Team:Lund/Results/Fermentor
Results
Results and Discussion
The cell pellets with belonging supernatant exposed with UV light from cultivation 1 is seen in fig 1. The distinct three layers are from the bottom: intact cells containing GFP and VHB, rupted cells and therefore a lower density (not being centrifuged down as fast) and supernatant containing GFP.
The bulk supernatant from cultivation 1 is shown in fig 2. It is clear that it contained GFP. A connection with the middle layer in Figure 1 was reasonable to make. If the bulk supernatant also contained GFP it was logical to think that the handling when resuspending the pellet did not cause that many cells to rupture since the GFP was already out of the cells before that.
fig. 3 is the same photo as in fig. 1 but for second cultivation. It was clearly seen that the ruptured cell amount was less. This might been because of the lower stress affecting the cells compared to the two protein-system with VHb-GFP.
The bulk supernatant of the second fermentation is shown in fig. 4. A not as prominent fluorescent light was seen as in the VHb-GFP, see fig. 2, indicating a probably lower number of ruptured cells.
In fig. 5 the SDS-PAGE gel is seen. Marked within the red rectangle are the GFP bands (between 25 and 30 kDa it is reasonable since GFP has a protein mass of 26.9 kDa) and the arrows indicate the bands of VHb.
According to the software analysis comparing the second and third GFP bands the VHb-GFP (lane 2) had an average intensity of 284 and GFP (lane 3) 370. This was calculated with the same amount of noise canceling for all lanes.
The pellet that was lysed for VHb-GFP was only the intact cell pellet. Meaning that a lot of GFP was not measured that ended up in the supernatant. From fig, 1 it was possible to see that there was approximately 4 mL intact cell pellet and 3 mL dead pellet. If the concentration was higher in the dead cells a higher average intensity could have been measured. This, combined with the risk of not lysing all cells, is a definite source of error.
How the fermentations progressed over time is seen in fig. 6. The cell concentrations (mg wet pellet/ml sample) increased for both cultivations until hour 10-15 where they flattened. Regarding the dissolved oxygen (DO) it decreased, as expected, with cell growth but never below approximately 7%. A constant stirrer speed was set.
The GFP concentration from the different fermentations using spectrophotometry.With the supernatant volumes of sonicated cells being 8.15 g/L(VHb-GFP) and 15.2 g/L (GFP) a cell productivity of 74.2 and 25.1 mg GFP/cell was calculated respectively.
According to the stated hypothesis, the productivity should be higher for the VHb-GFP- system. But this was not the case. If considering the results of GFP concentration for different headspace (75% and 20 %) the results are however not very surprising. BBa_K2602020, the composite part for VHb-GFP used in the up-scale fermentation showed less good results when it comes to the lower oxygen concentrations. The best one with 20 % headspace was in fact 2025. The production, and hence the sought effects on GFP production was not high enough. This is also indicated in fig. 5, where the faded bands of VHb, indicated with arrows, are quite faded.
Conclusion
According to the productivity of GFP per g of cells our hypothesis was not met. Mainly the sources of error could be that not all cells were lysed. If the dead cells contained more GFP, there was a problem in that the concentration of the supernatant was not taken into account. The system containing VHb-GFP was according to experiments, performed later than the first fermentation where this system was used, showed that the production of VHb was not as expected and thusly not giving the desired, or expected effects.
However, the possible stress on the cells may have caused a lower protein producing cell density for the system expressing both VHb and GFP. Also noted was the higher amounts of ruptured cells.
