Difference between revisions of "Part:BBa K4152008"
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[[File:mt8-pcr1'.png|400px]]<br>
 
[[File:mt8-pcr1'.png|400px]]<br>
 
'''Figure 3.'''  Fragments of mutated PK gene are PCR-amplified independently.<br>
 
'''Figure 3.'''  Fragments of mutated PK gene are PCR-amplified independently.<br>
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</p>
 
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<p style="text-align: center;">
 
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[[File:Mt8-pcr2'.png|250px]]<br>
 
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'''Figure 4.'''  Fragments of mutated PK gene are PCR-amplified independently.<br>
 
 
</p>
 
</p>
 
*2. Fuse the segments together in a subsequent reaction by High-fidelity thermostable DNA polymerase.
 
*2. Fuse the segments together in a subsequent reaction by High-fidelity thermostable DNA polymerase.
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
[[File:Mt8-pcr3'.png|250px]]<br>
 
[[File:Mt8-pcr3'.png|250px]]<br>
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'''Figure 5.'''  PCR Mutagenesis by Overlap Extension to obtain the mutated PK gene.<br>
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'''Figure 4.'''  PCR Mutagenesis by Overlap Extension to obtain the mutated PK gene.<br>
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</p>
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*3. Use restriction endonuclease XhoⅠ and EcoRⅠ to double digest our mutated PK gene and pPIC9.
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<p style="text-align: center;">
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[[File:Mt8-dd1'.png|250px]]<br>
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'''Figure 6.'''  Double digestion of mutated PK.<br>
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</p>
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<p style="text-align: center;">
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[[File:Mt8-dd2'.png|250px]]<br>
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'''Figure 7.'''  Double digestion of pPIC9.<br>
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</p>
 
</p>
 
*4. Use Ligase to link our mutated PK and pPIC9 after double digestion. <br>
 
*4. Use Ligase to link our mutated PK and pPIC9 after double digestion. <br>
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<p style="text-align: center;">
 
<p style="text-align: center;">
 
[[File:Mt8-dd3'.png|400px]]<br>
 
[[File:Mt8-dd3'.png|400px]]<br>
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'''Figure 8.'''  Double digestion verification of Recombinant pPIC9-PK.<br>
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'''Figure 5.'''  Double digestion verification of Recombinant pPIC9-PK.<br>
 
</p>
 
</p>
 
After verification, it was determined that the construction is successful. We transformed the constructed plasmid into competent DH5α cells to expand the plasmid largely<br>
 
After verification, it was determined that the construction is successful. We transformed the constructed plasmid into competent DH5α cells to expand the plasmid largely<br>
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<p style="text-align: center;">
 
<p style="text-align: center;">
 
[[File:Mt8-linearization.png|500px]]<br>
 
[[File:Mt8-linearization.png|500px]]<br>
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'''Figure 9.'''  Linearization of Recombinant pPIC9-PK.<br>
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'''Figure 6.'''  Linearization of Recombinant pPIC9-PK.<br>
 
</p>
 
</p>
 
'''Electrotransformation:'''<br>
 
'''Electrotransformation:'''<br>
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* 1. Use MD solid medium to screen positive GS115 cells which can grow without Histidine. (Because GS115 cannot grow at medium without Histidine except our gene was introduced in).<br>
 
* 1. Use MD solid medium to screen positive GS115 cells which can grow without Histidine. (Because GS115 cannot grow at medium without Histidine except our gene was introduced in).<br>
 
* 2. Extract the genomic DNA of recombinant GS115 and verify the sequence of Recombinant pPIC9-PK (from AOX1 promoter to AOX1 Terminator, about 1500bp).  
 
* 2. Extract the genomic DNA of recombinant GS115 and verify the sequence of Recombinant pPIC9-PK (from AOX1 promoter to AOX1 Terminator, about 1500bp).  
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<p style="text-align: center;">
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[[File:Mt8-genome pcr.png|300px]]<br>
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'''Figure 10.'''  Genome PCR genomic DNA in Recombinant GS115.<br>
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</p>
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* 3. Transfer the positive clones and preserve them in Glycerin (sterile), storing them at -80°C.<br>
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'''Express PK with Methanol:'''<br>
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Transfer some Glycerin recombinant GS115 to YPD, culture overnight. Then transfer some YPD culture to BMG, culture overnight. Transfer some BMG culture to BMM, add 0.6% Methanol daily, express PK for several days, then collect the supernatant and concentration it. At last, we do SDS-PAGE to make sure that the mutated PK has expressed successfully, and take the standard samples to do Western blot and quantitative analysis of stripes with ImageJ, then figure out the mass of PK.<br>
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<p style="text-align: center;">
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[[File:Mt8-SDS-PAGE-1.png|450px]]<br>
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'''Figure 11.'''  SDS-PAGE-1.<br>
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</p>
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<p style="text-align: center;">
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[[File:Mt8-SDS-PAGE-2.png|450px]]<br>
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'''Figure 12.'''  SDS-PAGE-2.<br>
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</p>
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===Enzyme activity and thermostability determination===
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We use Enzyme-labeled instrument to measure the Abs of OD<sub>660nm</sub> of the product of L-Tyrosine of the reaction. We use 1% Casein as our substrate, and Tris-HCl (pH8.0) as our Buffer, react at 55°C for several minutes. Then add trichloroacetic acid (TCA) to end the reaction, centrifuge to collect the supernatant where contains our product of L-Tyrosine. Next step, we use Na<sub>2</sub>CO<sub>3</sub> to provide alkaline environment, then add the supernatant and Folin-phenol reagant to colorate L-Trosine. In the end, we detect the Abs of OD<sub>660nm</sub> to assess the enzyme activity of our PK. <br>
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We store our PK at Room temperature for several days and detect the remains of it, then assess the thermostability of PK.
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<p style="text-align: center;">
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[[File:Mt8.png|300px]]<br>
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'''Figure 13.'''  Enzyme activity determination, compared with wild type.
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</p>
 
</p>
 
===Conclusion===
 
===Conclusion===
 
In conclusion,the thermostability of the Mutated PK has improved '''在这里填东西''' times with Ca<sup>2+</sup>, improved '''在这里填东西''' times without Ca<sup>2+</sup> compared with wild type of PK.
 
In conclusion,the thermostability of the Mutated PK has improved '''在这里填东西''' times with Ca<sup>2+</sup>, improved '''在这里填东西''' times without Ca<sup>2+</sup> compared with wild type of PK.

Revision as of 15:33, 8 October 2022


PK_MT8

To improve the performance of Proteinase K, we designed many Proteinase K mutant genes. PK_MT8 is a complicated mutation of Proteinase K, which contains 2 mutation sites: P175F-S197W.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 235
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 235
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 235
  • 1000
    COMPATIBLE WITH RFC[1000]


Origin(organism)

Tritirachium album limber

Structure Design

  • 1. Use PyMOL to mutate some residues of Proteinase K, analyse the possibility of the formation of new interaction forces like hydrogen bond, salt bond, disulfide bond and π-π interaction.
  • 2. Use AlphaFold v2.1.0 to predict the structure of the mutated PK.

Mt8-alphafold.png
Figure 1. The mutated PK structure compared to the Wild type PK.

  • 3. Use FoldX to calculate the Gibbs Free Energy compared to wild type PK with Ca2+. (PDB ID: 1ic6) The result of this mutated PK's ΔΔG is -13.81 kcal/mol.

Molecular cloning

We used the wild type Proteinase K(Hereinafter referred to as PK) DNA gene to overlap our mutated PK gene.

Mt8-total of pcr'.png
Figure 2. The process of PCR for our mutated PK gene.

  • 1. Use mutated PK primers to clone our small fragments.

Mt8-pcr1'.png
Figure 3. Fragments of mutated PK gene are PCR-amplified independently.

  • 2. Fuse the segments together in a subsequent reaction by High-fidelity thermostable DNA polymerase.

Mt8-pcr3'.png
Figure 4. PCR Mutagenesis by Overlap Extension to obtain the mutated PK gene.

  • 4. Use Ligase to link our mutated PK and pPIC9 after double digestion.
  • 5. Transform the constructed plasmid into competent DH5α cells to expand the plasmid largely
  • 6. Extract the recombinant pPIC9-PK and verify it by double digestion (XhoⅠ and EcoRⅠ), and sequence it for verication of mutation sites.

Mt8-dd3'.png
Figure 5. Double digestion verification of Recombinant pPIC9-PK.

After verification, it was determined that the construction is successful. We transformed the constructed plasmid into competent DH5α cells to expand the plasmid largely

Expression in Pichia Pastoris

Linearization of Recombinant pPIC9-PK:
We used restriction endonuclease SalⅠ to linearize our recombinant plasmid.

Mt8-linearization.png
Figure 6. Linearization of Recombinant pPIC9-PK.

Electrotransformation:
Add several μg linearized pPIC9-PK to GS115 competence cells, then use 1.5kV electric pulse to drill holes to let gene get in.
Screen positive colonies and culture preservation:

  • 1. Use MD solid medium to screen positive GS115 cells which can grow without Histidine. (Because GS115 cannot grow at medium without Histidine except our gene was introduced in).
  • 2. Extract the genomic DNA of recombinant GS115 and verify the sequence of Recombinant pPIC9-PK (from AOX1 promoter to AOX1 Terminator, about 1500bp).

</p>

Conclusion

In conclusion,the thermostability of the Mutated PK has improved 在这里填东西 times with Ca2+, improved 在这里填东西 times without Ca2+ compared with wild type of PK.