Difference between revisions of "Part:BBa K2762013"

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<partinfo>BBa_K2762013 short</partinfo>
 
<partinfo>BBa_K2762013 short</partinfo>
  
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===Usage and Biology===
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===Background===
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This composite part is composed of BBa_R0010, BBa_I719005, BBa_B0034, BBa_K2762003, BBa_K2762004, and BBa_B0015 which encodes phosphoribulokinase (PRK) and carbonic anhydrases/dehydratases (CcaA) derived from Synechococcus elongatus PCC7942 and Synechococcus elongatus PCC7002 respectively.
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This composite part is composed of BBa_R0010, BBa_I719005, BBa_B0034, BBa_K2762003, BBa_K2762004, and BBa_B0015 which encodes phosphoribulokinase (PRK) and carbonic anhydrases/dehydratases (CcaA) derived from <i>Synechococcus elongatus</i> PCC7942 and <i>Synechococcus elongatus</i> PCC7002 respectively.
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Enzyme digestion of finished construct
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==Characterization==
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===Enzyme Digestion of DNA Fragement===
  
 
[[File:T--NCKU Tainan--part BBa K2762013 new .png|200px|centre]]
 
[[File:T--NCKU Tainan--part BBa K2762013 new .png|200px|centre]]
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Fig. 1 Enzyme digestion of BBa K2762013
  
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===Total Solution Test===
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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.
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http://2018.igem.org/Team:NCKU_Tainan/Results
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===Function of CA===
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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 P<sub>lacI</sub> promoter and CA with P<sub>T7</sub> 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.
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[[File:T--NCKU_Tainan--Results_Results_Fig_14_a2.PNG|460px|left]]
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[[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.
  
 
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<partinfo>BBa_K2762013 parameters</partinfo>
 
<partinfo>BBa_K2762013 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.
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[2] Lindskog S. (1997) .Structure and mechanism of carbonic anhydrase. <i>Pharmacol Ther.</i>
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[3] Rowlett RS. (2010,Feb). Structure and catalytic mechanism of the β-carbonic anhydrases.<i>Biochimica et Biophysica Acta (BBA)</i>
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[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>
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[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


PlacI-B0034-prk-B0015-PT7-B0034-CcaA-B0015

Background

This composite part is composed of BBa_R0010, BBa_I719005, BBa_B0034, BBa_K2762003, BBa_K2762004, and BBa_B0015 which encodes phosphoribulokinase (PRK) and carbonic anhydrases/dehydratases (CcaA) derived from Synechococcus elongatus PCC7942 and Synechococcus elongatus PCC7002 respectively.

Characterization

Enzyme Digestion of DNA Fragement

T--NCKU Tainan--part BBa K2762013 new .png

Fig. 1 Enzyme digestion of BBa K2762013

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