Difference between revisions of "Part:BBa K2350003"

Revision as of 07:51, 21 October 2017

Intrinsic promoter of Rubisco large subunit (PrbcL)

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+In our project, we transfer six types of pigment related gene sequence (Melanin, Indigoidine, Astaxanthin, Zeaxanthin, and Lycopene) into our cyanobacteria. We expect to get six different colors of microalgae, so we could see whether changing the original color of microalgae would change wavelength absorbance and have better photosynthetic efficiencies. Due to better photosynthetic efficiencies, we could elevate oil accumulation in microalgae, which would have great benefit in both industry and scientific usage.
+To genetically engineer cyanobacteria, we chose Synechococcus elongatus PCC 7942 as our engineering host. Our main strategy is to embark on gene double-crossover homologous recombination in S. elongatus PCC 7942 genome, which is the first cyanobacterial strain to be transformed by exogenous DNAs and is reliably transformable through natural uptake of extracellular DNAs.
+First, we constructed a vector which is able to finish double-crossover homologous gene recombination in S. elongatus PCC 7942. The vector (pPIGBACK) contains 5’- and 3’-ends of the neutral site II (NSII) and an ampicillin resistance gene (AmpR) for antibiotic selection. Then we fused AmpR with double terminator, BBa_B0015, which is proved to be functional in cyanobacteria.
+Additionally, in order to easily manipulate DNAs for gene cloning and plasmid preparation in E. coli DH5a, the replication origin (ORI) of pBR322 was also introduced to make the plasmid vector replicable in E. coli.
+Then, 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. 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 chose PrbcL as the promoter of our pigment gene.
+The strategy we chose to construct the vector is to fuse B0015 and AmpR together first. Secondly, we fused 5’- and 3’-ends of the neutral site II (NSII) with PBR322 replication origin (ORI) together. At last, we ligated two parts together. The vector (pPIGBACK) is used to transform into PCC7942 with the inserted pigment gene in our experiment.
+After mass reproduction in E. coli DH5a, PCC7942 were transformed through the uptake of plasmid DNAs extracted from E. coli DH5a. The transformed strains (transformants) were usually successfully obtained after 2–3 weeks and survived the ampicillin treatment.