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| − | __NOTOC__
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| − | <partinfo>BBa_K4152008 short</partinfo>
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| − | 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.
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| − | <!-- Add more about the biology of this part here
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| − | ===Usage and Biology===
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K4152008 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K4152008 parameters</partinfo>
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| − | <!-- -->
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| − | ===Origin(organism)===
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| − | Tritirachium album limber
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| − | ===Structure Design===
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| − | *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.
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| − | *2. Use AlphaFold v2.1.0 to predict the structure of the mutated PK.
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| − | <p style="text-align: center;">
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| − | [[File:mt8-alphafold.png|400px]]<br>
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| − | '''Figure 1.''' The mutated PK structure compared to the Wild type PK.<br>
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| − | </p>
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| − | *3. Use FoldX to calculate the Gibbs Free Energy compared to wild type PK with Ca<sup>2+</sup>. (PDB ID: 1ic6) The result of this mutated PK's ΔΔG is '''-13.81''' kcal/mol.
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| − | ===Molecular cloning===
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| − | We used the wild type Proteinase K(Hereinafter referred to as PK) DNA gene to overlap our mutated PK gene.
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| − | <p style="text-align: center;">
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| − | [[File:mt8-total of pcr'.png|600px]]<br>
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| − | '''Figure 2.''' The process of PCR for our mutated PK gene.<br>
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| − | </p>
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| − | *1. Use mutated PK primers to clone our small fragments.
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| − | <p style="text-align: center;">
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| − | [[File:mt8-pcr1'.png|400px]]<br>
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| − | '''Figure 3.''' Fragments of mutated PK gene are PCR-amplified independently.<br>
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| − | </p>
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| − | *2. Fuse the segments together in a subsequent reaction by High-fidelity thermostable DNA polymerase.
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| − | <p style="text-align: center;">
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| − | [[File:Mt8-pcr3'.png|500px]]<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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| − | *4. Use Ligase to link our mutated PK and pPIC9 after double digestion. <br>
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| − | *5. Transform the constructed plasmid into competent DH5α cells to expand the plasmid largely <br>
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| − | *6. Extract the recombinant pPIC9-PK and verify it by double digestion (XhoⅠ and EcoRⅠ), and sequence it for verication of mutation sites.
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| − | <p style="text-align: center;">
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| − | [[File:Mt8-dd3'.png|400px]]<br>
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| − | '''Figure 5.''' Double digestion verification of Recombinant pPIC9-PK.<br>
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| − | </p>
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| − | 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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| − | ===Expression in <i>Pichia Pastoris</i>===
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| − | '''Linearization of Recombinant pPIC9-PK:'''<br>
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| − | We used restriction endonuclease SalⅠ to linearize our recombinant plasmid.
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| − | <p style="text-align: center;">
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| − | [[File:Mt8-linearization.png|500px]]<br>
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| − | '''Figure 6.''' Linearization of Recombinant pPIC9-PK.<br>
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| − | </p>
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| − | '''Electrotransformation:'''<br>
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| − | Add several μg linearized pPIC9-PK to GS115 competence cells, then use 1.5kV electric pulse to drill holes to let gene get in.<br>
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| − | '''Screen positive colonies and culture preservation:'''<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>
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| − | * 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>
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| − | ===Conclusion===
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| − | 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.
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