Part:BBa_K1465205:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

User Reviews

Yuru_chen
In 2018, AHUT_China iGEM team has changed the coding sequence (CDS) of the original part (BBa_K1465205) with codon-optimization and a histidine tag (His-Tag) added, forming a new Part (BBa_K2547003).

Contribution from iGEM2018 AHUT_China

The coding sequence of Carbonic anhydrase csoS3 from original part was codon-optimized, and also a His tag was added to the end, to ensure that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and retained potent carbonic anhydrase activity (Fig. 1).

Fig. 1 Map of Carbonic anhydrase csoS3-His-Tag expression vector <p>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. 2).

Fig. 2 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. 3, 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. 3 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.

</p>

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. 4. Then, the activity of csoS3 was measured via esterase method, and the enzyme activity was about 22.84 U/mL.

    

<center>Fig. 4 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 and activity than original part.

User Reviews

UNIQe93ef5d50294d718-partinfo-00000002-QINU UNIQe93ef5d50294d718-partinfo-00000003-QINU