Difference between revisions of "Part:BBa K2587024"
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<strong> Characterization using growth measurement by OD 600 </strong>
 
<strong> Characterization using growth measurement by OD 600 </strong>
 
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We analysed this construct by measuring the optical density of wild type <i> E.coli </i> BL21 (DE3) C43 cells, and the corresponding transformants harbouring the lysis construct.  
 
We analysed this construct by measuring the optical density of wild type <i> E.coli </i> BL21 (DE3) C43 cells, and the corresponding transformants harbouring the lysis construct.  
 
After one day incubation, cells were diluted to an OD of 0.1 in LB medium with 100µg/mL final ampicillin concentration. 200µl were then added to a 96 well plate and OD600 has been measured at 37°C at 200 rpm shaking for a total of 24 hours.
 
After one day incubation, cells were diluted to an OD of 0.1 in LB medium with 100µg/mL final ampicillin concentration. 200µl were then added to a 96 well plate and OD600 has been measured at 37°C at 200 rpm shaking for a total of 24 hours.
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https://static.igem.org/mediawiki/parts/3/35/T--Duesseldorf--lysis_construct.PNG
 
https://static.igem.org/mediawiki/parts/3/35/T--Duesseldorf--lysis_construct.PNG
 
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<strong> Figure 2: Growth of <i>E.coli</i> wildtype and <i>E.coli</i> with the lysis plasmid. Cell lysis was not directly measured, but derived from the OD600, which is a measurement of intact cells in the suspension.  
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<strong> Figure 2: Growth of <i>E.coli</i> wildtype and <i>E.coli</i> with the lysis plasmid.</strong> Cell lysis was not directly measured, but derived from the OD600, which is a measurement of intact cells in the suspension.  
 
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<strong>Characterisation using further induction with synthetized N-(3-Oxohexanoyl)-L-homoserine lactone</strong>
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To check whether more cell lysis and as a consequence less cells were present upon induction with further quorum sensing molecule, a further test was performed. Here cells were induced with 0.05mM 3-oxo-C6-HSL prior incubation. Here as well, incubation was performed at 37°C, 200rpm and 24 hours.
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<strong>Results</strong> Growth is only slightly influenced and only at the end of the stationary phase as measured by the optical density at 600nm.
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https://static.igem.org/mediawiki/parts/7/78/T--Duesseldorf--%2BAHL.PNG
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<strong>Figure 3: Optical density of E.coli wild type cells, E.coli with lysis construct and E.coli with lysis construct and AHL.</strong>
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2587024 parameters</partinfo>
 
<partinfo>BBa_K2587024 parameters</partinfo>
 
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Revision as of 08:56, 1 October 2018


luxI_luxR_Plux_E

This part is an assembly of different constructs, which then yield a final plasmid, able to self lyse Escherichia coli cells. It is a construct based on the Quorum sensing system of Vibrio fischeri. With the addition of a lysis gene it will induce cell lysis upon synthesis of the quorum sensing molecule acyl homoserine lactone. Upon synthesis of this by the LuxI gene, it will bind to the regulator LuxR and then to the promoter pLux, which will activate synthesis of the lysis protein E from the bacteriophage phiX174E.


Usage and Biology


  • Acyl homoserine lactone synthase : LuxI
  • Regulator: LuxR
  • Promoter: pLux, inducible by the quorum sensing molecule acyl homoserine lactone bound to LuxR
  • Lysis gene: Lysis gene E from bacteriophage phiX174E
  • Lysis protein induces host cell lysis by inhibiting host translocase MraY activity, which is necessary for catalysis of lipid I , a factor for cell wall synthesis (Cytolysis) (1)
  • This construct is able to modify E.coli´s growth, by slowing it down
  • This part contains Type II S restriction sites and can be further used for scarless assembly with other parts

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
    Illegal NheI site found at 829
    Illegal NheI site found at 852
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1082
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 892
    Illegal BsaI site found at 1681
    Illegal BsaI site found at 1832
    Illegal BsaI.rc site found at 1523
    Illegal BsaI.rc site found at 1790


Characterization


Characterisation of promoter by fluorescence measurement of GFP
To check whether the promoter, pLux, is really induced by the binding of the acyl homoserine lactone to the LuxR, a further construct (BBa_K2587027) is created which has the lysis gene E exchanged by a GFP gene. Here the relative fluorescence units are measured in comparison to the wild type E.coli cells.

Results It can clearly be observed, that fluorescence increases upon growth of the cells. Wild type cells, as a negative control, show little or no fluorescence at all.

T--Duesseldorf--promoter_functionality.PNG
Figure 1: Relative fluorescence measurement of lysis construct and E.coli wild type cells (negative control). Exctinction: 485nm; Emission: 520nm. Cells were measured only within a time span of 24h.



Characterization using growth measurement by OD 600

We analysed this construct by measuring the optical density of wild type E.coli BL21 (DE3) C43 cells, and the corresponding transformants harbouring the lysis construct. After one day incubation, cells were diluted to an OD of 0.1 in LB medium with 100µg/mL final ampicillin concentration. 200µl were then added to a 96 well plate and OD600 has been measured at 37°C at 200 rpm shaking for a total of 24 hours.

Results Growth is clearly influenced by the presence of the lysis plasmid as compared to the wild type E.coli cells. Cells harbouring this plasmid grow slower. Observation of their growth shows a remissive exponential phase, reaching the stationary phase with a lower cell density than wild type.

T--Duesseldorf--lysis_construct.PNG
Figure 2: Growth of E.coli wildtype and E.coli with the lysis plasmid. Cell lysis was not directly measured, but derived from the OD600, which is a measurement of intact cells in the suspension.