Part:BBa_K2762004

CcaA (formerly icfA)

Background

This biobrick is a cloning intermediate of the carboxysome from Synechococcus elongatus PCC7002. The carboxysomal carbonic anhydrase (CA) (EC 4.2.1.1) of Synechococcus elongatus PCC7002, CcaA is needed for the CO₂ fixation in the working carboxysome as it converts incoming hydrogen carbonate into carbon dioxide inside of the carboxysome. This step is essential for the CO₂ fixation since it can increases the intracellular CO₂ concentration, indirectly affect the CO₂ fixation rate. CcaA is one of the beta-class CA which can be found in plants, algae, bacteria, and archaea, and is far more diverse in sequence than the other two classes, with only five residues (three forming the zinc ligand) being completely conserved. It is a zinc-containing enzyme that catalyzes the reversible hydration of CO₂. It has a tertiary fold, with a central 10-stranded beta-sheet as the dominating secondary structure element. The zinc ion is located in a cone-shaped cavity and coordinated to three histidyl residues and a solvent molecule.

User Reviews

Contribution from iGEM2018 AHUT_China

yu Zhang

In 2019, AHUT_China iGEM team has characterized the output of this part in E. coli BL21(DE3) and tested its activity by esterase method which was different form the original method. The result was documented in the experience page and the main page of BBa_K2762004.

The sequence of BBa_K2762004 was synthesized and cloned it into the expression plasmid pET-30a(+) to obtain the recombinant expression vector. Then, The plasmid containing CcaA was introduced into E. coli BL21(DE3) for culturing in the medium containing kanamycin, and IPTG was added to induce CcaA expression for 4h. The CcaA Protein was extracted from the bacterial lysates followed by identification via SDS-PAGE gel electrophoresis.(Fig. 1)

Fig. 1 SDS-PAGE analysis of CcaA protein extracted from lysates of expressed in E.coli BL21(DE3) strain

After protein purification, the activity of the enzyme was determined by esterase method different from the original method. As carbonic anhydrase can catalyze the hydrolysis of p-nitrophenyl acetate. Therefore, we intended to use the esterase activity of carbonic anhydrase to catalyze the p-nitrophenyl acetate, and obtain the enzyme activity data by the change of absorbance before and after a certain reaction time. The result showed that CcaA also showed high enzyme activity. (Fig. 2)

Fig 2 The enzyme activity of CcaA analyzed by esterase method

Characterization

Colony PCR of finished construct

After finishing the CA biobrick construction, colony PCR is run to check the success of ligation. The length of the DNA is verified with agarose gel electrophoresis.

Protein expression of CcaA

The SDS-PAGE of this construct is done to prove the expression of the protein. The cells were harvested by centrifuging at 10,000×g for 10 min., and then washed with deionized water for 2 times. The cell density was adjusted to an O.D.600 of 5 as the sample of whole cell (WC, whole cell catalyst). Finally, WC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 15% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R-250 and were scanned with an Image scanner. The result is shown below.

Enzyme activity measurement

An activity test is also conducted to determine the catalytic rate of the CA. CA activity was determined using the Wilbur-Anderson assay [1]. Briefly, 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer and 0.2 mL enzyme were mixed and transferred to a 20 mL sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of ice-cold CO₂-saturated solution was added immediately into the sample bottle and the time course (in sec) of pH decrease from 8.3 to 6.3 was recorded. CA activity was calculated using a Wilbur–Anderson unit (WAU) per millilitre of sample. The definition for WAU is (T₀-T)/(T₀) in which T₀ and T was the time required for the pH drop from 8.3 to 6.3, with and without CA, respectively.

Total Solution Test

We use total solution test to determine the function of CA. To view more details about the total solution test, please check the results page of 2018_NCKU_TAINAN. http://2018.igem.org/Team:NCKU_Tainan/Results

Function of CA

From the above results, we discovered that although Rubisco and PRK alone can enhance the utilization rate of carbon dioxide, the growth and utilization ability didn’t meet our expectations. The third important enzyme came into play: CA enzyme. We cloned Rubisco (BBa_K2762011) into pSB1C3 and cloned PRK with PLacI promoter and CA with PT7 promoter(BBa_K2762013) into pSB3K3. Two plasmids are then co-transformed into BL21(DE3). We measured the XUI of this strain and compare with the previous strain that only contains PRK and Rubisco. We found out that CA can raise the growth and lower the XUI. We infer that CA can enhance the intracellular CO₂ concentration and thus increase the carbon flux of the bypass pathway. The efficiency of the bypass pathway is thus been increased.

Fig. 3 Shows the growth and XUI comparison of each strain. All the tested strains are incubated in 5% CO₂ incubator for 12 hr. 0.1mM of IPTG was added to induce the protein expression. We can observe that growth speed of the construction has been increased with the CA, and the XUI of the strain that contains complete three enzymes was the lowest compared to the strain without plasmid or the strain that only contains PRK and Rubisco, stating that three enzymes are required to optimized the carbon fixing bypass pathway. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Source

Codon oprimized Synechococcus elongatus PCC7002.

Reference

[1]M. Wilbur, N.G. Anderson.(1948, Oct.1) Electrometric and colorimetric determination of carbonic anhydrase, J. Biol. Chem. 147–154.

[2] Lindskog S. (1997) .Structure and mechanism of carbonic anhydrase. Pharmacol Ther.

[3] Rowlett RS. (2010,Feb). Structure and catalytic mechanism of the β-carbonic anhydrases.Biochimica et Biophysica Acta (BBA)

[4] Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO₂-fixing Escherichia coli reveals great potential of heterotrophic CO₂ fixation. Biotechnology for Biofuels.

[5] Shih-I Tana, Yin-Lung Han, You-Jin Yua, Chen-Yaw Chiuc, Yu-Kaung Chang,Shoung Ouyanb, Kai-Chun Fanb, Kuei-Ho Lo, and I-Son Ng.( 2018,October) Efficient carbon dioxide sequestration by using recombinant carbonic Anhydrase.Process Biochemistry