Difference between revisions of "Part:BBa K2762008"

Revision as of 18:18, 15 October 2018

CcaA (formerly icfA)

Usage and Biology

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.

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.

SDS-PAGE 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. Finally, the sample 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.

Carbonic anhydrase activity assay

We then ran the activity test of CA. In our bypass pathway, the function of CA is to convert proton and bicarbonate into water and carbon dioxide. CA activity was determined using the Wilbur-Anderson assay. 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 (sec) of pH decrease from 8.3 to 6.3 was recorded. CA activity was calculated using a Wilbur–Anderson unit (WAU) per milliliter 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

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. 3Shows 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