Difference between revisions of "Part:BBa K2762006"
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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 then 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 then did the SDS-PAGE to confirm the present of the RbcX and RbcS protein. | ||
| − | [[File:T--NCKU Tainan--part BBa K2762006. | + | [[File:T--NCKU Tainan--part BBa K2762006 new.png|200px|centre]] |
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Revision as of 11:54, 14 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 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 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
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]