SEVAtile: a standardised DNA assembly method optimised for Pseudomonas

Abstract

To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non-model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs-based assembly approach, which enables the rapid and standardised assembly of genetic partsor tilesto create genetic circuits in the established SEVA-vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA-and non-SEVA. To prove the efficiency of the SEVAtile method, a threevector system was successfully generated to independently co-express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self-replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field.

Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

“…terminator of phage T7 between the two genes, as described earlier (46,51). Relative to the control, all phage terminators show reduced levels of downstream mCherry expression and, therefore, significant termination activity (Student's t-test, p < 0.05), confirming their functionality in vivo (Figure 6e).…”

“…As a separate validation, the activity of a subset of the late LUZ7 promoters was evaluated in vivo using a promoter trap system using the SEVAtile-based expression systems (46). For this, genetic constructs containing a late phage promoter, a standard ribosome binding site (RBS), and the green fluorescent reporter protein (msfGFP) gene were constructed with the SEVAtile assembly method, followed by transformation to P. aeruginosa and E. coli .…”

“…To this end, we constructed a terminator trap using the SEVAtile assembly method, in which the phage terminators are inserted between two fluorescent reporter genes, msfGFP and mCherry , whose expression is driven by a constitutive promoter (46). To quantify termination activity, the intensity of msfGFP and mCherry expression was measured for all samples, and compared to control constructs containing either no terminator sequence or the native T7 terminator of phage T7 between the two genes, as described earlier (46, 51). Relative to the control, all phage terminators show reduced levels of downstream mCherry expression and, therefore, significant termination activity (Student’s t-test, p < 0.05), confirming their functionality in vivo (Figure 6e).…”

“…The functionality of selected LUZ7 late promoters was confirmed experimentally in vivo using our recently established SEVAtile-based expression systems. For this, adaptors (generated by overlapping primer pairs) encoding putative promoters were cloned upstream of a standardized ribosomal binding site (BCD2) and an msfGFP reporter gene in a pBGDes vector using SEVAtile assembly (46). In addition, a vector lacking a promoter sequence (pBGDes BCD2-msfGFP) and a vector with a constitutive promoter (pBGDes P EM7 -BCD2-msfGFP) was created to serve as a negative and positive control, respectively.…”

“…The next day, the cultures were diluted in fresh M9 medium with the appropriate antibiotic in a Corning® 96 Well Black Polystyrene Microplate with Clear Flat Bottom and incubated with shaking. After 3.5 hours, msfGFP and OD 600 levels were measured on the CLARIOstar® Plus Microplate Reader (BMG Labtech, Ortenberg, Germany), as described previously (46). The msfGFP fluorescence units were normalized for the associated OD 600 values and converted to absolute units using an independent calibrant, 5(6)- carboxyfluorescein (5(6)-FAM) (Sigma Aldrich) (46, 48).…”