Part:BBa_K4202004

TSLV-BS-CA

Usage and Biology

Biomineralization can be used to deposit calcium carbonate on the surface of microbial cells, filling cracks in stone artifacts. This part is the coding sequence (CDS) of Carbonic anhydrase (CA) encoding a zinc-containing enzyme, α-carbonic anhydrase, which efficiently catalyzes the reversible hydration of CO₂ to rapidly produce bicarbonate (HCO₃^-) and protons (H⁺).

Chemical reaction equation: H₂O+CO₂↔HCO₃^-+H⁺

Bicarbonate (HCO₃^-) can be transported down the concentration gradient to the outside of the cell. When we provide calcium ions (Ca²⁺) in the extracellular medium, the bicarbonate can combine to the Ca²⁺ to form calcium carbonate precipitates. In our project, the calcium carbonate precipitate can accumulate in the tiny cracks of the stone artifacts, filling the cracks and providing support.

This part derivers from BBa_K2232000 We replace codons in the original mRNA sequence with codons that are used frequently in Bacillus subtilis to ensure that the codons in the newly designed mRNA sequence are more compatible with the codon usage bias of Bacillus subtilis, avoiding the emergence of rare codons

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 540
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 166
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 535
  

Characterization

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 BBa_K2232000 as CA1, while name the carbonic anhydrase expressed by 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).

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

<p>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).

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.

Determination the ability of TSLV-BS-CA to catalyze the hydration of CO₂

To measure the activity of CA, we used the modified Wilbur-Anderson's method. As we know, CA can catalyze CO₂ hydration and at the same time release H⁺ reducing the pH. According to that, we chose bromothymol blue, an acid-base indicator that appears yellow when pH≤6 and blue when pH > 7.6. Therefore, the color development of bromothymol blue can indirectly reflect the change of pH from 8.0 to 6.0 by CA.

After adding ice-saturated CO₂ solution for 10min, the color of the tubes containing crude enzyme solution CA1 and CA2 began to change, indicating that the pH of the solution began to decrease. After 10min, the tubes containing the crude enzyme solution showed significant discoloration, and after 5 days, the tubes containing the CA1 crude enzyme solution turned completely yellow, implying that the pH had decreased from 8.0 to 6.0 due to the formation of H⁺ during CO₂ hydration(Fig.1-3).

The activity of CA1 and CA2 were verified in this experiment, but the enzyme activities were weak, possibly due to low enzyme expression or insufficient concentration of unpurified enzyme. In addition, the catalytic rate of CA2 crude enzyme solution was lower than that of CA1.

Fig 1-3 The activity of CA detected by the Wilbur-Anderson's method. From left to right, the four tubes were pH=6.0 Tris-HCl buffer with bromothymol blue indicator, reaction system with blank WB600 lysate, CA2 crude enzyme solution, CA1 crude enzyme solution. A: Initial reaction solution（0min); B: 10min after adding ice-saturated CO₂ solution; C: 5d after adding ice-saturated CO₂ solution.

Detection of esterase activity of CA

The common α-CA is a secreted monomeric protein with esterase activity that can catalyze the catalyzes the formation of p-nitrophenol from p-nitrophenyl acetate and show the color(yellow). Although it was reported that the BhCA naturally produced by Bacillus Halodurans TSLV1 had no esterase activity, we still wanted to test whether the recombinant CA expressed in Bacillus subtilis WB600 had esterase activity. In this part, we used the Nanodrop and microplate reader to measure the color reaction rates of the CA crude enzyme solutions. We also measured the esterase activity of purified CA solution in order to avoid influence of the other proteins in the cell lysis solution.

The system with crude enzyme solution which quickly changed from colorless to yellow had an absorption peak at about 400nm(Figure 1-4 A). Absorption-time curve at 405nm showed that CA1 and CA2 crude enzyme solutions had high esterase activity (Figure 1-4B). For purified CA solution, the results showed that purified CA1 and CA2 both still had strong esterase activity (Fig. 1-4 C), which was inconsistent with the literature we checked. This may be because the recombinant CA expressed in Bacillus subtilis WB600 has both monomer and dimer forms, and some monomer CA own esterase activity.

Fig 1-4 Detection of esterase activity of CA. A: absorbance of the system with crude enzyme solution at different wavelengths, B: esterase activity of CA1 and CA2 crude enzyme solution, x: Time (min), Y: A405, Blank: no bacterial split solution, WB600: lysate of blank WB600 was added, CA1: the lysate of transformant-CA1, CA2:the lysate of transformant-CA2, C: esterase activity of purified CA1 and CA2 enzyme eluent, x: Time (min), Y: A405, Blank: no enzyme solution, WB600: lysate of blank WB600, CA1: purified CA1 enzyme solution, CA2: purified CA2 enzyme solution.

Determination the ability of TSLV-BS-CA to catalyze the precipitation of CaCO₃

To test the ability of engineered Bacillus subtilis WB600 to precipitate CaCO₃, we cultured the engineered bacteria in 30ml of LB medium at 25℃ for 3 days and added 5 ml of 100mM CaCl₂ solution on the first and second days. We filtered the culture medium through a Whatman membrane filter paper to separate the bacteria and CaCO₃. The bacteria and CaCO₃ were dried and weighed, respectively. We can calculate CaCO₃ precipitation capacity of engineered bacteria by the formula:CaCO₃ dry weight (mg)/cell dry weight (g).

After three days of cultivation, we could clearly see that the culture medium of the CA1 and CA2 transformant became turbid(Fig.1-5 A). The precipitate was filtered and dried.(Fig.1-5B). According to the the foemula:CaCO₃ production capacity = CaCO₃ dry weight (mg)/cell dry weight (g), we found that the the precipitation efficiency of CA1 was higher than that of CA2(Fig.1-5C).

Fig 1-5 A: The turbidity of the culture solution. B: Filtered and dried sediment products. C: CaCO₃ productions by CA1 and CA2 for 3d.