We learned that two of the strains that we were planning to use didn’t perform well when ammonia was the only source of nitrogen. This experiment helped us to exclude them for further tests as they weren’t suitable for our purpose (see Description page). Four strains proved to grow in different concentrations of ammonia, with strain ySB76 (S. cerevisiae) being the fastest grower overall. We could not observe a clear tendency that would suggest the optimal ammonia concentration for growing the cells. This result is relevant for our project, since the concentration of this nutrient doesn't necessarily need to be kept constant, which allows for more flexibility in ammonia concentration of the cell culture and thus more flexibility in how much ammonia the MOF should be able to capture. As we wanted to test different chassis in the optimization process, after this experiment we decided to express alpha amylase in yYS76, yYB77, ySB78 and ySB85, which correspond with 3 S. cerevisiae strains and 1 S. paradoxus strain.

GFP screening smoothened the process, considering the amount of samples that we needed to assemble. Generally, all the transformed E.coli with cassette plasmids (64 plates) showed a high efficiency of the Golden Gate assembly; a few countable green colonies were observed in contrast to hundreds of white colonies. One sample (SP007) didn’t show any white colonies, the sample was excluded for future experiments.

The A260/280 ratio resembles the purity of the DNA, an ideal sample would have a value of approximately 1.8. Due to large variation in DNA concentration and A260/280 ratio, 20ul of each cassette plasmid was used in the Saccharomyces spp. transformation instead of 5ul. Overall the concentration of DNA in the samples after transformation and plasmid isolation were good and ensured the continuation with Saccharomyces spp. transformations.

Generally the results were quite successful and no major mutations were observed. However, two clear missense mutations were identified in alpha amylases from B. amyloliquefaciens and B. licheniformis that may have an effect on the protein functionality. It would have been nice if we had the time to restart the cloning from the beginning, unfortunately, the cassette plasmids were already assembled and the time was very limited. In Part 3b full coverage of the gene couldn’t be achieved, repeating the sequencing was planned but sadly we didn’t have the time.

All in all, the mutations found will be taken into account for further results of those enzymes.

After the digestion of all the constructs it can be seen that the expected sizes of the engineered plasmids match the sizes of the bands shown in the gel. The efficiency of the Golden Gate assembly is high. There are two samples in which a band of the size of the GFP-drop out is observed, SP012 and SP058, besides the expected size. This result may be due to contamination while pipetting. Nevertheless the expected band can be observed, the samples were used in follow up experiments, consideration on alpha amylase activity will be taken into account. Additionally, some samples (SP006, SP011, SP022, SP023, SP044, SP046, SP047, SP052, SP053, SP055) show faint bands, likely evidencing that the concentration of DNA was low in the first place. The faint bands have the expected size, and considering the high efficiency in the rest of the digests we conclude that our Golden Gate assemblies and further screening was highly successful. No samples were excluded after digestion.

As visible in Figure 6, a larger halo was observed in the cells’ lysate samples. Halos were also visible in most of the supernatant samples.This implies that alpha-amylase is present extra- and intracellular after cell lysis. We were able to confirm that this activity was only produced in the strains that had taken up the created constructs, as the non engineered strains didn’t show any halo at all. Overall we see a difference in the alpha amylase activity when samples were treated differently, suggesting that the secretion of the enzyme varies between constructs. More specifically to our project, we planned to isolate the protein from the supernatant, this way the processing of the cells is minimal and lysating is not directly necessary. Therefore it was interesting to find that lysating some of the samples did produce a bigger halo, since this could change our proposed way of extracting alpha-amylase. Only few samples of the library were tested through this essay, therefore general conclusions can not be drawn, but confirmation of alpha amylase activity could be assessed.

Alpha-amylase assay kit

We used a colorimetric assay to obtain quantitative data of the activity of the alpha amylase produced by the engineered yeast, allowing for comparison between samples and for quantitative data to use in our model.

The assay is based on the capacity of alpha amylase to cleavage ethylidene-pNP-G7, resulting in p-nitrophenol, a product that can be read at a wavelength of 405 nm, more details on the Experiments page. A testing round was first performed (Figure 7) in duplicates per sample to check how the experiment was done and identify possible steps that may need to be optimized. This round also helped to exclude samples SP003, SP004 and SP036 for further analysis as we were tight on reagents and the results weren’t promising. 

During the high throughput experiments, we tested the capacity to produce functional alpha amylase of all the samples which were not excluded from the subset of the combinatorial library (the excluded samples are listed on the Model page, section Accounting for lost samples). Activity of alpha amylase in triplicates of each sample can be found in Figure 8. Supernatants from overnight cultures were incubated with ethylidene-pNP-G7 in 96 well plates and the appearance of p-nitrophenol was measured every 5 min during the first hour, then once after 2h and 3h. If alpha amylase was in the supernatant an increase of absorbance was expected due to the appearance of p-nitrophenol, an indirect measurement of alpha amylase activity. Activity (y-axis of Figure 8) was therefore measured as the increase of absorbance (quantity of p-nitrophenol) that appears per time unit (d[p-nitrophenol]/dt).

After having excluded the samples in which the variance between replicates deviated too much from the average variance, a table summarizing the mean activity of each sample can be found below. The different combinations from the subset library are also displayed in Table 6.

Alpha amylase activity of the construct from the subset of combinations. The different components from each sample are also displayed.