Fig 1.
Plasmid maps of the cloned constructs generated using the HiFi DNA Assembly Kit.
A) The Hcotba-1 cDNAs, either with an intact ORF or a scrambled ORF, were inserted into the pLEXSY_hygR vector backbone. B) Similarly, the Hcotbb-1 cDNAs, with an intact ORF or a scrambled ORF, were inserted into the pLEXSY_satR vector backbone. These plasmids do not contain functional promoters for L. tarentolae.
Fig 2.
Chromosomal 18S rRNA locus (ssu(18S)) after the integration of the Hcotba-1 and Hcotbb-1 genes into the expression site.
Transcription of the Hcotba-1 and Hcotbb-1 genes is driven by the strong RNA polymerase I, regulated by a chromosomal ribosomal promotor (Pr) [28]. The 5’-ssu(18S) and 3’-ssu(18S) are homology arms for homologous recombination. The selection markers satR and hygR confer resistance to nourseothricin and hygromycin B. The arrows indicate the positions of the forward and reverse primers used in the conventional PCRs.
Fig 3.
Selection curves of transgenic LEXSY P10 after Amaxa electroporation in BHI+ medium.
Each group consisted of three replicates. The intact ORF group was able to express HcoTBA-1 and HcoTBB-1, the scrambled ORF group was unable to express HcoTBA-1 and HcoTBB-1, and the control group was a mock transfection without DNA.