Gels were prepared with the agarose (Lonza, Seakem®) content of 1.5% and mixed in solution with TAE buffer (in house). The standard gel used was 100ml of TAE buffer with 1.5g of agarose which was heated till a clear solution was seen. Once the mixture was cool to touch (5-10 minutes), 10µl of 10,000 x at 1mg/ml ethidium bromide (in house) was added before the mixture was poured into the insert with a 16 toothed comb. The gel was then left to set until a cloudy colour was seen. Gel and insert were placed into the tank (Bio Rad) with the comb removed and 600ml of TAE buffer was added. Finally, after the 5µl samples had been added to each well and 5µl of 100bp ladder (NEB) was added on the first well, the gel was run for 20 minutes at 100V using a power pack (Bio Rad).
The gels were imaged using Bio Rad Gel Cloc machine. Restriction Enzymes (RE) Test The check the PCR product size was correct; each gene was cut once with an applicable restriction enzyme. Once the genes had been amplified through standard PCR, 10µl of amplified cDNA was added to the RE mixture. For a final volume of 50µl RE mixture: 10µl of PCR product, 1µl of restriction enzyme (NEB); 0.5µl of BSA, if applicable or water (NEB); 5µl of NEB Buffer (NEB), 33.5µl of distilled water. The specific enzymes and buffer used can be seen in table 1. The RE mixture then went on a heat block at 37OC for one hour. This was followed by standard gel electrophoresis.
The gene size for the cbhA (without introns) is 475bp and the bands present are between the 400bp and 500/575bp marks on the ladder. The expression of cbhA is induced at 9 hours. Between 9 and 24 hours, cbhA expression is at a consistent level but after glucose is added to the media, the level of expression has decreased (figure 1A). The gene size for the cbhB (without introns) is 208bp and the bands present are close to the 200bp mark on the ladder. The expression of cbhB is weakly induced at 6 hours. From 9 hours until the end of the time course, cbhB expression is maintained at a constant level, even after glucose is added back into the solution (Figure 1B).
In figure 2, the expression of eglA is induced at 9 hours and maintained up to 24 hours. After glucose has been added, the expression of eglA appears to have decreased. eglA gene expression seems to correlate with the expression of cbhA. The gene size for the EGLA is 201bp and the bands present are close to the 200bp mark on the ladder (Figure 1A). Expression of yefC (figure 1C) and glaA (figure 3) are mostly the same. Expression of yefC is not affected if A.niger is undergoing starvation and has a carbon source available for breakdown as it is a known house-keeping gene so its expression will be maintained regardless, of the nutritional status of the fungus. The gene size for the yefC is 717bp and the bands present are close to the 700bp mark (figure 1C). glaA’s gene size is 241bp (without introns) and all bands are between the 200bp and 300bp (figure 3).
When A.niger has wheat straw as carbon source, it is able to hydrolyse the polysaccharides in the plant cell walls to produced glucose as a food source. However, if there is no carbon source, the induction of may be genes may to be different. When comparing figures 1A and 4A, the expression of cbhA is significantly lower when A.niger does not have a carbon source. A basal level of expression of cbhA is seen throughout the time-course as cbhA expression is not induced through starvation and is under the control of XInR.
In figure 4B, partial induction of cbhB is seen at 3 hours and the gene is fully induced at 6 hours. From 6 hours till the end of the time-course, its expression level does not change until 24 hours where expression has decreased. Figure 4C shows that expression of yefC is at a consistent level throughout the time-course. This is the same for glaA (figure 6). In figure 5, induction of eglA is seen at 6 hours and is maintained until the 12 hour time point but, expression levels at 24 hours have dropped.
All the band sizes in all figures correspond to the correct size of each gene. The restriction digest of eglA and glaA were unsuccessful. In figure 10, there is one band present at around 250bp which close normal gene size of eglA (299bp) with introns. In figure 11, there is only one band present just above the 200bp marker which represents the gene size of glaA (201bp). Figure 7 shows that cbhA was successfully cut by MfeI into its two fragments as two bands are present representing the 145bp and 330 fragment. In figure 8, two light bands representing the 50bp and 158 fragments from the MfeI digestions of the cbhB. Partial digestion of yefC is seen in figure 9. There are three bands present representing the uncut yefC at 717bp and the two cut fragments of yefC, 565bp and 152bp.