Coding

Part:BBa_K2762001

Designed by: Ting-Shou Liu   Group: iGEM18_NCKU_Tainan   (2018-09-29)
Revision as of 14:23, 12 October 2023 by Rhea3957 (Talk | contribs)


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.

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.

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