Difference between revisions of "Part:BBa K1736300"
 
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[[File:sydney_laciq1.png | 800px | thumb | center | LacI construct with LacIQ1 strong constitutive promoter and wild-type RBS.]]
 
[[File:sydney_laciq1.png | 800px | thumb | center | LacI construct with LacIQ1 strong constitutive promoter and wild-type RBS.]]
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=Background=
  
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As explained in our project's [http://2015.igem.org/Team:Sydney_Australia/project_overview description & results] page, the P<sub>LacIQ1</sub> part was added to the project to tackle the issue of possible toxicity to the host cells due to overexpression of our main enzyme of interest (ethene monooxygenase). This enzyme is known to be very difficult to clone and express in heterologous hosts, and since our primary cloning vector (pBBR1MCS-2) only had the Plac promoter but no LacI repressor, we needed to add the latter part separately.  
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P<sub>LacIQ1</sub> was utilised in our project (see [http://2015.igem.org/Team:Sydney_Australia/project_overview description & results]) to tackle the issue of possible toxicity to host cells due to overexpression of our main enzyme of interest (ethene monooxygenase). This enzyme is known to be very difficult to clone and express in heterologous hosts, and since our primary cloning vector (pBBR1MCS-2) only had the Plac promoter but no LacI repressor, we needed to add the latter part separately.  
  
 
In order to ensure that we had strong expression and activity of LacI, we placed the LacIQ1 promoter <sup>1</sup> upstream of the ''E. coli'' LacI gene . In this version of the promoter, the -35 sequence has been modified to make it identical to the ''E.coli'' consensus sequence TTGACA, thus making the promoter much stronger than usual. The following Parts are already deposited for P<sup>LacIQ1</sup>: [https://parts.igem.org/Part:BBa_K091112 BBa_K091112] and [https://parts.igem.org/Part:BBa_K091131 BBa_K091131], while for LacI itself, many parts for the gene have also been deposited, such as [https://parts.igem.org/Part:BBa_C0012 BBa_C0012]. However, to date there is no composite part where the LacI gene and LacIQ1 have been placed together, making this a valuable new addition to the registry, which will be useful for future teams and experiments relying on strong expression control of genes under ''lac'' regulation.  
 
In order to ensure that we had strong expression and activity of LacI, we placed the LacIQ1 promoter <sup>1</sup> upstream of the ''E. coli'' LacI gene . In this version of the promoter, the -35 sequence has been modified to make it identical to the ''E.coli'' consensus sequence TTGACA, thus making the promoter much stronger than usual. The following Parts are already deposited for P<sup>LacIQ1</sup>: [https://parts.igem.org/Part:BBa_K091112 BBa_K091112] and [https://parts.igem.org/Part:BBa_K091131 BBa_K091131], while for LacI itself, many parts for the gene have also been deposited, such as [https://parts.igem.org/Part:BBa_C0012 BBa_C0012]. However, to date there is no composite part where the LacI gene and LacIQ1 have been placed together, making this a valuable new addition to the registry, which will be useful for future teams and experiments relying on strong expression control of genes under ''lac'' regulation.  
  
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To confirm that the new composite part is functional, we ran a qualitative experiment to check for the activity of LacI via its control of the reporter gene LacZ based on hydrolysis of the colourless reagent X-gal to yield a blue dye . This system was comprised of three parts: the P<sub>LacIQ1</sub>-LacI part cloned in pSB1C3 (CmR), the lacZ alpha fragment in the plasmid pBR1MCS-2 (KmR) under the control of Plac, and the lacZ omega fragment in the chromosome of ''E. coli'' JM109, also under control of Plac.  
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=Experimental Validation=
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To confirm that the composite part is functional, we ran a qualitative experiment to check for the activity of LacI via its control of the reporter gene LacZ based on hydrolysis of the colourless reagent X-gal to yield a blue dye . This system was comprised of three parts: the P<sub>LacIQ1</sub>-LacI part cloned in pSB1C3 (CmR), the lacZ alpha fragment in the plasmid pBR1MCS-2 (KmR) under the control of Plac, and the lacZ omega fragment in the chromosome of ''E. coli'' JM109, also under control of Plac.  
  
 
[[File:sydney_lac_operon.png | center | 800px | thumb | Schematic of the experimental setup for testing LacI activity in ''E.coli''. Note that part of lacZ is coded on a plasmid, and another part is coded on the chromosome.]]
 
[[File:sydney_lac_operon.png | center | 800px | thumb | Schematic of the experimental setup for testing LacI activity in ''E.coli''. Note that part of lacZ is coded on a plasmid, and another part is coded on the chromosome.]]
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[[File:sydney_parts_laci_psb1c3.png | 700px | thumb | center | Structure of the pSB1C3-PlacIQ1-LacI plasmid construct submitted to the registry. ]]
 
[[File:sydney_parts_laci_psb1c3.png | 700px | thumb | center | Structure of the pSB1C3-PlacIQ1-LacI plasmid construct submitted to the registry. ]]
  
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=References=
 
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<sup>1</sup> Glascock CB. ''et al.'' Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli. Gene. 1998 Nov 26;223(1-2):221-31.
 
  
 
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<partinfo>BBa_K1736300 parameters</partinfo>
 
<partinfo>BBa_K1736300 parameters</partinfo>
 
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=References=
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<sup>1</sup> Glascock CB. ''et al.'' Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli. Gene. 1998 Nov 26;223(1-2):221-31.

Latest revision as of 14:04, 18 September 2015

LacI-LacIQ1

LacI construct with LacIQ1 strong constitutive promoter and wild-type RBS.

Background

PLacIQ1 was utilised in our project (see [http://2015.igem.org/Team:Sydney_Australia/project_overview description & results]) to tackle the issue of possible toxicity to host cells due to overexpression of our main enzyme of interest (ethene monooxygenase). This enzyme is known to be very difficult to clone and express in heterologous hosts, and since our primary cloning vector (pBBR1MCS-2) only had the Plac promoter but no LacI repressor, we needed to add the latter part separately.

In order to ensure that we had strong expression and activity of LacI, we placed the LacIQ1 promoter 1 upstream of the E. coli LacI gene . In this version of the promoter, the -35 sequence has been modified to make it identical to the E.coli consensus sequence TTGACA, thus making the promoter much stronger than usual. The following Parts are already deposited for PLacIQ1: BBa_K091112 and BBa_K091131, while for LacI itself, many parts for the gene have also been deposited, such as BBa_C0012. However, to date there is no composite part where the LacI gene and LacIQ1 have been placed together, making this a valuable new addition to the registry, which will be useful for future teams and experiments relying on strong expression control of genes under lac regulation.

Experimental Validation

To confirm that the composite part is functional, we ran a qualitative experiment to check for the activity of LacI via its control of the reporter gene LacZ based on hydrolysis of the colourless reagent X-gal to yield a blue dye . This system was comprised of three parts: the PLacIQ1-LacI part cloned in pSB1C3 (CmR), the lacZ alpha fragment in the plasmid pBR1MCS-2 (KmR) under the control of Plac, and the lacZ omega fragment in the chromosome of E. coli JM109, also under control of Plac.

Schematic of the experimental setup for testing LacI activity in E.coli. Note that part of lacZ is coded on a plasmid, and another part is coded on the chromosome.

E.coli JM109 cells containing both plasmids were grown on a variety of media to test the interactions between lacI and Plac in this system. These media were:

1. LB-Cm-Km-Xgal (repressed, expect white colonies due to lack of IPTG)

2. LB-Cm-Km-Xgal-glucose (strongly repressed, expect white colonies due to lack of IPTG and presence of glucose)

3. LB-Cm-Km-Xgal-IPTG (induced, expect blue colonies due to presence of IPTG)

4. LB-Km-Xgal-IPTG (strongly induced, expect blue colonies due to presence of IPTG and loss of plasmid carrying lacI repressor gene)

Plates of the four different media mentioned above to test for the activity of PLacIQ1-LacI.

The results of this experiment were not clear cut (above). Cells grown on LB-Cm-Km-Xgal-glucose were completely white, as expected, and cells grown on LB-Km-Xgal-IPTG were predominantly blue, as expected, but the other two media yielded a mix of white and blue cells, mostly white. It was notable that addition of IPTG was not sufficient to give uniform positive induction of lacZ. Our interpretation is that in this experimental setup, lacI repression is exceedingly strong. We believe this is because the amount of LacI protein expressed from the strong promoter on a high-copy plasmid will completely repress expression of the chromosomally-encoded omega fragment of LacZ, even when IPTG is present.

Structure of the pSB1C3-PlacIQ1-LacI plasmid construct submitted to the registry.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1 Glascock CB. et al. Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli. Gene. 1998 Nov 26;223(1-2):221-31.