Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

BackgroundThe unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a photoautotrophic host for synthetic biology studies. However, as a green chassis to capture CO2 for biotechnological applications, the genetic toolbox for Synechocystis 6803 is still a limited factor.ResultsWe systematically characterized endogenous genetic elements of Synechocystis 6803, including promoters, ribosome binding sites, transcription terminators, and plasmids. Expression from twelve native promoters was compared by measuring fluorescence from the reporter protein EYFP in an identical setup, exhibiting an 8000-fold range of promoter activities. Moreover, we measured the strength of twenty native ribosome binding sites and eight native terminators, indicating their influence on the expression of the reporter genes. In addition, two shuttle vectors, pCA-UC118 and pCB-SC101, capable of replication in both Synechocystis 6803 and E. coli were constructed. Expression of reporter proteins were significantly enhanced in cells containing these new plasmids, thus providing superior gene expression platforms in this cyanobacterium.ConclusionsThe results of this study provide useful and well characterized native tools for bioengineering work in the model cyanobacterium Synechocystis 6803.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0897-8) contains supplementary material, which is available to authorized users.

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters

“…Nevertheless, the vast majority of the available synthetic biology tools have been developed for heterotrophic chassis like E. coli or Saccharomyces cerevisiae (6,7), and most of the regulatory elements characterized in E. coli function rather poorly or not at all in cyanobacteria (8,9). Consequently, a considerable effort has been placed on the design and construction of efficient, predictable and easy-to-use molecular tools for cyanobacteria (8,(10)(11)(12)(13)(14). Within this context, native promoters such as P psbA2 , P nrsB or the 'super strong' P cpcG560 have been previously used to drive gene expression for specialty chemicals production (11,15,16).…”

Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria

“…Therefore, it was not obvious that all strong E. coli terminators would work well in Synechocystis . During preparation of this manuscript the characterisation of seven putative terminators found in the Synechocystis genome was reported [12] , with moderate termination efficiencies observed. In our study, non-native and synthetic sequences were included and twelve strong Rho-independent terminators identified.…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Synechocystis sp PCC 6803 (Huang et al, 2010;Huang and Lindblad, 2013;Englund et al, 2016;Ferreira et al, 2018;Liu and Pakrasi, 2018) Synechococcus sp PCC 7002 (Markley et al, 2015;Ruffing et al, 2016;Zess et al, 2016) S. elongatus PCC 7942 Qiao et al, 2018) S. elongatus UTEX 2973 Nostoc sp PCC 712mix0 (Videau et al, 2016;Wegelius et al, 2018) Vectors and Sandardized Assembly…”

“…Pcpc560 (Huang et al, 2010;Zhou et al, 2014;Englund et al, 2016;Ruffing et al, 2016;Ferreira et al, 2018;Li et al, 2018;Liu and Pakrasi, 2018 Ptrc (Huang et al, 2010;Guerrero et al, 2012;Oliver et al, 2013;Markley et al, 2015;Ferreira et al, 2018;Li et al, 2018) TetR (aTc Inducible) L03 (Huang and Lindblad, 2013;Zess et al, 2016;Ferreira et al, 2018) efficient transformation using conjugation and electroporation (compared to homologous recombination); (2) avoidance of the need for time-consuming segregation to achieve incorporation of the transgenic sequence in all copies of the genome; and (3) increased orthogonality of the heterologous sequences, meaning no insertion in genomic loci that may not be neutral. A hybrid strategy that combines the advantages of using a shuttle vector and the stability of genomic insertion involves using native cyanobacterial plasmids, either as insertion sites (Ng et al, 2015) or as true shuttle vectors after isolation and modification (Jin et al, 2018).…”

“…Synechocystis sp. PCC 6803 (Huang et al, 2010;Huang and Lindblad, 2013;Englund et al, 2016;Liu and Pakrasi, 2018;Ferreira et al, 2018) Synechococcus sp. PCC 7002 (Markley et al, 2015;Ruffing et al, 2016;Zess et al, 2016) Synechococcus elongatus PCC 7942 Qiao et al, 2017) Synechococcus elongatus UTEX 2973 Nostoc sp.…”