pSyn-6 Synechococcus Protein Expression Plasmid Vector蓝细菌聚球藻属蛋白表达载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

pSyn-6 Synechococcus Protein Expression Plasmid Vector蓝细菌聚球藻属蛋白表达载体质粒

BioVector® pSyn-6 Synechococcus Protein Expression Vector
For expression of recombinant proteins in Synechococcus elongatusIntroduction The GeneArt™ Synechococcus Protein Expression System is a prokaryotic
photosynthetic model system based on the unicellular cyanobacterium Synechococcus
elongatus PCC 7942 (http://cyanobacteria.web.pasteur.fr), which offers a simplified
approach for cloning and expressing genes of interest for the study of circadian
rhythms, nutrient regulation, environmental response, lipid metabolism, and
protein expression.
Synechococcus
elongatus PCC 7942
Synechococcus elongatus PCC 7942 is a freshwater unicellular cyanobacterium.
Cyanobacteria, sometimes referred to as blue-green algae, are prokaryotes that are
able to obtain their energy through photosynthesis.
S. elongatus has a rod-shaped appearance and is oligotrophic, having the ability to
survive in freshwater environments with low nutrients. S. elongatus genome
consists of one chromosome (chromosome 1) and possibly up to two plasmids that
have been sequence verified (Holtman et al. 2005, Chen et al. 2008, and Van der
Plas et al. 1992). The circular chromosome of S. elongatus is ~2.7 Mb in length (fully
sequenced) with 55.5% GC content (https://www.ncbi.nlm.nih.gov/genome/430).
718 of the organism's 2,723 genes have been identified as essential for survival
under laboratory conditions (Rubin et al., 2017).
The cyanobacterium S. elongatus PCC 7942 is an excellent synthetic biology chassis
and a model system for studying prokaryotic circadian rhythms, nutrient
regulation, environmental responses, and lipid metabolism because of its small
genome size and the ease with which it can be genetically manipulated by natural
transformation or conjugation from E. coli (Atsumi et al., 2009; Ducat et al., 2011;
Lan & Liao, 2011; Min & Golden, 2000; Simkovsky et al., 2012; Taniguchi et al., 2012).
Transformation of
Synechococcus
elongatus PCC 7942
The transformation of S. elongatus PCC 7942 relies on homologous recombination
between the cell’s chromosome and exogenous DNA that is not autonomously
replicating and containing sequences homologous to the chromosome.
The location of integration into the chromosome (neutral site, NS1) has been
developed as a cloning locus (Clerico et al., 2007) as it can be disrupted without any
aberrant phenotype, thus allowing the homologous recombination of ectopic
sequences.
When transformed with vectors containing an antibiotic resistance cassette and
neutral site sequences, a double homologous recombination event occurs between
the neutral site vector and the S. elongatus chromosome.
The selective marker (Spectinomycin) and the gene of interest driven by a
promoter are inserted into the neutral site and the vector backbone (pUC) is lost,
allowing the expression of recombinant genes in S. elongatus PCC 7942.

pSyn_6 vector The pSyn_6 vector (4461 bp) is designed to facilitate rapid cloning of your gene of
interest (GOI) for expression in Synechococcus elongatus PCC 7942. Some of the
features of the vector are listed below. For a map of the vector, see page 17.
• NS1 (neutral site 1) homologous recombination sites for the integration of the
vector into the Synechococcus elongatus genome.
• The strong constitutive promoter of psbA gene (encoding photosystem II
protein D1) from Synechococcus elongatus PCC 7942 driving the high level
expression of your gene of interest.
• An N-terminal polyhistidine tag followed by a TEV recognition site for TEV
protease-dependent cleavage of the N-terminal tag from your recombinant
protein.
• A C-terminal V5 epitope followed by a polyhistidine tag for detection and
purification of your recombinant protein.
• Two multiple cloning sites (MCS) that provide the flexibility to include either
or both or none of the N-terminal and C-terminal tags using seamless,
Type IIs, or restriction enzyme digestion-based cloning methods.
Note: When cloning, make sure that your gene of interest is in frame with the
tags you wish to include. For
• Spectinomycin resistance gene for selection in E. coli and Synechococcus
elongatus PCC 7942.
• rrnB sequences for strong transcription termination.
• pUC origin for maintenance in E. coli.
• RP4/RK2 bom site (basis of mobility or oriT) for conjugation.
Experiment Outline
Workflow The table below describes the major steps needed to clone and express your gene of
interest (GOI) in Synechococcus elongatus PCC 7942. For details, refer to the pages
indicated.
Step Action Page
1 Clone your gene of interest (GOI) directly into pSyn_6 vector 5
2 Transform One Shot™ TOP10 E. coli with the pSyn_6 construct
containing your GOI and select the transformants on LB plates
containing spectinomycin
8
3 Analyze transformants by restriction digestion or PCR 9
4 Thaw and resuscitate Synechococcus elongatus PCC 7942 cells 12
5 Transform Synechococcus elongatus PCC 7942 cells and select
transformants
14
7 Perform colony PCR to screen the transformed Synechococcus
elongatus PCC 7942 colonies for full integration of the GOI


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