Part:BBa_K3971030
Cassette to insert spp gene in the Neutral Site 3 of the S. elongatus UTEX 2973 genome.
spp codes for Sucrose-phosphate phosphatase (SPP), an enzyme involved in the sucrose biosynthesis pathway in the cyanobacteria Synechocystis PCC 6803 [1].
This composite part has been designed to insert the spp gene into the Neutral Site 3 of the S. elongatus UTEX 2973 genome.
The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another. Thus one can study the effect of different promoters on sucrose production efficiency.
Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid, which is a Cpf1 based CRISPR plasmid [2], which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.
We aimed to overexpress the spp gene in order to increase the amount of sucrose produced by S. elongatus when exposed to salt stress.
References
1:Lin, Po-Cheng, Fuzhong Zhang, and Himadri B. Pakrasi. "Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973." Scientific reports 10.1 (2020): 1-8.
2: Du, Wei, et al. "Exploring the photosynthetic production capacity of sucrose by cyanobacteria." Metabolic engineering 19 (2013): 17-25.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 635
Illegal PstI site found at 2220 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 635
Illegal PstI site found at 2220 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 635
Illegal PstI site found at 2220 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 635
Illegal PstI site found at 2220 - 1000COMPATIBLE WITH RFC[1000]
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