Isolation of a strong constitutive promoter from Euglena gracilis
thesisposted on 2022-03-28, 11:36 authored by Bishal Khatiwada
Euglena gracilis is a unicellular protist currently used to produce nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organisms requires the development of genetic engineering tools. Chloroplast transformation using the non-native promoter (CaMV 35S) has been reported, but there is no published information about the use of native promoters for recombinant gene expression in Euglena. The aim of this research was to isolate a strong constitutive promoter with a view to establish an expression vector for E. gracilis. Proteins produced during dark cultivation were separated using 2D SDS-PAGE and identified by mass spectrometry. One of the highly expressed proteins was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The gapC gene encoding GAPDH was identified using BLAST. The 5’ UTR region of the gene was amplified by PCR using gDNA of E. gracilis as a template and inserted into a promoterless E. coli and yeast plasmids containing a reporter gene (β-gal or eGFP). Promoter activity was verified by successful expression of EGFP in yeast. Sequence analysis of the putative Euglena promoter revealed the presence of eukaryotic promoter elements like CAAT box, GC Box and myb-like transcriptional activator. Final confirmation of the promoter activity will be carried out in Euglena after a suitable transformation method has been established.