Difference between revisions of "Part:BBa K4152200"

Latest revision as of 14:43, 11 October 2022

XhoⅠ+Linker a+propeptide+PK_WT+His-Tag+Terminator+EcoRⅠ

These elements are the target PK gene we constructed, which would be used to insert the Proteinase K gene into pPIC9.

Sequence and Features

Origin(organism)

Tritirachium album limber

Structure

Figure 1. Structure of wild-type Proteinase K (PDB ID=1ic6).

Molecular cloning

We used plasmids with Proteinase K (PK) synthesized by the company to do our next expression.

• 1. Use PK primers to clone our fragments which would be inserted into pPIC9.

Figure 2. Fragments of wild-type PK gene are PCR-amplified independently.

• 2. Use restriction endonuclease XhoⅠ and EcoRⅠ to double digest our PK gene and pPIC9.

Figure 3. Double digestion of pPIC9 and wild-type PK.

• 3. Use Ligase to link our wild-type PK and pPIC9 after double digestion.
  
• 4. Transform the constructed plasmid into competent DH5α cells to expand the plasmid largely
  
• 5. Extract the recombinant pPIC9-PK, verify it by double digestion (XhoⅠ and EcoRⅠ), and sequence it to verify.

Figure 4. 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 5. Linearization of Recombinant pPIC9-PK.

Electrotransformation:
Add several μg linearized pPIC9-PK to GS115 competence cells, then use a 1.5kV electric pulse to drill holes to let the 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).

Figure 6. Genome PCR genomic DNA in Recombinant GS115.

• 3. Transfer the positive clones and preserve them in Glycerin (sterile), storing them at -80°C.
  

Express PK with Methanol:
Transfer some Glycerin recombinant GS115 to YPD, and 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 concentrate it. At last, we do SDS-PAGE to make sure that the wild-type 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.

Figure 7. SDS-PAGE-1.

Figure 8. SDS-PAGE-2.

Enzyme activity and thermostability determination

We use an Enzyme-labeled instrument to measure the Abs of OD_660nm of the product of L-Tyrosine of the reaction. We use 1% Casein as our substrate, and Tris-HCl (pH8.0) as our Buffer, and react at 55°C for several minutes. Then add trichloroacetic acid (TCA) to end the reaction, and centrifuge to collect the supernatant containing our L-Tyrosine product. Next step, we use Na₂CO₃ to provide the alkaline environment, then add the supernatant and Folin-phenol reagent to colorate L-Tyrosine. In the end, we detect the Abs of OD_660nm to assess the enzyme activity of our PK.
We store our PK at Room temperature for several days and detect the remains of it, then assess the thermostability of PK.

Figure 9. Enzyme activity determination curve.

Conclusion

Wild-type Proteinase K shows poor thermostability at Room Temperature.