Part:BBa_K3930026

3,6-nonadienal induction system and expression in S. elongatus (pCONCOMBRE) Sequence and Features

Assembly Compatibility:

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Introduction

The pCONCOMBRE part (BBa_K3930026) enables the production of 3,6-nonadienal from linolenic and linoleic acids, and is composed of:

- the RA (BBa_K3930027) and LA (BBa_K3930028) integration sites in the NSI locus of S.elongatus genome (based on the plasmid pAM4951 from Easyclone Marker free kit (Taton et al. 2014)).

- the Nb-9-LOX (BBa_K3930030), Cm-9-HPL (BBa_K3930031) and LacI genes (BBa_C0012), for the production of 3,6-nonadienal and LacI repressor. The sequences of the Nb-9-LOX and the Cm-9-HPL were codon optimized for expression into S. elongatus.

- the IPTG and theophylline inducible promoter Trc-theoE-riboswitch (BBa_K3930029), driving the expression of Nb-9-LOX and Cm-9-HPL.

- the resistance marker SpecR (BBa_K3930032) to select for cyanobacteria integrants.

Construction

IDT performed DNA synthesis and delivered the part as gBlock. The construction was cloned with the In-Fusion Takara kit into the pAM4951 plasmid and then transformed into E.coli Dh5α strain. Figure 1 shows the restriction map of the correct resulting clone.

Figure 1: pCONCOMBRE assembly

pCONCOMBRE restriction profile from clone A5 was checked by digestion and visualized on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel).

pCONCOMBRE insert was then linearized by inverse PCR. The amplicon was integrated into the genome of S. elongatus strain following the triparental conjugation protocol of Gale et al. (2019). Figure 2 shows the electrophoresis gel of colony PCR to verify integrants genotype. Most expected sizes were obtained, but one amplicon for the left arm (LA) had not the right size. However, all genes from the insert were detected.

Figure 2: Integration of pCONCOMBRE insert in the cyanobacterium genome

pCONCOMBRE insert integration from clone D2 was checked by PCR visualised on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel).

We concluded pCONCOMBRE insert integration at the NSI locus was nonetheless successful. The integrant strain was named Synecho-pCONCOMBRE and saved as glycerol stock.

Conclusion and Perspectives

The pCONCOMBRE insert was successfully integrated into the S. elongatus genome. Nonetheless, the characterization of the production of 3,6-nonadienal has not been conducted, future iGEM teams will have to perform their own tests to verify the functionality of the construct.

References

1. Gale GAR, Osorio AAS, Puzorjov A, Wang B, McCormick AJ. 2019. Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit. JoVE (Journal of Visualized Experiments).(152):e60451. doi:10.3791/60451.
2. Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepes J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, et al. 2014. Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res. 42(17):e136. doi:10.1093/nar/gku673.