Difference between revisions of "Part:BBa K4202004:Experience"

(→‎Purification of TSLV-BS-CA)
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As Part:BBa_K4202004 is improved by codon optimization from Part:BBa_K2232000, we have done a series of control experiments to compare the function of two parts. Both of the two parts encodes carbonic anhydrase, so we name the carbonic anhydrase expressed by  Part:BBa_K2232000 as <b>CA1</b>, while name the carbonic anhydrase expressed by Part:BBa_K4202004 as <b>CA2</b>.
 
===Expression of BBa_K4202004===
 
===Expression of BBa_K4202004===
  

Revision as of 15:19, 25 September 2022


As Part:BBa_K4202004 is improved by codon optimization from Part:BBa_K2232000, we have done a series of control experiments to compare the function of two parts. Both of the two parts encodes carbonic anhydrase, so we name the carbonic anhydrase expressed by Part:BBa_K2232000 as CA1, while name the carbonic anhydrase expressed by Part:BBa_K4202004 as CA2.

Expression of BBa_K4202004

After chemical transformation of plasmid with this part, the transformed Bacillus subtilis WB600 were cultured in optimized LB and SMM medium, and obtained crude enzyme solution by centrifugation and ultrasonic disruption.Then we detected the molecular mass by SDS-PAGE and coomassie blue staining.
SDS-PAGE displayed bands of 37kDa and 74kDa for CA monomer and dimer, which didn' t exist in the control group(Fig.1-1).

Liu Junyi 1-1.png


Fig 1-1 SDS-PAGE and Coomassie brilliant blue staining results of whole protein lysates of strain CA1, strain CA2 and blank WB600. Lane 1: Protein Ladder; Lane2: CA2 strain grown in LB medium, Lane3: CA2 strain grown in SMM medium, Lane4: CA1 strain grown in LB medium, Lane5: CA1 strain grown in SMM medium, Lane6: blank WB600 strain.

Purification of TSLV-BS-CA

We added a 6X His tag to the N-terminus of CA on the vector, and the CA was purified by agarose-nickel column affinity chromatography. SDS-page analysis was performed on the flowing fluid, washing fluid and eluent during the purification of CA1 and CA2 by Ni-column. The eluate of CA1 and CA2 had two obvious bands at 37kDa and 74kDa, which were monomer CA and dimer CA, respectively. However, the Ni column had much non-specific protein binding, we can add a small amount of imidazole to reduce non-specific protein binding(Fig.1-2).


Liu Junyi 1-2.png


Fig 1-2 A: SDS-PAGE analysis of CA1 protein purified by Ni column. Lane1: flowing fluid, Lane2-4: the 1-4 washing fluid, Lane5-9: the 1-5 eluate, Lane10: protein Maker B:SDS-PAGE analysis of CA2 protein purified by Ni column. Lane1-5: the 1-5 eluent, Lane6-8: the 1, 2 and 4 washing fluid, Lane9: the flowing fluid, Lane10: protein Maker.

Hydrated CO2 activity of CA

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