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| Backbone Design | | Backbone Design |
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| <p> To genetically engineer cyanobacteria, we chose <font class='mark_backbone'><i>Synechococcus elongatus PCC 7942</i></font> as our engineering host. | | <p> To genetically engineer cyanobacteria, we chose <font class='mark_backbone'><i>Synechococcus elongatus PCC 7942</i></font> as our engineering host. |
| Our main strategy is to embark on <font class='mark_backbone'><b>gene double-crossover homologous recombination</b></font> in <i>S. elongatus PCC 7942</i> genome, which is the first cyanobacterial strain to be transformed by exogenous DNAs and is reliably transformable through natural uptake of extracellular DNAs. | | Our main strategy is to embark on <font class='mark_backbone'><b>gene double-crossover homologous recombination</b></font> in <i>S. elongatus PCC 7942</i> genome, which is the first cyanobacterial strain to be transformed by exogenous DNAs and is reliably transformable through natural uptake of extracellular DNAs. |
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| The transformed strains (transformants) were usually successfully obtained after 2 to 3 weeks and survived the ampicillin treatment. | | The transformed strains (transformants) were usually successfully obtained after 2 to 3 weeks and survived the ampicillin treatment. |
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Pigments
In our project, we transfer five types of pigment-related gene sequence (Indigoidine, Zeaxanthin, Melanin, Astaxanthin 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.
Backbone Design
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 DH5α, 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.1
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 experiments.
After mass reproduction in E. coli DH5α, PCC7942 were transformed through the uptake of plasmid DNAs extracted from E. coli DH5α.
The transformed strains (transformants) were usually successfully obtained after 2 to 3 weeks and survived the ampicillin treatment.
Cyan pigment (Indigoidine)
Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In nowadays studies, an Indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment2. The IndB gene codes for a putative phosphatase and the IndC gene codes for Indigoidine synthase. Together, these enzymes convert L-glutamine into Indigoidine. Recently, it has been shown that IndC alone can produce Indogoidine, and the inclusion of IndB expression in the system will increase yields significantly3.
As we know, L-Glutamine is the direct biosynthetic precursor of Indigoidine, and it is a key amino acid in primary metabolism and thus naturally exists in S. elongatus PCC7942. Because glutamine related products are already existed in S. elongatus PCC7942, we only need to activate the expression of Sc-IndC in S. elongatus PCC7942 which leads to the production of Indigoidine. However, due to the access difficulties of Streptomyces chromofuscus ATCC 49982, we decided to use the previous part for IndC, which has been submitted to the iGEM Parts Registry (BBa_K1152008)4. According to the part design, our Indigoidine gene comes from Photorhabdus luminescens laumondii TT01 (DSM15139).