Team:AHUT China/Improve

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Improvement

1.Characterization of an existing BioBrick Part BBa_K2232000 (TSLV1-CA)

For characterization, we have demonstrated the output of this part BBa_K2232000, the coding sequence (CDS) of Carbonic anhydrase (CA) from the polyextremophilic bacterium Bacillus halodurans TSLV1 (MTCC 10961, 16S rDNA Acc. No. HQ235051), in our chassis E. coli BL21(DE3).

The sequence of BBa_K2232000 was synthesized and cloned it into the expression plasmid pET-30a(+) to obtain the recombinant expression vector (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, the TSLV1-CA expression plasmid was transformed into E. coli BL21 (DE3) strain, and positive clones were screened by kanamycin resistance. The positive clones were further propagated and induced with IPTG (isopropyl thiogalactoside), followed by protein extraction from lysates of bacterial solution. The expression of TSLV1-CA was identified by Western blot analysis. The results are shown in Fig. 2, indicating that the coding sequence of BBa_K2232000 can be expressed in our chassis E. coli BL21 (DE3).




Fig. 2 Western blot analysis of protein extracted from lysates of TSLV1-CA expressed E.coli BL21(DE3) strain








2. Improve a Previous Part

We have changed the sequences of the existing part Carbonic anhydrase (csoS3) of the carboxysome of Halothiobacillus neapolitanus (BBa_K1465205), and have generated a new Part BBa_K2547003 (Carbonic anhydrase (csoS3)-His) (Fig. 3)

Fig. 3 Map of Carbonic anhydrase csoS3-His 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, the original coding sequence of csoS3 and the coding sequence with codon optimization were synthesized, and cloned into the pET-30a (+) expression vectors, respectively. The correctness of the two recombinant plasmids was verified by PCR (Fig. 4).




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




Subsequently, the expression of two csoS3 plasmids in E. coli was detected via SDS-PAGE and Coomassie blue staining. As shown in Fig. 5, the result presented that the expression of csoS3 original part in E. coli was relatively low, and the expression of codon-optimized csoS3 new part in E. coli was higher than original part.




Fig. 5 SDS-PAGE and Coomassie blue staining of Carbonic anhydrase csoS3 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.




To further demonstrate the activity of our new part, new part of csoS3 carbonic anhydrase was purified through Ni-chelating affinity chromatography and detected by SDS-PAGE and Coomassie blue staining, as shown in Fig. 6. Then, the activity of csoS3 was measured via esterase method, and the enzyme activity was about 22.84 U/mL.




Fig. 6 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.




In conclusion, our results demonstrated that the function of csoS3 new part has been improved with higher expression than original part and activity retained.