Difference between revisions of "Team:AHUT China/Improve"

1.Characterization：

     Characterization of parts BBa_K2232000 (TSLV1-CA) This part is the coding sequence (CDS) of Carbonic anhydrase (CA) from The polyextremophilic bacterium Bacillus halodurans TSLV1 (MTCC 10961, 16S rDNA Acc. No. HQ235051).CA is a metalloenzyme with zinc, which is highly efficient and one of the fastest enzymes catalyzes the reversible hydration of CO2 forming bicarbonate and protons rapidly.
    We synthesized the sequence of BBa_K2232000 and cloned it into the expression plasmid pET-30a(+) to obtain the recombinant expression plasmid (Fig. 1).

    

Fig. 1 Agarose Gel Electrophoresis of TSLV1-CA recombinant plasmid and its identification by PCR. Lane M: DL marker; Lane 1: TSLV1-CA recombinant plasmid; Lane 2: PCR band of TSLV1-CA, the length was 894 bp.

        Then, it was transformed into E. coli BL21 (DE3), and positive clones were screened by kanamycin resistance. The positive clones were expanded and IPTG (isopropyl thiogalactoside) was induced to lyse and extract proteins. The expression of carbonic anhydrase was identified by SDS-PAGE and Coomassie blue staining. The results are shown in Figure 2, indicating that the coding sequence of BBa_K2232000 can be expressed in our chassis E. coli BL21 (DE3).

Figure 2 is missing

2.Improvement：

      We sequenced the existing part Carbonic anhydrase (csoS3) of the carboxysome of Halothiobacillus neapolitanus (BBa_K1465205) to generate a new PART (BBa_K2547003 (Carbonic anhydrase (csoS3)-His-Tag) (Fig. 1)

    

Fig. 1 Map of Carbonic anhydrase csoS3-His-Tag expression vector

      Specifically, the coding sequence of Carbonic anhydrase csoS3 was codon-optimized, and His-tag was added to the end, so that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and had good carbonic anhydrase activity.
    First, we synthesized the original coding sequence of csoS3 and the coding sequence after codon optimization, and cloned into the expression vector pET-30a(+) respectively. The correctness of the two plasmids was verified by PCR (Fig. 2).

    

Fig. 2 Agarose Gel Electrophoresis of Carbonic anhydrase csoS3-His-Tag expression vector and its identification by PCR. Lane M: DL marker; Lane 1: expression vector of csoS3 original part; Lane 2: PCR band of expression vector of csoS3 original part, the length was 1620 bp; Lane 3: expression vector of csoS3 new part; Lane 4: PCR band of expression vector of csoS3 new part, the length was 1620 bp.

          Subsequently, we examined the expression of two csoS3 in E. coli. The results are shown in Figure 3. The expression of the codon-optimized plasmid in E. coli is very low, and the codon-optimized csoS3 is in E. coli. The expression increased significantly.

    

Fig. 3 SDS-PAGE analysis of Carbonic anhydrase csoS3-His-Tag plasmids expressed in E. coli BL21(DE3) strains. The arrow indicated was the bands of csoS3. Lane 1: Negative control (cell lysate without IPTG induction) of new part; Lane 2: Cell lysate with induction for 6 h at 37 ℃ of new part; Lane 3: Negative control (cell lysate without IPTG induction) of original part; Lane 4: Cell lysate with induction for 6 h at 37 ℃ of original part.

    On this basis, we further purified E. coli expressing new part csoS3 to obtain purified csoS3 carbonic anhydrase (Fig. 4), and carried out enzyme activity assay by esterase method. The enzyme activity assay showed that csoS3 had certain The enzyme activity was 22.84 U/mL.

    

Fig. 4 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.