Question: Put the figures in the right order and analyse result of each figure
Figure in picture 1. Membrane transport characteristics of Xenopus laevis oocytes transformed with clone#3992. mRNA encoding orf3992 was transcribed in vitro from plasmid pEP(clone#3992) and transformed into Xenopus laevis oocytes. (A) Sugar transport into oocytes in the presence and absence of 10 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Extracellular medium supplemented with 1 mM sugar was left with oocytes for 1 hour and then intracellular concentration of sugar was measured. (B) Measurement of beemolulose transport kinetics into oocytes in the presence of different sugars.
Figure In picture 2. Modifications to orf3992 within A. candicus. (A) Schematic showing CRISPR/Cas9 with guide RNA (gRNA) that targets orf3992 co-transformed into A. candicus. (B) Effect of CRISPR/Cas9 disruption of orf3992 gene on A. candicus growth characteristics. Sugars were added to minimal growth medium and A. candicus was grown for 12 hours in minimal medium plus sugar. (C) Schematic showing transformation of expression plasmid encoding orf3992 into A. candicus. (D) Effect of orf3992 overexpression on A. candicus growth in minimal medium plus sugars.
Figure in picture 3. Screening Austral candicus cDNA library. (A) cDNA library preparation diagram. mRNA was extracted from A. candicus, converted to cDNA via reverse-transcription, followed by cloning into an expression plasmid with promoter constitutively active within S. cerevisiae. Following transformation of plasmid library into S. cerevisiae, 109 clones were plated onto minimal medium with all amino acids added lacking any sugar but beemolulose. Resulting colonies were picked and grown in 96-well liquid format with the same medium as used for plating colonies. Clone #3992 was one of 20 colonies that showed growth in the 96-well plate, had the most robust growth, and was chosen for further study. (B) Growth of yeast strains on different sugars. Growth was in liquid minimal medium supplemented with sugar(s) indicated. In the right panel, equimolar combinations of beemolulose plus indicated sugar were supplemented into minimal growth medium.
Figure in picture 4. Identification and cellular localisation of ORF3992 protein. (A) Sequencing of clone #3992 reveals an open reading frame (ORF3992) of 700 amino acids long with hydropathy plotting potentially showing transmembrane domains. (B) Adding FLAG-tag to C-terminus of ORF3992 followed by transformation into yeast strain W303. (C) SDS-PAGE analysis of cytoplasm, membrane, and nucleus fractions from (-) yeast cells alone and (+) yeast cells carrying pEP(clone #3992). (D) Western blot of cytoplasm, membrane, and nucleus fractions from (-) yeast cells alone and (+) yeast cells carrying pEP(clone #3992). Probed with a rabbit anti-FLAG tag primary antibody, followed by anti-rabbit secondary antibody, and visualised with enhanced chemiluminescence (ECL) reagent.
