Difference between revisions of "Part:BBa K1381012"

 
 
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<partinfo>BBa_K1381012 short</partinfo>
 
<partinfo>BBa_K1381012 short</partinfo>
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<br><br><i>cheZ</i> ([https://parts.igem.org/Part:BBa_K629003 BBa_K629003]) combined with an RBS ([https://parts.igem.org/Part:BBa_B0034 BBa_B0034]) and constitutive promoter ([https://parts.igem.org/Part:BBa_J23114 BBa_J23114]). This part was created to see if expression of <i>cheZ</i> in a <i>cheZ</i>-knockout strain would restore its motility. Similar parts are: [https://parts.igem.org/Part:BBa_K1381011 BBa_K1381011] and [https://parts.igem.org/Part:BBa_K1381013 BBa_K1381013].
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<br><br>
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<b><i>cheZ</i></b><br>
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Chemotaxis is the cell’s system for directed movement which allows the cell to move towards nutrition sources as well as away from toxic substances. The chemotactic system is a complex chain of phosphorylation reactions. At the end of the chain is the flagellum-binding protein CheY. The phosphorylated CheY (CheY-p) binds the flagella and causes the cell to tumble in place, essentially remaining immotile. The phosphatase CheZ dephosphorylates CheY, causing CheY to unbind to the flagella, resulting in directed movement of the cell [1].
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<b>Characterisation</b><br>
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All motility tests were carried out on tryptone swarm plates (10g/L bactotryptone, 5g/L NaCl, 2,5g/L agar) with chloramphenicol. The bacteria was incubate directly from frozen stock samples, the controlls were taken from re-streaked colonies. Further experiments should be done with liquid cultures at specific OD to ensure same amount of initial cells. 
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<br><br>
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https://static.igem.org/mediawiki/2014/9/91/CheZswarmassay_Uppsala14.PNG
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<br><i>Figure 1. Swarmplate assays incubated for 2 days in room temperature<br><br>
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The following strains are present in the figure:
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<br>
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1)positive control (RP437 with empty pSB1C3 plasmid) [220] <br>
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2)negative control (UU2685 with empty pSB1C3 plasmid) [216] <br>
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3)B0034_J23100-cheZ (UU2685) [170] <br>
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4)B0034_J23113-cheZ (UU2685) [171] <br>
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5)B0034_J23114-cheZ (UU2685) [172] <br>
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<br>
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On all plates presented in figure 2, positive controls (strain 1) swarmed, while negative controls (strain 2) did not swarm. The altered protocol for the swarm plate assay was thus able to distinguish motile strains from non-motile strains.
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<br><br>
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Strain 3, containing a cheZ expression cassette with the J23100 promoter, swarmed all six times when it was tested, though not to the same extent as the positive control. Strain 4, containing a cheZ expression cassette with the J23113 promoter, also showed to be motile, although it only swarmed to a small extent.
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<br><br>
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The result of the strain containing the cheZ expression cassette including the J23114 promoter (strain 5) is not as clear as that of the other two constructs. The structures that have grown upon incubation resemble the negative control. For strain 5, these structures include cloudy formations, however, which is most clearly visible in figure 2C.
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<br><br>
  
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<b>References</b><br>
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[1]  <i>Madigan MT, Martinko JM, Stahl DA, Clark DP. 2010. Brock biology of microorganisms. 13th ed. Benjamin Cummings, San Francisco.</i><br>
 
<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 18:04, 15 October 2014

J23114-B0034-CheZ

cheZ (BBa_K629003) combined with an RBS (BBa_B0034) and constitutive promoter (BBa_J23114). This part was created to see if expression of cheZ in a cheZ-knockout strain would restore its motility. Similar parts are: BBa_K1381011 and BBa_K1381013.

cheZ
Chemotaxis is the cell’s system for directed movement which allows the cell to move towards nutrition sources as well as away from toxic substances. The chemotactic system is a complex chain of phosphorylation reactions. At the end of the chain is the flagellum-binding protein CheY. The phosphorylated CheY (CheY-p) binds the flagella and causes the cell to tumble in place, essentially remaining immotile. The phosphatase CheZ dephosphorylates CheY, causing CheY to unbind to the flagella, resulting in directed movement of the cell [1].

Characterisation
All motility tests were carried out on tryptone swarm plates (10g/L bactotryptone, 5g/L NaCl, 2,5g/L agar) with chloramphenicol. The bacteria was incubate directly from frozen stock samples, the controlls were taken from re-streaked colonies. Further experiments should be done with liquid cultures at specific OD to ensure same amount of initial cells.

CheZswarmassay_Uppsala14.PNG
Figure 1. Swarmplate assays incubated for 2 days in room temperature

The following strains are present in the figure:
1)positive control (RP437 with empty pSB1C3 plasmid) [220]
2)negative control (UU2685 with empty pSB1C3 plasmid) [216]
3)B0034_J23100-cheZ (UU2685) [170]
4)B0034_J23113-cheZ (UU2685) [171]
5)B0034_J23114-cheZ (UU2685) [172]

On all plates presented in figure 2, positive controls (strain 1) swarmed, while negative controls (strain 2) did not swarm. The altered protocol for the swarm plate assay was thus able to distinguish motile strains from non-motile strains.

Strain 3, containing a cheZ expression cassette with the J23100 promoter, swarmed all six times when it was tested, though not to the same extent as the positive control. Strain 4, containing a cheZ expression cassette with the J23113 promoter, also showed to be motile, although it only swarmed to a small extent.

The result of the strain containing the cheZ expression cassette including the J23114 promoter (strain 5) is not as clear as that of the other two constructs. The structures that have grown upon incubation resemble the negative control. For strain 5, these structures include cloudy formations, however, which is most clearly visible in figure 2C.

References
[1] <i>Madigan MT, Martinko JM, Stahl DA, Clark DP. 2010. Brock biology of microorganisms. 13th ed. Benjamin Cummings, San Francisco.
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]