Difference between revisions of "Part:BBa K2762010"

Latest revision as of 14:22, 17 October 2018

PlacI-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 gene are from cynobacteria Synechococcus elongatus PCC 7002. We designed the P_lacI (BBa_R0010) promoter for the gene and cloned gene in DH5 alpha in order to test the function while consecutive expression.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 PlacI 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_K2762012.

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 W3110. We than did the SDS-PAGE to confirm the present of the rbcX and rbcS protein.

Sequence and Features

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-Hartl^1,*. (2007, APR. 25). Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric Rubisco. Cell

[5] Cuimin Liu^1*, Anna L. Young^2*, 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