Difference between revisions of "Part:BBa K2762002"

Revision as of 13:28, 17 October 2018

Small subunit of the ribulose-bisphosphate carboxylase/oxygenase rbcS

Background

The rbcS part encode the rbcS subunit of RubisCO. The rbcS affect the activity of RubisCO.

Mechanism

The 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: RubisCO.

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

Source

Codon oprimized Synechococcus elongatus PCC7002.

References

[1] Janet Newman 1t and Steven Gutteridge2*. (1994,JUNE.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. Doi:https://doi.org/10.1016/S0969-2126(00)00050-2