Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002

Abstract

Background: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations.

Results: We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly.

Conclusions: The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications.

Keywords: BioBrick; Cyanobacteria; Genetic engineering; Neutral integration sites; Synechococcus sp. PCC7002; Synthetic biology; Transformation.

Fig. 1

Schematic representation of the construction of neutral integration site strains. The genes in green arrows represent the genetic sites of interest. The BioBrick Prefix and Suffix are indicated in blue and the kanamycin resistance cassette in red. a, c, e Schematic representation of the genetic organization of wildtype chromosomal DNA Synechococcus sp. PCC 7002 at three NISs (not to scale). b Chromosomal DNA after homologous recombination replacing SYNPCC7002_A2842 with a kanamycin resistance cassette flanked by the BioBrick Prefix and Suffix. d Chromosomal DNA after homologous recombination inserting a kanamycin resistance cassette flanked by the BioBrick Prefix and Suffix between SYNPCC7002_A0935 and SYNPCC7002_A0936. f Chromosomal DNA after homologous recombination resulting in a disruption of SYNPCC7002_A0159 by inserting a kanamycin resistance cassette flanked by the BioBrick Prefix and Suffix

Fig. 2

Results of the transformation efficiency assessment by optical density measurements at 730 nm. Synechococcus sp. PCC 7002 strains were transformed with a kanamycin resistance cassette containing either 100, 200, 400 or 800 nt homology with the targeted genetic site in the chromosome to induce homologous recombination. Three different approaches were used to target three sites: (1) Insertion in the non-coding region between SYNPCC7002_A0935 and SYNPCC7002_A0936 (noted as A0935), (2) gene disruption of the coding region of desB (noted as A0159) and (3) gene replacement of glpK (noted as A2842). The optical density at 730 nm (OD₇₃₀) of the constructed mutants were measured after transformation and antibiotic selection and normalized to the highest measured OD₇₃₀. Data points are the average of three separate experiments, and the error bars are indicative of the standard error

Fig. 3

Results of the transformation efficiency assessment by camera-based plate imaging. Synechococcus sp. PCC 7002 strains were transformed with a kanamycin resistance cassette containing either 100, 200, 400 or 800 nt homology with the targeted genetic site in the chromosome to induce homologous recombination. Three different approaches were used to target three sites: (1) Insertion in the non-coding region between SYNPCC7002_A0935 and SYNPCC7002_A0936 (noted as A0935), (2) gene disruption of the coding region of desB (noted as A0159) and (3) gene replacement of glpK (noted as A2842). The intensities of spotted transformant colonies on plates of the constructed mutants were measured after transformation and antibiotic selection and analyzed with a camera-based method. Data points are the average of three separate experiments, and the error bars are indicative of the standard error

Fig. 4

Growth densities of Synechococcus sp. PCC 7002 wildtype and A0159, A0935 and A2842 strains. Growth density was calculated based on data obtained either by spectrophotometric measurements or by measuring spot intensities and related to the Synechococcus sp. PCC 7002 wildtype growth density. Statistical tests were performed with the unpaired t-test provided by Prism version 6 (GraphPad). Shown are the average of technical triplicates and error bars indicate the standard error. a Maximal growth density of A0159, A0935 and A2842 strains as a percentage of wildtype density in liquid medium. Mutated strains and wildtype Synechococcus sp. PCC 7002 were grown in liquid medium and OD₇₃₀ measurements were performed every 24 h. Two-tailed P-values were 0.0018 (A2842), 0.0060 (A0935) and 0.0034 (A0159). b Maximal growth density A0159, A0935 and A2842 strains as a percentage of wildtype density on solid medium. Cultures of Synechococcus strains A0159, A0935 and A2842 were spot-plated and the relative change in intensity of the spots was assessed by a camera-based method. Wildtype Synechococcus sp. PCC 7002 was used as a control