Difference between revisions of "Part:BBa K2762008"

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===Usage and Biology===
 
===Usage and Biology===
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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 CO<sub>2</sub> fixation in the working carboxysome as it converts incoming hydrogen carbonate into carbon dioxide inside of the carboxysome. This step is essential for the CO<sub>2</sub> fixation since it can increases the intracellular CO<sub>2</sub> concentration, indirectly affect the CO<sub>2</sub> fixation rate.
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This biobrick is a cloning intermediate of the carboxysome from <i>Synechococcus elongatus</i> PCC7002. The carboxysomal carbonic anhydrase (CA) (EC 4.2.1.1) of <i>Synechococcus elongatus</i> PCC7002, CcaA is needed for the CO<sub>2</sub> fixation in the working carboxysome as it converts incoming hydrogen carbonate into carbon dioxide inside of the carboxysome. This step is essential for the CO<sub>2</sub> fixation since it can increases the intracellular CO<sub>2</sub> concentration, indirectly affect the CO<sub>2</sub> 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 CO<sub>2</sub>. 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.
 
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 CO<sub>2</sub>. 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.
  
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===Colony PCR of finished construct===
 
===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.
 
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.
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[[File:T--NCKU Tainan--part BBa K2762008 new .png|500px|centre]]
 
  
 
===SDS-PAGE of CcaA===
 
===SDS-PAGE of CcaA===
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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).
+
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.
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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.
+
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.
 
The result is shown below.
  
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===Carbonic anhydrase assay===
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[[File:T--NCKU Tainan--part BBa K2762008 white final.png|500px|centre]]
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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 CO<sub>2</sub>-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 (T<sub>0</sub>-T)/(T<sub>0</sub>) in which T<sub>0</sub> and T was the time required for the pH drop from 8.3 to 6.3, with and without CA, respectively.
+
  
 +
===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 CO<sub>2</sub>-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 (T<sub>0</sub>-T)/(T<sub>0</sub>) in which T<sub>0</sub> and T was the time required for the pH drop from 8.3 to 6.3, with and without CA, respectively.
  
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<!-- Add more about the biology of this part here
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===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 CO<sub>2</sub> concentration and thus increase the carbon flux of the bypass pathway. The efficiency of the bypass pathway is thus been increased.
  
  
 +
[[File:T--NCKU_Tainan--Results_Results_Fig_14_a2.PNG|460px|left]]
 +
[[File:T--NCKU_Tainan--Results_Results_Fig_of_xui_ca.PNG|460px|right]]
  
  
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Fig. 3 Shows the growth and XUI comparison of each strain. All the tested strains are incubated in 5% CO<sub>2</sub> 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.
 +
<!-- Add more about the biology of this part here
 +
 +
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Fig. 3  Shows the growth and XUI comparison of each strains. All the tested strains are incubated in 5% CO<sub>2</sub> 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.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2762004 parameters</partinfo>
 
<partinfo>BBa_K2762004 parameters</partinfo>
 
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====Reference====
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[1]M. Wilbur, N.G. Anderson.(1948, Oct.1) Electrometric and colorimetric determination of carbonic anhydrase,<i> J. Biol. Chem. </i>147–154.
 +
 +
[2] Lindskog S. (1997) .Structure and mechanism of carbonic anhydrase. <i>Pharmacol Ther.</i>
 +
 +
[3] Rowlett RS. (2010,Feb). Structure and catalytic mechanism of the β-carbonic anhydrases.<i>Biochimica et Biophysica Acta (BBA)</i>
 +
 +
[4] Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO<sub>2</sub>-fixing Escherichia coli reveals great potential of heterotrophic CO<sub>2</sub> fixation.<i> Biotechnology for Biofuels.</i>
 +
 +
[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.<i>Process Biochemistry</i>

Latest revision as of 14:12, 17 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.

T--NCKU Tainan--part BBa K2762008 white final.png

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

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.


T--NCKU Tainan--Results Results Fig 14 a2.PNG
T--NCKU Tainan--Results Results Fig of xui ca.PNG


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]


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