Part:BBa_K4277006
pET28a-PKC
pET28a-PKC
Characterization by Shanghai_United
BBa_K4277006
Name: pET28a-PKC
Base Pairs: 5515 bp
Origin: Pseudomonas aeruginosa, genome
Properties: Providing the method for feed degradation
Usage and Biology
The alkaline cellulase is from Pseudomonas aeruginosa (PKC), and used to degrade the insoluble cellulose to soluble glucose. Cellulase with high alkaline stability, active under low temperature is used to as additive.
Construct design
The alkali-cellulase process the biomass into glucose. The gene of alkali-cellulase referred as PKC was integrated into pET28a vector, shown as Figure 1.
Experimental approach
1.1 The colony PCR of pET28a-PKC in competent cells DH5α
PKC gene amplified by PCR, double-enzyme digested by NheI and HindIII, and inserted into the same cohesive site of pET28a (+) vector, to obtain the plasmid pET28a-PKC. Then, the pET28a-PKC transformed into DH5α. Colony PCR results showed that the correct band of PKC, and confirmed that the recombinant plasmid of pET28a-PKC is successful (Figure 2).
1.2 Sequencing result of plasmid pET28a-PKC
The recombinant plasmid further confirmed by sequencing. The sequence alignment result exhibited the no mutation or mismatch in the plasmid pET28a-PKC, shown in Figure 3.
1.3 The colony PCR of pET28a-PKC in competent cells BL21
The plasmid pET28a-PKC was extracted from E. coli DH5α, and transformed it into E. coli BL21(DE3). The colony PCR results showed that it was successful (Figure 4).
Functional assay
2.1 Protein expression of PKC
In order to obtain the protein PKC, we transformed the plasmid pET28a-PKC into E. coli BL21(DE3). The colony was inoculated and cultured in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and cultivation, the cells were collected and lysed by ultrasonication to release the intracellular proteins. Next, we used nickel resin to purify the interested protein. The molecular weights of PKC were 48.27 KD.
SDS-page showed the protein PKC in lane S, indicating that it was successfully expressed in E. coli BL21 (DE3). The protein PKC was also found in lane P, possibly due to the inactivation of a small number of proteins.
2.2 Determination of PKC enzymatic activity
Determination of reducing the sugar by DNS method: The absorbance OD540 value of the purified enzyme solutions (PKC) was measured after color reaction with DNS. The activity of the enzyme can be converted by the amount of sugar consumed and the working time.
The enzymatic activity of PKC is about 0.26 U/mL according to DNS method (Table 1 and Figure). The results indicated that the protein PKC has active enzyme activity. It also proved that PKC had the function of degrading cellulose.
Improvement of existing data
The old part BBa_K4096002 of the iGEM2021_Shanghai_Metro_HS team attempted PKC protein expression, but the enzymatic activity was not detected. Compared with 2021, in our BBa_K4277006 the PKC was integrated into pET28a rather than pET-25b, and the functional assay contains not only protein expression but also successfully measured enzyme activity. pET28a enables the soluble protein over-expressing under strong bacteriophage T7 promotor. Thus, we chose the vector to express the PKC protein in order to measure the enzymatic activities. In addition, this part provides extra experimental data of PKC and a reference for the future iGEM team.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4402
Illegal XhoI site found at 4430 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 2782
Illegal NgoMIV site found at 4370 - 1000COMPATIBLE WITH RFC[1000]
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