Difference between revisions of "Part:BBa K1736300"

 
 
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As explained in the project description & results page, the LacI-LacIQ1 was a late addition to the project to tackle the issue of toxicity to the expressing cells as a results of overexpression or over-synthesis of the enzymatic products. In this way, we add a layer of control to the expression of our ethene MO enzymes to ensure that they are expressing at appropriate and safe levels. Furthermore, its inactivation during the lab preparatory procedures of transformation and developing competent cells can aid the cell to deal with the immense amount of physiological stress imposed upon it and avoid an impending apoptosis.
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[[File:sydney_laciq1.png | 800px | thumb | center | LacI construct with LacIQ1 strong constitutive promoter and wild-type RBS.]]
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In order to ensure that we have a constitutively strong expression and activity of LacI, we placed the LacIQ1 promoter upstream of the E. coli LacI ORF, which is highly homologous and similar across bacteria. The following parts are already deposited for LacIQ1: BBa_K091112 and BBa_K091131. As for LacI, many parts for the gene have been deposited such as BBa_C0012. However, there is no composite part where the LacI gene and LacIQ1 have been placed together, making a new addition to the registry which will be useful for future and teams and experiments relying on strong expression control of gene under lac operon regulation.
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=Background=
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To confirm that the composite part is functional, we ran an experiment to qualitative check for the activity of LacI via the reporter gene LacZ and it's hydrolysis of X-gal (derivative of lactose which turns blue upon hydrolysis by B-galatosidase encoded by LacZ-alpha, and is not taken up by the bacteria). E. coli competent cells containing the LacZ-alpha gene (placed under the control of lac promoter) were transformed with LacI-LacIQ1 and placed in media containing IPTG and no IPTG to induce expression of LacZ (B-galactosidase), with both having equal amounts of X-gal.
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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.  
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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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=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.
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[[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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''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:
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1. LB-Cm-Km-Xgal (repressed, expect white colonies due to lack of IPTG)
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2. LB-Cm-Km-Xgal-glucose (strongly repressed, expect white colonies due to lack of IPTG and presence of glucose)
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3. LB-Cm-Km-Xgal-IPTG (induced, expect blue colonies due to presence of IPTG)
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4. LB-Km-Xgal-IPTG (strongly induced, expect blue colonies due to presence of IPTG and loss of plasmid carrying lacI repressor gene)
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[[File:sydney_iptg_laci_plates.png | center | 800px | thumb | Plates of the four different media mentioned above to test for the activity of P<sub>LacIQ1</sub>-LacI.]]
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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.
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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. ]]
  
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After the induction, there were blue colonies on the IPTG media as expected, however, some blue colonies (to a lesser extent qualitatively) in the media containing no IPTG. This is unexpected and hypothesised to be due to the genomic native host lac operon system being responsible for the B-galactisidase activity and expression. This explanation becomes more credible considering that the host contains a lac operon containing both fragments of B-galactosidase enzyme LacZ-alpha and LacZ-beta, where each are under the control of a separate lac promoter. Hence, due to the high-copy presence of LacZ-alpha in a recombiannt vector in the cells and the higher expression of repressor proteins (also due to the high copy number of recombinant vectors containing LacI-LacIQ1), most of the repressor protein block the LacZ-alpha on the vectors, not the genomic ones. Furthermore, it could also be possible that the LacI repressor cannot control the genomic LacZ-beta expression, which can still be expressed and perform X-gal hydrolysis. Hence, while we know this system should work due to countless studies including a paper on LacIQ1 by Glascock CB et al. 3, every user of this construct has to check for the absence of LacZ subunits in the genome which could breakaway from the claws of LacI repressor control.
 
  
 
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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.