Difference between revisions of "Part:BBa K2762005"
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<partinfo>BBa_K2762005 short</partinfo>
 
<partinfo>BBa_K2762005 short</partinfo>
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===Usage===
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===Background===
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Ribulose-1,5-biphosphate carboxylase/oxygenase catalyzes the first reaction of the Calvin cycle, converting the combination of Ribulose-1,5-biphosphate (RuBP) and carbon dioxide to two 3-phosphoglycerate molecular. The rbcL part encodes the large subunit of the RubisCO enzyme, which also contain the active site of the enzyme. In our carbon fixing  pathway, the RubisCO enzyme is the most important enzyme catalyzing the reaction of RuBP and CO2.  
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Ribulose-1,5-biphosphate carboxylase/oxygenase catalyzes the first reaction of the Calvin cycle, converting the combination of Ribulose-1,5-biphosphate (RuBP) and carbon dioxide to two 3-phosphoglycerate molecular. The rbcL part encodes the large subunit of the RubisCO enzyme, which also contain the active site of the enzyme. In our carbon fixing  pathway, the RubisCO enzyme is the most important enzyme catalyzing the reaction of RuBP and CO2.
  
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===Biology===
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===Mechanism===
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The <i>rbcL</i> gene is from  cynobacteria <i>Synechococcus elongatus PCC 7002</i>. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21 (DE3) to ensure the high expression rate because the RbcL protein is the most important protein as previous description. We also coden optimized the gene to ensure successful expression. However, the activation of RubisCO will be maximize with the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011.
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The <i>rbcL</i> gene is from  cynobacteria <i>Synechococcus elongatus</i> PCC7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21 (DE3) to ensure the high expression rate because the RbcL protein is the most important protein as previous description. We also coden optimized the gene to ensure successful expression. However, the activation of RubisCO will be maximize with the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011.
  
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===Characterization===
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==Characterization==
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We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion. Second, we cloned the plasmid into BL21 (DE3) and induced the promoter with IPTG. We than did the SDS-PAGE to confirm the present of the RbcL.
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===Expression in <i>E. coli</i>===
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We inserted the part on pSB1C3 and transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion.  
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[[File:T--NCKU Tainan--part BBa K2762005 new .png|200px|centre]]
  
 
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<partinfo>BBa_K2762005 parameters</partinfo>
 
<partinfo>BBa_K2762005 parameters</partinfo>
 
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====References====
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[1] Janet Newman 1t and Steven Gutteridge2*. (1994,JUN.15). Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate.<i> Cell</i>
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[2] Inger Andersson <sup>a,</sup>*, Anders Backlund <sup>b</sup>. (2007, DEC.20). Structure and function of Rubisco. <i>Plant Physiology and Biochemistry</i>.
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[3] 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>

Latest revision as of 14:10, 17 October 2018


PT7-B0034-rbcL-B0015

Background

Ribulose-1,5-biphosphate carboxylase/oxygenase catalyzes the first reaction of the Calvin cycle, converting the combination of Ribulose-1,5-biphosphate (RuBP) and carbon dioxide to two 3-phosphoglycerate molecular. The rbcL part encodes the large subunit of the RubisCO enzyme, which also contain the active site of the enzyme. In our carbon fixing pathway, the RubisCO enzyme is the most important enzyme catalyzing the reaction of RuBP and CO2.

Mechanism

The rbcL gene is from cynobacteria Synechococcus elongatus PCC7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21 (DE3) to ensure the high expression rate because the RbcL protein is the most important protein as previous description. We also coden optimized the gene to ensure successful expression. However, the activation of RubisCO will be maximize with the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011.

Characterization

Expression in E. coli

We inserted the part on pSB1C3 and transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion.

T--NCKU Tainan--part BBa K2762005 new .png

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1198
    Illegal AgeI site found at 301
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Janet Newman 1t and Steven Gutteridge2*. (1994,JUN.15). Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate. Cell

[2] Inger Andersson a,*, Anders Backlund b. (2007, DEC.20). Structure and function of Rubisco. Plant Physiology and Biochemistry.

[3] 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.