Difference between revisions of "Part:BBa K1590006"
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Figure 1: Detection of His-tagged LSS in whole cells of E.coli. Single colonies of E.coli strain M15 pREP4 harbouring LSS. Cells were used to inoculate 5ml of LB growth medium supplemented with 100ug/ml ampicillin and 50ug/ml Kanamycin. Once the OD600 reached 0.7 the cells were then induced with IPTG, as indicated. Cells were then grown for a further 4 hours at 37oC, 1ml aliquots were pelleted and cells reuspended in 100ul laemmli buffer and 20ul of samples were separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose membrane and probed with anti-His antibody.
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Figure 1: Detection of His-tagged LSS in whole cells of <i>E.coli</i>. Single colonies of <i>E.coli</i> strain M15 pREP4 harbouring LSS. Cells were used to inoculate 5ml of LB growth medium supplemented with 100µg/ml ampicillin and 50µg/ml Kanamycin. Once the OD<sub>600</sub> reached 0.7 the cells were then induced with IPTG, as indicated. Cells were then grown for a further 4 hours at 37<sup>o</sup>C, 1ml aliquots were pelleted and cells reuspended in 100ul laemmli buffer and 20µl of samples were separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose membrane and probed with anti-His antibody.
  
  
 
The enzyme was purified by Ni-affinity chromatography. The eluted fractions under the peak were run on an SDS gel (12% acrylamide). Furthermore, western blotting against 6-His - LS was done. Surprisingly the eluted protein showed a molecular mass of only 37 kDa, whereas 83 kDa were expected.
 
The enzyme was purified by Ni-affinity chromatography. The eluted fractions under the peak were run on an SDS gel (12% acrylamide). Furthermore, western blotting against 6-His - LS was done. Surprisingly the eluted protein showed a molecular mass of only 37 kDa, whereas 83 kDa were expected.
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https://static.igem.org/mediawiki/parts/a/a6/LSDundee20153.jpg
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https://static.igem.org/mediawiki/2015/a/a1/Finger-fig5.png
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<br>
 
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Figure 2: Purification of lanosterol synthase by nickel affinity chromatography. A) Chromatogram showing the purification profile of the His-tagged haptoglobin. The fractions labelled (A8-A12) were further analysed by SDS page gel. B) 10ul of the labelled fractions were mixed with 10ul laemmli buffer and loaded onto a 12.5% SDS-PAGE gel and stained with Coomassie Blue.  
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Figure 2: Purification of lanosterol synthase by nickel affinity chromatography. A) Chromatogram showing the purification profile of the His-tagged haptoglobin. The fractions labelled (A8-A12) were further analysed by SDS page gel. B) 10µl of the labelled fractions were mixed with 10µl laemmli buffer and loaded onto a 12.5% SDS-PAGE gel and stained with Coomassie Blue.  
  
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In order to avoid the enzyme destroying vital evidence at a crime scene, the next step would be to mutate one or more of the active site residues(positions 232 and 455) in order to inhibit the catabolic activity of the enzyme while still retaining binging.
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Lanosterol synthase was expected to be 83kDa, however the majority of protein purified is at 37kDa. This was further analysed by carrying out a western blot to detect for any full length LSS- His purified.
  
  
 +
https://static.igem.org/mediawiki/2015/9/90/LSpurificationIMACDundee2015.jpg
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Figure 3. Elutions corresponding to the peaks from the nickel IMAC were collected ran on a 12.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane and probed with an anti-His antbody. The band detected in the blot corresponds to the thick banding detected in Fig 2, at 37kDa.
 +
 +
The results obtained from the blot showed no presence of lanosterol synthase at 83kDa, suggesting we are only detecting a degraded version of it instead of the whole enzyme. The purification conditions will have to be optimized in order to continue with our fingerprint aging device.
 +
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In order to avoid the enzyme destroying vital evidence at a crime scene, the next step would be to mutate one or more of the active site residues(positions 232 and 455) in order to inhibit the catabolic activity of the enzyme while still retaining binging.
  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1590006 parameters</partinfo>
 
<partinfo>BBa_K1590006 parameters</partinfo>

Latest revision as of 11:47, 19 September 2015

Human Lanosterol Synthase

Lanosterol Synthase catalyses the reaction from 2,3-oxido-squalene (squalene epoxide) to Lanosterol. It is one of the enzymes in the enzymatic cascade that converts squalene to cholesterol through stepwise modifications of the substrate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 819
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 666
    Illegal AgeI site found at 145
    Illegal AgeI site found at 283
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Lanosterol synthase (LSS) an oxidosqualene cyclase (OSC) enzyme that specifically binds to squalene epoxide (2,3- oxidosqualene), which is present in fingerprints. Our modelling showed us that squalene epoxide is the compound with the most distinct degradation pattern in fingerprints, and it was hence selected as an appropriate target for approximating the age of a fingerprint.

Western blot analysis of the 6-His - LS shows successful expression of the enzyme.

Finger-fig4.png
Figure 1: Detection of His-tagged LSS in whole cells of E.coli. Single colonies of E.coli strain M15 pREP4 harbouring LSS. Cells were used to inoculate 5ml of LB growth medium supplemented with 100µg/ml ampicillin and 50µg/ml Kanamycin. Once the OD600 reached 0.7 the cells were then induced with IPTG, as indicated. Cells were then grown for a further 4 hours at 37oC, 1ml aliquots were pelleted and cells reuspended in 100ul laemmli buffer and 20µl of samples were separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose membrane and probed with anti-His antibody.


The enzyme was purified by Ni-affinity chromatography. The eluted fractions under the peak were run on an SDS gel (12% acrylamide). Furthermore, western blotting against 6-His - LS was done. Surprisingly the eluted protein showed a molecular mass of only 37 kDa, whereas 83 kDa were expected. LSDundee20153.jpg
Figure 2: Purification of lanosterol synthase by nickel affinity chromatography. A) Chromatogram showing the purification profile of the His-tagged haptoglobin. The fractions labelled (A8-A12) were further analysed by SDS page gel. B) 10µl of the labelled fractions were mixed with 10µl laemmli buffer and loaded onto a 12.5% SDS-PAGE gel and stained with Coomassie Blue.

Lanosterol synthase was expected to be 83kDa, however the majority of protein purified is at 37kDa. This was further analysed by carrying out a western blot to detect for any full length LSS- His purified.


LSpurificationIMACDundee2015.jpg


Figure 3. Elutions corresponding to the peaks from the nickel IMAC were collected ran on a 12.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane and probed with an anti-His antbody. The band detected in the blot corresponds to the thick banding detected in Fig 2, at 37kDa.

The results obtained from the blot showed no presence of lanosterol synthase at 83kDa, suggesting we are only detecting a degraded version of it instead of the whole enzyme. The purification conditions will have to be optimized in order to continue with our fingerprint aging device.

In order to avoid the enzyme destroying vital evidence at a crime scene, the next step would be to mutate one or more of the active site residues(positions 232 and 455) in order to inhibit the catabolic activity of the enzyme while still retaining binging.

Functional Parameters