Results
Introduction
In this page the main findings and interpretations of the results of our project can be found. In our project we aimed to achieve four different goals. These four proof of concepts are summarized in Figure 1.
Ammonia as the only nitrogen source
As we plan on feeding our GMO with ammonia extracted from the MOF, an essential part of our project is that our GMO should be able to grow in the presence of ammonia as its only nitrogen source. We therefore grew 6 different Saccharomyces strains under different ammonia concentrations to see if the presence of ammonia would affect their growth. In the end we were able to identify the best performing strains. The protocol can be found in the Experiments page.
As can be seen from Figure 2 there are 4 strains that outperformed in the experiment, those strains were chosen for further experimentation. The doubling time of the chosen strains can be seen in Table 1.
Strain | TD (min) | ||
---|---|---|---|
0.05 g/L | 5 g/L | 7.5 g/L | |
ySB76 | 51,71 | 70,71 | 69,3 |
ySB77 | 130,75 | 90 | 60,78 |
ySB78 | 101,91 | 94,93 | 88,85 |
ySB85 | 103,43 | 91,18 | 108,28 |
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.
Engineering success
For our second proof of concept, we wanted to demonstrate that it is possible to use Golden Gate Assembly to clone heterologous genes for alpha-amylase in Saccharomyces spp. Going from the gene of interest to the cassette plasmid expressed in Saccharomyces spp. is a long process, through it there were some checkpoints that we used to confirm the success of the cloning: GFP screening; DNA concentration measurements with a Nanodrop; basic parts sequencing; and digestion of the final cassette plasmids.
GFP screening
The empty plasmid (pRS426__ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan) used in the Golden Gate cassette plasmid assemblies contains a GFP drop-out. This way, when the assembled plasmid is inserted correctly, the GFP insert will drop-out and white colonies will be formed. Whenever the plasmid was not inserted correctly the GFP drop-out will still be present and colonies will appear green when examined with UV-light (Figure 3). By this simple technique we make sure that the chosen colonies have uptaken the assembled plasmid without the need of performing sequencing or digestion experiments prior to plasmid isolation. This GFP screening was used both for assembling our part plasmids and cassette plasmids.
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.
DNA concentrations
After purification of cassette plasmids amplified in E. coli, DNA concentrations were measured in order to assure a successful purification prior to Saccharomyces spp. transformations, results can be found in Table 2. These measurements were performed using a nanodrop spectrophotometer, measuring the DNA concentration at a spectrum of wavelengths.
Sample number | ng/ul | A260/280 ratio | Sample number | ng/ul | A260/280 ratio |
---|---|---|---|---|---|
SP001 | 22,6 | 1,823 | SP034 | 80 | 0,001 |
SP002 | 15,6 | 1,803 | SP035 | 35,5 | 1,529 |
SP003 | 29 | 1,598 | SP036 | 15 | 1,523 |
SP004 | 19,7 | 1,662 | SP037 | 29,4 | 1,699 |
SP005 | 54,3 | 2,027 | SP038 | 48,5 | 1,960 |
SP006 | 53,5 | 1,508 | SP039 | 14,6 | 2,021 |
SP008 | 69,5 | 1,853 | SP040 | 18,3 | 1,649 |
SP009 | 16,1 | 2,414 | SP041 | 25,1 | 1,668 |
SP010 | 12,1 | 1,898 | SP042 | 45,1 | 1,488 |
SP011 | 13,3 | 1,430 | SP043 | 15 | 2,150 |
SP012 | 58,9 | 1,812 | SP044 | 29,9 | 1,472 |
SP013 | 19,5 | 2,058 | SP045 | 78,4 | 1,708 |
SP014 | 29,1 | 1,473 | SP046 | 33 | 1,451 |
SP015 | 28,3 | 1,461 | SP047 | 62,3 | 1,489 |
SP016 | 52,3 | 1,458 | SP048 | 97,6 | 0,002 |
SP017 | 72,1 | 1,553 | SP049 | 13,4 | 1,949 |
SP018 | 34,7 | 1,627 | SP050 | 119 | 0,002 |
SP019 | 79,8 | 1,788 | SP051 | 21,2 | 1,612 |
SP020 | 15,7 | 1,627 | SP052 | 56,5 | 1,471 |
SP021 | 17,9 | 1,729 | SP053 | 6,3 | 2,117 |
SP022 | 71 | 1,514 | SP054 | 39,2 | 1,798 |
SP023 | 13,2 | 1,688 | SP055 | 25,7 | 1,490 |
SP024 | 10,6 | 2,078 | SP056 | 50,7 | 2,069 |
SP025 | 82,7 | 0,002 | SP057 | 73,3 | 1,806 |
SP026 | 9,5 | 1,602 | SP058 | 51,7 | 1,987 |
SP027 | 47,2 | 1,512 | SP059 | 30,6 | 1,363 |
SP028 | 64,2 | 1,544 | SP060 | 61,6 | 1,936 |
SP029 | 10,5 | 2,297 | SP061 | 62,4 | 1,848 |
SP030 | 16,6 | 1,892 | SP062 | 82,4 | 1,741 |
SP031 | 51,3 | 1,507 | SP063 | 66 | 1,547 |
SP032 | 45 | 2,067 | SP064 | 58,8 | 1,718 |
SP033 | 48,7 | 1,617 |
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.
DNA sequencing
To ensure that our ordered G-blocks for part 3 and part 3B we made were correct, we sequenced the basic parts (4 full alpha amylases and 4 alpha amylases without the native secretion peptide) assembled in the entry vector (more on this on the Engineering page). pYTK001 (entry vector) with Part 3 and 3b was sequenced using two primers flanking the gene, the vector is used to compose the cassettes plasmids in further experiments. All sequencing results are summarized in Table 3.
Part | Alpha amylase Gene | Mutations | Comments |
---|---|---|---|
Part 3 | From A. oryzae | - | Good quality sequence, 100% gene coverage. |
From B. amyloliquefaciens | c.301C>Tp.T101I | The amino acid is not in the substrate binding site of the protein. 100 % gene coverage. | |
From B. licheniformis | c.690G>Tp.D231Y | Mutations in the flanking aspartate have shown a decrease in thermostability [1]. No data about our mutation. 100% gene coverage. | |
From B. subtilis | - | Good quality sequence, 100% gene coverage. | |
Part 3b | From A. oryzae | - | Good forward sequencing, 63% gene coverage. |
From B. amyloliquefaciens | - | Good forward sequencing, 52% | |
From B. licheniformis | - | Good forward sequencing, 66% gene coverage. | |
From B. subtilis | - | Good forward sequencing, 50% gene coverage. |
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.
Constructs digestion
We used the restriction enzyme BsmBI to check for the correct ligation of the different constructs of the library. There are two sites for the enzyme BsmBI in the vector employed (pRS246) for our constructs, the sites are in part 1 and part 5, on both sides of the insert of interest (Figure 4). The use of BsmBI allows us to check for the size (Table 4) of the construct and assess the success of the assembly. The results of the digestion can be found in Figure 5.
Sample | Expected sizes A (bp) | Expected sizes B (bp) | Sample | Expected sizes A (bp) | Expected sizes B (bp) |
---|---|---|---|---|---|
SP001 | 3729 | 3387 | SP034 | 3729 | 2983 |
SP002 | 3729 | 2869 | SP035 | 3729 | 2970 |
SP005 | 3729 | 3387 | SP037 | 3729 | 3387 |
SP006 | 3729 | 2953 | SP038 | 3729 | 3387 |
SP008 | 3729 | 3237 | SP039 | 3729 | 2982 |
SP009 | 3729 | 3237 | SP040 | 3729 | 2970 |
SP010 | 3729 | 3387 | SP041 | 3729 | 2802 |
SP011 | 3729 | 2776 | SP042 | 3729 | 2982 |
SP012 | 3729 | 2755 | SP043 | 3729 | 2953 |
SP013 | 3729 | 3381 | SP044 | 3729 | 2886 |
SP014 | 3729 | 2982 | SP045 | 3729 | 2982 |
SP015 | 3729 | 2869 | SP046 | 3729 | 2866 |
SP016 | 3729 | 2790 | SP047 | 3729 | 2755 |
SP017 | 3729 | 2869 | SP048 | 3729 | 2970 |
SP018 | 3729 | 2880 | SP049 | 3729 | 2749 |
SP019 | 3729 | 2796 | SP050 | 3729 | 2970 |
SP020 | 3729 | 2886 | SP051 | 3729 | 2970 |
SP021 | 3729 | 3387 | SP052 | 3729 | 2982 |
SP022 | 3729 | 2982 | SP053 | 3729 | 2755 |
SP023 | 3729 | 2970 | SP054 | 3729 | 3291 |
SP024 | 3729 | 2886 | SP055 | 3729 | 2982 |
SP025 | 3729 | 2802 | SP056 | 3729 | 2796 |
SP026 | 3729 | 2982 | SP057 | 3729 | 2970 |
SP027 | 3729 | 3237 | SP058 | 3729 | 2755 |
SP028 | 3729 | 2982 | SP059 | 3729 | 2886 |
SP029 | 3729 | 2982 | SP060 | 3729 | 3237 |
SP030 | 3729 | 2866 | SP061 | 3729 | 2970 |
SP031 | 3729 | 2886 | SP062 | 3729 | 2976 |
SP032 | 3729 | 2970 | SP063 | 3729 | 2982 |
SP033 | 3729 | 3387 | SP064 | 3729 | 2869 |
pRS426-GFP | 3729 | 1338 |
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.
Alpha amylase production
For our third proof of concept, we wanted to show that our GMO does indeed show alpha-amylase activity after taking up our construct. We aimed to measure the Alpha amylase production of 64 samples listed at our Engineering page. This was tested with two main experiments. The principal one being an alpha amylase kit assay used to perform high throughput experiments and obtain quantitative data, needed for our model (see Model page). Additionally, a qualitative assay was employed to confirm the production of alpha amylase in some of the samples. To understand more how this assay works, see the Experiments page.
Alpha-amylase starch breakdown
Functionality of alpha amylase can qualitatively be proved by observing the breaking down of starch. The ability to break down starch is only available when amylase is produced, which in the case of S. cerevisiae and S. paradoxus doesn’t happen natively (Figure 6e). However, the assembled constructs should be able to break down starch. In order to confirm this, selective media plates without uracil (specific for the assembled constructs) were created with the addition of 1% starch. Several samples were cultured ON and treated before being plated on the starch plates, as described in the notebook (wk 39), after which pictures were taken (Figure 6) of the with/without iodine treated starch plates. Samples tested with this assay can be found in Table 5.
Sample | Yeast Strain | Promoter | Gene | Terminator |
---|---|---|---|---|
SP001 | ySB77 | pPGK1 | αMF + BS | tTDH1 |
SP010 | ySB77 | pHHF1 | αMF + BS | |
SP021 | ySB77 | pPGK1 | αMFΔ + BA | |
SP026 | ySB76 | pHHF1 | αMF + BL | |
SP027 | ySB76 | pRNR1 | BS | |
SP034 | ySB76 | pTEF1 | αMF + BL | |
SP035 | ySB76 | pPAB1 | αMF_no_EAEA + BL | |
SP051 | ySB76 | pRNR1 | αMF_no_EAEA + BL | |
SP063 | ySB76 | pRNR2 | αMF + BL |