Part:BBa_K2350003:Design
Intrinsic promoter of Rubisco large subunit (PrbcL)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
In order to overexpress foreign genes in the cyanobacteria, the intrinsic promoter of Rubisco large subunit (PrbcL) was chosen as the target for vector construction, which is retrieved from pBR322 in our experiment. PrbcL regulates the expression of the most abundant proteins in photosynthetic species and has been proven to have a high activity to express foreign genes, so we choose PrbcL as the promoter of our pigment gene. To insert PrbcL with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting site in PrbcL nucleotide sequence.
Design Notes
1.Using fusion PCR, we fused PrbcL with desired pigment gene together.
Fusion PCR primers for PrbcL Forward:AATTC TGATGGAAAAAGCACTGTAA Reverse:tgtgtaatattattttctaa CATGTCGTCTCTCCCTAGAG
2.All Pst1 cutting sites in PrbcL were site-directed mutated.
Site-directed primers: Forward: GGACTTGCGCTGTGGGACTGGAGCTTTACAGGCTCCCCCT Reverse: GGACTTGCGCTGTGGGACTGGAGCTTTACAGGCTCCCCCT
3.Fusion PCR protocol of PrbcL and IndC
94 2min
94 20sec X5
51 30sec
68 4min30sec
Pause and add 2ul Fusion Primers
94 20sec
32.6 30sec
68 4min30sec
68 10min
Source
pBR322 gene sequence
References
Pei-Hong Chen, Hsien-Lin Liu, Yin-Ju Chen, Yi-Hsiang Cheng, Wei-Ling Lin, Chien-Hung Yeh and Chuan-Hsiung Chang (2012). Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria