Difference between revisions of "Part:BBa K4152001"

Revision as of 03:12, 8 October 2022

PK_MT1

To improve the performance of Proteinase K, we designed many Proteinase K mutant genes. PK_MT1 is a complicated mutation of Proteinase K, which contains 5 mutation sites: N257C-S173Y-T16C-T20D-V198F.

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]

@@ Line 14: / Line 14: @@
 <!-- Uncomment this to enable Functional Parameter display
-===Functional Parameters===
-<partinfo>BBa_K4152001 parameters</partinfo>
-<!-- -->
-===Functional Parameters===
-<partinfo>BBa_K4152011 parameters</partinfo>
-<!-- -->
-===Functional Parameters===
-<partinfo>BBa_K4152011 parameters</partinfo>
-<!-- -->
 ===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:mt1-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. <br>
+We used the wild type Proteinase K(Hereinafter referred to as PK) DNA gene to overlap our mutated PK gene.
-*1. We used mutated PK primers to clone our small fragments.
+<p style="text-align: center;">
+[[File:mt1-total of pcr'.png|600px]]<br>
+'''Figure 2.'''   The process of PCR for our mutated PK gene.<br>
+</p>
+*1. Use mutated PK primers to clone our small fragments.
 <p style="text-align: center;">
 [[File:mt1-pcr1'.png|400px]]<br>
-'''Figure 1.'''   The first time PCR for our small fragments-1.<br>
+'''Figure 3.'''   Fragments of mutated PK gene are PCR-amplified independently.<br>
 </p>
 <p style="text-align: center;">
 [[File:Mt1-pcr2'.png|250px]]<br>
-'''Figure 2.'''   The first time PCR for our small fragments-2.<br>
+'''Figure 4.'''   Fragments of mutated PK gene are PCR-amplified independently.<br>
 </p>
-*2. We overlapped the small fragments 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;">
 [[File:Mt1-pcr3'.png|250px]]<br>
-'''Figure 3.'''   The overlap PCR for our entire PK fragment.<br>
+'''Figure 5.'''   PCR Mutagenesis by Overlap Extension to obtain the mutated PK gene.<br>
 </p>
-*3. Use restriction enzyme XhoⅠ and EcoRⅠ to double digest our mutated PK gene and pPIC9.
+*3. Use restriction endonuclease XhoⅠ and EcoRⅠ to double digest our mutated PK gene and pPIC9.
 <p style="text-align: center;">
 [[File:Mt1-dd1'.png|250px]]<br>
-'''Figure 4.'''   Double digestion of mutated PK.<br>
+'''Figure 6.'''   Double digestion of mutated PK.<br>
 </p>
 <p style="text-align: center;">
 [[File:Mt1-dd2'.png|250px]]<br>
-'''Figure 5.'''   Double digestion of pPIC9.<br>
+'''Figure 7.'''   Double digestion of pPIC9.<br>
 </p>
 *4. Use Ligase to link our mutated PK and pPIC9 after double digestion. <br>
-*5. Then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely. <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:Mt1-dd3'.png|400px]]<br>
-'''Figure 6.'''   Double digestion verification of Recombinant pPIC9-PK.<br>
+'''Figure 8.'''   Double digestion verification of Recombinant pPIC9-PK.<br>
 </p>
-After verification, it was determined that the construction is successful. We converted the plasmid to E. coli DH5α 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>
 ===Expression in <i>Pichia Pastoris</i>===
 '''Linearization of Recombinant pPIC9-PK:'''<br>
-We used restriction enzyme SalⅠ to linearize our recombinant plasmid.
+We used restriction endonuclease SalⅠ to linearize our recombinant plasmid.
 <p style="text-align: center;">
 [[File:Mt1-linearization.png|500px]]<br>
-'''Figure 7.'''   Linearization of Recombinant pPIC9-PK.<br>
+'''Figure 9.'''   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>
+* 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 genome 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).
 <p style="text-align: center;">
 [[File:Mt1-genome pcr.png|300px]]<br>
-'''Figure 7.'''   Genome PCR verification of Recombinant GS115.<br>
+'''Figure 10.'''   Genome PCR genomic DNA in Recombinant GS115.<br>
 </p>
-* 3. transfer the positive clonies and preserve it in Glycerin (steriled), store it at -80°C.<br>
+* 3. Transfer the positive clones and preserve them in Glycerin (sterile), storing them at -80°C.<br>
 '''Express PK with Methanol:'''<br>
-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 our PK has expressed successfully, and take the standard samples to do grey analysis, then figure out the mass of PK.<br>
+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>
 <p style="text-align: center;">
-[[File:Mt1-SDS-PAGE-1.png|300px]]<br>
+[[File:Mt1-SDS-PAGE-1.png|450px]]<br>
-'''Figure 8.'''   SDS-PAGE-1.<br>
+'''Figure 11.'''   SDS-PAGE-1.<br>
 </p>
 <p style="text-align: center;">
-[[File:Mt1-SDS-PAGE-2.png|300px]]<br>
+[[File:Mt1-SDS-PAGE-2.png|450px]]<br>
-'''Figure 9.'''   SDS-PAGE-2.<br>
+'''Figure 12.'''   SDS-PAGE-2.<br>
 </p>
 ===Enzyme activity and thermostability determination===
-We use ELIASA 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>
+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>
 We store our PK at Room temperature for several days and detect the remains of it, then assess the thermostability of PK.
 <p style="text-align: center;">
 [[File:Mt1.png|300px]]<br>
-'''Figure 10.'''   Enzyme activity determination, compared with wild type.
+'''Figure 13.'''   Enzyme activity determination, compared with wild type.
 </p>
 ===Conclusion===
-In conclusion,the thermostability of this mutated PK has improved '''在这里填东西''' times, compared with WT(wild type).
+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.