Carbonic anhydrase

Introduction

In our project, we use biomineralization to deposit calcium carbonate on the surface of microbial cells, filling cracks in stone artifacts. So we choose to use and improve part BBa_K2232000 , a CDS encoding α-carbonic anhydrase, which efficiently catalyzes the reversible hydration of CO₂ to rapidly produce bicarbonate (HCO₃^-) and protons (H⁺).

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.

We created a new part BBa_K4202004 deriving fromBBa_K2232000 by replacing codons in the original mRNA sequence with codons that are used frequently in Bacillus subtilis , in order 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.

Based on this work, we added the new part BBa_K4202004 to the Registry, and documented our new part as a contribution on the original page of BBa_K2232000.

Materials and Methods

We have done a series of control experiments to compare the function of the 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 Carbonic anhydrase

After chemical transformation of plasmid with this part to Basillus subtilis WB600, 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.

Determination the ability of Carbonic anhydrase to hydrate 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.

Add 5mL of 20mM pH 8.0 Tris-HCl buffer, 3 drops of bromothymol blue, 0.3mL of crude enzyme solution (lysate of Carbonic anhydrase producing bacterial), 2mL of ddH₂O (2.3mL ddH₂O for control) to a 15mL centrifuge tube. Screw the cap on tightly and ice bath for 15min. Then slowly add 5mL icy saturated CO₂ solution to the centrifuge tube, screw the lid tightly. Leave the mixed system at room temperature and record the time taken for the indicator to change from blue to yellow.

Determination the ability of Carbonic anhydrase to precipitate CaCO₃

To test the ability of engineered Bacillus subtilis WB600 to precipitate CaCO₃, we cultured the engineered bacteria in 30ml of LB medium at 20^oC 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).

Results

Expression of Carbonic anhydrase

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.

Determination the ability of Carbonic anhydrase to catalyze the hydration of CO₂

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 1-2).

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 1-2 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.

Determination the ability of Carbonic anhydrase to catalyze the precipitation of CaCO₃

After three days of cultivation, we could clearly see that the culture medium of the CA1 and CA2 transformant became turbid(Fig 1-4 A). The precipitate was filtered and dried.(Fig 1-4B). 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 1-4C).

Fig 1-3 A: The cultures after induction. B: Filtered and dried sediment products. C: CaCO₃ productions by CA1 and CA2 for 3d.

Conclusions

1. We constructed our improved part by codon optimization to fit the codon usage bias of Bacillus subtilis, avoiding the emergence of rare codons.

2. We demonstrated that the modified carbonic anhydrase Part has the ability to express carbonic anhydrase. The expressed carbonic anhydrase has the ability to catalyze the hydration of CO₂ as well as the precipitation of CaCO₃

3. However, the catalytic rate of CA2 was lower than that of CA1 in both experiments, indicating that the expression of carbonic anhydrase in Bacillus subtilis has not been increased by our modification.

Although our modification did not increase the expression of carbonic anhydrase in Bacillus subtilis, we confirmed that the codon-optimized carbonic anhydrase expressed by Bacillus subtilis was still biologically active, thus providing an evidence for expression of carbonic anhydrase in other prokaryotic chassis after codon optimization. We also confirmed the role of carbonic anhydrase in promoting biomineralisation, which will provide inspiration for future iGEM teams who want to exploit biomineralisation.

Engineered Sucrose-sensitive Promoter

Introduction

Inducible promoter is a regulated promoter that allows transcription of its associated genes only in certain circumstances. They are central regulatory component in synthetic biology, metabolic engineering, and production for laboratory and commercial uses [1]. In the Part Registry, there is a wide variety of inducible promoters that can be used in E. coli, of which the commonly used ones is the T7-Lac promoter. However, for B. subtilis, the inducible promoters submitted to the registry are either exogenous promoters that require exogenous protein expression (e.g. promoter hyper-spank BBa_K143015, which requires LacI expression) and put unquantifiable metabolic burden on the living system, or endogenous weak promoters (e.g. PsacB BBa_K4043004), which had low expression activity as well as low induction activity [2].

To address the problems of endogenous inducible promoters in Bacillus subtilis, we construct an engineered promoter based on the endogenous promoter PsacB with the aim of achieving lower leaky expression and higher induction sensitivity.

In this study, we introduced the B. subtilis endogenous strong promoter Pveg to improve the expression efficiency of the sucrose-sensitive promoter PsacB ( BBa_K4043004). Previous studies have shown that the regulation of the PsacB promoter is mainly dependent on the leader RNA sequence upstream of the sacB gene with the SacY and the LicT protein. Under different concentrations of sucrose induction, SacY protein and LicT protein can bind to the anti-terminator RNA hairpin that overlaps with an intrinsic terminator and regulate the balance of anti-termination/termination to influence the mRNA transcription efficiency of the downstream gene [3, 4].

For more information about the sensitivity of PsacB promoter to sucrose and sucralose (sucrose analogs), please turn to the main page of BBa_K4043004.

Design

We speculated that the low induction activity of PsacB is due to the low transcriptional initiation activity of its core promoter sequence. Therefore, we introduced the Pveg promoter sequence to replace the PsacB core promoter sequence. Also, to reduce the leaky expression caused by the increased transcription activity of the promoter, we duplicated the regulatory region of PsacB (leader RNA sequence, BBa_K4202019) multiple times (in our experiment, we designed one, two, three and five replications). Theoretically, the engineered sucrose-sensitive promoter will have lower or equivalent leaky expression compared to the PsacB promoter, and the activity after induction will be much higher than the PsacB promoter, too.

Vector construct

We separately amplified the Pveg promoter (from BBa_K823003 ) as well as different numbers of repeated leader RNA (separated by spacer) sequences and constructed the new vector with the above components with the spoVG RBS and the GFP fluorescence gene via simple one-step recombination based on the pHY300PLK backbone. Finally we constructed four promoters:(BBa_K4202025 with single leader RNA repeat, BBa_K4202026 with double leader RNA repeats, BBa_K4202027 with triple leader RNA repeats, and BBa_K4202028 with penta leader RNA repeats). Then we transformed the recombinant measurement plasmids into Bacillus subtilis WB600 for the characterization.

Fig 2-1 The sequence of PsacB (A, BBa_K4043004) and one of our engineered promoter (B, Pveg-2×leader RNA, BBa_K4202026)

Result

Our engineered promoters were induced by sucrose characterized using the flow cytometer. Two of our engineered promoters BBa_K4202026 and BBa_K4202027showed tolerance to high sucrose concentrations(the fluorescence intensity didn't decrease when the sucrose concentration was 2%) compared to the other promoters and BBa_K4202028 has the highest activity after induction, which is twice as high as control.

Fig 2-2 The characterization result of our engineered promoters