Difference between revisions of "Part:BBa K4152008"

Revision as of 03:08, 9 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

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.

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.

Figure 2. The process of PCR for our mutated PK gene.

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

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.

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.

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.

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).

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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.