does infact increase the motility of the cells. We believe this is caused by hyperflagellation. <br><br>
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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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The growth assay showed no significant difference between the wild type and the cells containing either of the two plasmids.<br>
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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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For further information, raw data and background of the assay see [http://2010.igem.org/Team:SDU-Denmark/project-p#K343004 characterization of K343004] <br><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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For further information, raw data and background of the assay see [http://2010.igem.org/Team:SDU-Denmark/K343004 characterization of K343004]. <br><br>
== Retinal ==
== Retinal ==
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Sequencing results (as .ab1 files):<br>
Sequencing results (as .ab1 files):<br>
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[http://2010.igem.org/wiki/images/a/ab/Sequencing_K343006.ab1| Sequencing results for K343006]
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[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8025| Sequencing results for K343006]
<br><br>
<br><br>
When comparing the theoretical sequence with the sequence done on the part K343006 the two sequences was identical.
When comparing the theoretical sequence with the sequence done on the part K343006 the two sequences was identical.
=== Effect ===
=== Effect ===
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The growth assay of ''E. coli'' strain MG1655 transformed with plasmids containing the NinaB brick shows no significant difference from the wild type ''E. coli'' strain MG1655, we did however remove and outlier from the [http://2010.igem.org/Team:SDU-Denmark/project-p#3._Growth_measurement: graph]. [http://2010.igem.org/Team:SDU-Denmark/raw raw data] <br>
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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 [http://2010.igem.org/Team:SDU-Denmark/K343006 characterization of K343006]  

Latest revision as of 02:22, 28 October 2010