Difference between revisions of "Part:BBa K4152008"

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<partinfo>BBa_K4152008 short</partinfo>
 
 
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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===Usage and Biology===
 
 
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<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4152008 SequenceAndFeatures</partinfo>
 
 
 
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===Functional Parameters===
 
<partinfo>BBa_K4152008 parameters</partinfo>
 
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===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.
 
<p style="text-align: center;">
 
[[File:mt8-alphafold.png|400px]]<br>
 
'''Figure 1.'''  The mutated PK structure compared to the Wild type PK.<br>
 
</p>
 
*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.
 
===Molecular cloning===
 
We used the wild type Proteinase K(Hereinafter referred to as PK) DNA gene to overlap our mutated PK gene.
 
<p style="text-align: center;">
 
[[File:mt8-total of pcr'.png|600px]]<br>
 
'''Figure 2.'''  The process of PCR for our mutated PK gene.<br>
 
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*1. Use mutated PK primers to clone our small fragments.
 
<p style="text-align: center;">
 
[[File:mt8-pcr1'.png|400px]]<br>
 
'''Figure 3.'''  Fragments of mutated PK gene are PCR-amplified independently.<br>
 
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*2. Fuse the segments together in a subsequent reaction by High-fidelity thermostable DNA polymerase.
 
<p style="text-align: center;">
 
[[File:Mt8-pcr3'.png|250px]]<br>
 
'''Figure 4.'''  PCR Mutagenesis by Overlap Extension to obtain the mutated PK gene.<br>
 
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*4. Use Ligase to link our mutated PK and pPIC9 after double digestion. <br>
 
*5. Transform the constructed plasmid into competent DH5α cells to expand the plasmid largely <br>
 
*6. Extract the recombinant pPIC9-PK and verify it by double digestion (XhoⅠ and EcoRⅠ), and sequence it for verication of mutation sites.
 
<p style="text-align: center;">
 
[[File:Mt8-dd3'.png|400px]]<br>
 
'''Figure 5.'''  Double digestion verification of Recombinant pPIC9-PK.<br>
 
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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>
 
===Expression in <i>Pichia Pastoris</i>===
 
'''Linearization of Recombinant pPIC9-PK:'''<br>
 
We used restriction endonuclease SalⅠ to linearize our recombinant plasmid.
 
<p style="text-align: center;">
 
[[File:Mt8-linearization.png|500px]]<br>
 
'''Figure 6.'''  Linearization of Recombinant pPIC9-PK.<br>
 
</p>
 
'''Electrotransformation:'''<br>
 
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>
 
'''Screen positive colonies and culture preservation:'''<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).
 
 
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===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.
 

Latest revision as of 13:10, 9 October 2022