Difference between revisions of "Part:BBa K2762001"

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===Expression in <i>E. coli</i>===
 
===Expression in <i>E. coli</i>===
 
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.
 
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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===Additional documentation on Rbc X===
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The subunit RbcX of the RUBISCO protein structure is an integral part of the protein. While it plays no part in the protein folding process [3]. It is present in both the cytoplasm as well as within a carboxysome. Therefore for extraction, a simple crude cell lysis will be sufficient [4]. It is so integral that delayed post translational addition of RbcX resulted in the rapid loss of RbcL assembly and in
 +
the formation of insoluble RbcL. This indicates that RbcX has a specific chaperone function in preventing RbcL misassembly downstream of chaperonin [5]. To ensure this delay in transcription does not occur, it is suggested that a strong promoter is added upstream of the RbcX coding sequence to prevent any delays in translation and thereby prevent any missassembly of the other Rbc subunits.
  
 
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<partinfo>BBa_K2762001 parameters</partinfo>
 
<partinfo>BBa_K2762001 parameters</partinfo>
 
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===Source===
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Codon oprimized <i>Synechococcus elongatus</i> PCC7002.
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====References====
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[1] 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>
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[2] 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>
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[3]Huang, F., Vasieva, O., Sun, Y., Faulkner, M., Dykes, G. F., Zhao, Z., & Liu, L. N. (2019). Roles of RbcX in carboxysome biosynthesis in the cyanobacterium Synechococcus elongatus PCC7942. Plant Physiology, 179(1), 184-194.
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[4]https://www.uniprot.org/uniprotkb/Q44177/entry#subcellular_location
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[5]Saschenbrecker, S., Bracher, A., Rao, K. V., Rao, B. V., Hartl, F. U., & Hayer-Hartl, M. (2007). Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell, 129(6), 1189-1200.

Latest revision as of 14:25, 12 October 2023


Assembly chaperone of ribulose-bisphosphate carboxylase/oxygenase rbcX

Background

The rbcX part encode the the chaperon of rbcX RubisCO. The rbcX contribute to the correct folding of the whole RubisCO enzyme.

Mechanism

The rbcX 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. We also did the coden optimization for the gene to ensure successful expression. To get more information, see our composite part: BBa_K2762011.

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 than did the SDS-PAGE to confirm the present of the RbcX and RbcS protein.


Additional documentation on Rbc X

The subunit RbcX of the RUBISCO protein structure is an integral part of the protein. While it plays no part in the protein folding process [3]. It is present in both the cytoplasm as well as within a carboxysome. Therefore for extraction, a simple crude cell lysis will be sufficient [4]. It is so integral that delayed post translational addition of RbcX resulted in the rapid loss of RbcL assembly and in the formation of insoluble RbcL. This indicates that RbcX has a specific chaperone function in preventing RbcL misassembly downstream of chaperonin [5]. To ensure this delay in transcription does not occur, it is suggested that a strong promoter is added upstream of the RbcX coding sequence to prevent any delays in translation and thereby prevent any missassembly of the other Rbc subunits.

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]


Source

Codon oprimized Synechococcus elongatus PCC7002.

References

[1] 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

[2] 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

[3]Huang, F., Vasieva, O., Sun, Y., Faulkner, M., Dykes, G. F., Zhao, Z., & Liu, L. N. (2019). Roles of RbcX in carboxysome biosynthesis in the cyanobacterium Synechococcus elongatus PCC7942. Plant Physiology, 179(1), 184-194.

[4]https://www.uniprot.org/uniprotkb/Q44177/entry#subcellular_location

[5]Saschenbrecker, S., Bracher, A., Rao, K. V., Rao, B. V., Hartl, F. U., & Hayer-Hartl, M. (2007). Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell, 129(6), 1189-1200.