Difference between revisions of "Part:BBa K2762006"
 
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__NOTOC__
 
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<partinfo>BBa_K2762006 short</partinfo>
 
<partinfo>BBa_K2762006 short</partinfo>
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===Usage===
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===Background===
 
The <i>rbcXS</i> part encode the RbcS subunit of RubisCo and the chaperon RbcX. The RbcS affect the activity of RubisCO and RbcX contribute to the correct folding of the whole RubisCO enzyme.  
 
The <i>rbcXS</i> part encode the RbcS subunit of RubisCo and the chaperon RbcX. The RbcS affect the activity of RubisCO and RbcX contribute to the correct folding of the whole RubisCO enzyme.  
  
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===Biology===
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===Mechanism===
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The <i>rbcX</i> and <i>rbcS</i> genes are from cyanobacteria Synechococcus elongatus PCC 7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21  to ensure the high expression rate. It has reported that the expression of RubisCO could not be detectable while only add single promoter of the RbcL-RbcX-RbcS gene fragment. Therefore we added another T7 promoter on the upstream of <i>rbcX-rbcS</i> gene and named the part as <i>rbcXS</i>. We also did the coden optimization for the gene to ensure successful expression. The activation of RubisCO will be maximize after the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011
+
The <i>rbcX</i> and <i>rbcS</i> genes are from cyanobacteria <i>Synechococcus elongatus</i> PCC7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21  to ensure the high expression rate. It has reported that the expression of RubisCO could not be detectable while only add single promoter of the RbcL-RbcX-RbcS gene fragment. Therefore we added another T7 promoter on the upstream of <i>rbcX-rbcS</i> gene and named the part as <i>rbcXS</i>. We also did the coden optimization for the gene to ensure successful expression. The activation of RubisCO will be maximize after 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 cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. 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 RbcX and RbcS protein.
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===Expression in <i>E. coli</i>===
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https://static.igem.org/mediawiki/parts/1/1f/T--NCKU_Tainan--part_BBa_K2762006.jpg|350px|centre
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We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. Second, we cloned the plasmid into BL21(DE3) and induced the promoter with IPTG. We then did the SDS-PAGE to confirm the present of the RbcX and RbcS protein.
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[[File:T--NCKU Tainan--part BBa K2762006 new.png|200px|centre]]
  
 
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<partinfo>BBa_K2762006 parameters</partinfo>
 
<partinfo>BBa_K2762006 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>
 +
 +
[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>.
 +
 +
[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>
 +
 +
[4] Sandra Saschenbrecker,<sup>1,2</sup> Andreas Bracher,<sup>1,2,*</sup> Karnam Vasudeva Rao,<sup>1</sup> Bharathi Vasudeva Rao,<sup>1</sup> F. Ulrich Hartl,<sup>1,*</sup> and Manajit Hayer-Hartl<sup>1,*</sup>. (2007, APR. 25). Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric Rubisco. <i>Cell</i>
 +
 +
[5] Cuimin Liu<sup>1*</sup>, Anna L. Young<sup>2*</sup>, Amanda Starling-Windhof<sup>1</sup>, Andreas Bracher<sup>1</sup>, Sandra Saschenbrecker<sup>1</sup>, Bharathi Vasudeva Rao<sup>1</sup>, Karnam Vasudeva Rao<sup>1</sup>, Otto Berninghausen<sup>2</sup>, Thorsten Mielke<sup>3</sup>, F. Ulrich Hartl<sup>1</sup>, Roland Beckmann<sup>2</sup> & Manajit Hayer-Hartl<sup>1</sup>. (2010, JAN. 4). Coupled chaperone action in folding and assembly of hexadecameric Rubisco. <i>Nature</i>

Latest revision as of 14:22, 17 October 2018


PT7-B0034-rbcX-B0034-rbcS-B0015

Background

The rbcXS part encode the RbcS subunit of RubisCo and the chaperon RbcX. The RbcS affect the activity of RubisCO and RbcX contribute to the correct folding of the whole RubisCO enzyme.

Mechanism

The rbcX and rbcS genes are from cyanobacteria Synechococcus elongatus PCC7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21 to ensure the high expression rate. It has reported that the expression of RubisCO could not be detectable while only add single promoter of the RbcL-RbcX-RbcS gene fragment. Therefore we added another T7 promoter on the upstream of rbcX-rbcS gene and named the part as rbcXS. We also did the coden optimization for the gene to ensure successful expression. The activation of RubisCO will be maximize after the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011

Characterization

Expression in E. coli

We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. Second, we cloned the plasmid into BL21(DE3) and induced the promoter with IPTG. We then did the SDS-PAGE to confirm the present of the RbcX and RbcS protein.

T--NCKU Tainan--part BBa K2762006 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
    COMPATIBLE WITH RFC[25]
  • 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.

[4] Sandra Saschenbrecker,1,2 Andreas Bracher,1,2,* Karnam Vasudeva Rao,1 Bharathi Vasudeva Rao,1 F. Ulrich Hartl,1,* and Manajit Hayer-Hartl1,*. (2007, APR. 25). Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric Rubisco. Cell

[5] Cuimin Liu1*, Anna L. Young2*, Amanda Starling-Windhof1, Andreas Bracher1, Sandra Saschenbrecker1, Bharathi Vasudeva Rao1, Karnam Vasudeva Rao1, Otto Berninghausen2, Thorsten Mielke3, F. Ulrich Hartl1, Roland Beckmann2 & Manajit Hayer-Hartl1. (2010, JAN. 4). Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature