Difference between revisions of "Part:BBa K2762005"
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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 transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion. 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 RbcL. | We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion. 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 RbcL. | ||
| − | [[File:T--NCKU Tainan--part BBa K2762005.png|200px|centre]] | + | [[File:T--NCKU Tainan--part BBa K2762005 new .png|200px|centre]] |
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Revision as of 11:57, 14 October 2018
PT7-B0034-rbcL-B0015
Background
Ribulose-1,5-biphosphate carboxylase/oxygenase catalyzes the first reaction of the Calvin cycle, converting the combination of Ribulose-1,5-biphosphate (RuBP) and carbon dioxide to two 3-phosphoglycerate molecular. The rbcL part encodes the large subunit of the RubisCO enzyme, which also contain the active site of the enzyme. In our carbon fixing pathway, the RubisCO enzyme is the most important enzyme catalyzing the reaction of RuBP and CO2.
Mechanism
The rbcL gene is from cynobacteria Synechococcus elongatus PCC 7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21 (DE3) to ensure the high expression rate because the RbcL protein is the most important protein as previous description. We also coden optimized the gene to ensure successful expression. However, the activation of RubisCO will be maximize with 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 transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion. 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 RbcL.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1198
Illegal AgeI site found at 301 - 1000COMPATIBLE WITH RFC[1000]