Our chosen method of inserting genes into chlamy was to use electroporation to insert linearized DNA into chlamy (see our Protocols for more details). Once the DNA was inserted the chlamy could then be placed on plates containing spectinomycin. This media could then kill off any cells that were not transformed with at least part of our DNA
Our plasmids were designed so that all the genes we wanted to insert were bookended by a gene for spectinomycin resistance at one end (Aada) and Psr1-nanoLuc at the other end. This means that any cells growing on spec plates after electroporation have at least part of the plasmid they were electroporated with. The presence of the rest of the plasmid in these spec resistant chlamy colonies could then be verified with a simple luciferase assay.
This was first done with a wild-type strain of chlamy (CC-1690, row A) and a strain mutant for the Amt4 gene (CC-4039, row C) electroporated with a small plasmid containing only Aada and Psr1-nanoLuc.
From this experiment six of the most expressive strains were kept, three from CC-1690 and 3 from CC-4039.
This experiment was repeated once more after chlamy, transformed with De-Slimer, had formed colonies. Because the De-Slimer plasmid is much larger than the one used for the previous electroporation we sampled nearly 200 colonies. Very few of these colonies showed luciferase expression, and the lumens read by the spec ranged from 13-112 for those expressing luciferase. For future testing, we grew any of the cell lines that resulted in over 40 lumens being read. This resulted in six cell lines being preserved, all of which came from CC-4039, and one of which had a variant of De-slimer that used a nosZ gene derived from D. denitrificans. The other five had nosZ derived from P. stutzeri, which are the cell lines used in future experiments.
To see if nosZ-mCherry was being expressed by our transformed chlamy, we checked for fluorscence, but unfortunately, did not detect any. We also attempted to detect mCherry with a Western blot, but were also unsuccessful. We are continuing additional studies to determine if nosZ-mCherry is expressed in any of our colonies.
We also performed RT-qPCR for Ptc expression to see if our amiRNA was able to reduce levels of the transcript. We found that two of our colonies transformed with De-Slimer (Ddenit 3 and Pstu 2) did appear to have lower levels of Ptc expression than untransformed chlamy of the same strain (CC-4039).
Our first phosphate assay was performed to test if there was a difference in phosphate uptake between untransformed chlamy CC-4039, and chlamy transformed to express Psr1 continually. We performed the experiment using TAP media, which has a relatively high amount of phosphate, and uses ammonium for nitrogen content.
In our first experiment, we observed a max uptake rate of 11.264 mg P/L in our transformed strain. Another group (Slocombe et al. 2023) previously published the results of a similar experiment. Their results showed a maximum phosphorus uptake rate of 21 mg/L in their transformed strain. Compared to the rate seen by Slocombe et al, our transformed chlamy had about half the maximum rate of uptake that theirs did, and we are actively working to try to figure out why this happened.
This is a t-test of the media phosphorus concentration values per day in triplicate. The highlighted portion shows a statistical difference on the third day with a p-value less than 0.05 between our transformed vs untransformed strain. By day 4, the large variation in phosphate concentrations among our transformed samples made the difference non-significant, and we also observed an increase in phosphate concentrations in the media after this point.
In our second phosphate assay our goal was to observe any difference in phosphate uptake between two untransformed strains of chlamy CC-4039, and CC-1690, and strains of CC-4039 transformed with PSR1 and De-Slimer (Pstu5).
Because these chlamy were also transformed with De-Slimer, we also wanted to examine whether nitrate uptake was changed in the transformed strains. TAP media does not have nitrate in it, so the media used was a mixture of TAP and Allen media. Compared to TAP, Allen media has a lower amount of phosphate in it, and uses nitrate for nitrogen content.
In this experiment, we saw that chlamy transformed with Psr1-nanoLuc gene had a max uptake of 4.3 mg/L Phosphorus. This was not significantly different from the rate of the untransformed strain of chamy which was 4.001mg/L.
Despite not seeing a difference in phosphate uptake in the time frame of the experiment, what we hope to happen in our transformed strain compared to the untransformed strain is the modified strain will continuously uptake phosphorus rather than "turn off" the Psr1 gene responsible for uptake once the cell's needs are met.
Notably, we found that chlamy transformed with the De-Slimer (Pstu 5) grew more slowly compared to ones transformed with Psr1-nanoLuc alone, and we are currently working to optimize growth conditions. This slower rate of growth could help explain why our deSlimer transformed strain had the smallest max rate of phosphorus intake.
t-test's between the various media phosphorus concentration values per day in triplicate were done between each strain. The highlighted portion shows a statistical difference between our two transformed strains, Psr1 and De-Slimer (Pstu 5).
Our second phosphate assay was performed in tandem with our first nitrate assay. The primary focus of this experiment was to see if chlamy, transformed with De-Slimer, would uptake nitrate at a faster rate than chlamy that did not contain the nosZ gene.
Unfortunately, we did not observe any reduction of nitrate levels in our media by the unmodified control, or Chlamy engineered with De-Slimer. There could be several potential explanations for this: First, this could be due to the inherent difficulty in detecting increased nitrate uptake, as “chlamy” already uses nitrates as a nutrient. Second, the presence of nosZ may not influence the uptake of nitrates. Measuring changes in nitrogen gas levels is extremely challenging, given the high background levels of nitrogen in the atmosphere. Small amounts of nitrogen production by the engineered “chlamy” may be difficult to detect. Future experiments will need to focus on nitrous oxide measurements in the water to determine the efficacy of nosZ, which is beyond the current capabilities of our lab. We are currently trying to detect expression of nosZ through a combination of fluorescence microscopy and Western blots for the mCherry tag.
The goal of our final assay was to try and improve some aspects of our previous experiments. Initially chlamy was cultured in 15 ml conical tubes, while this was a convenient way to house chlamy it also resulted in large pellets forming in the bottom of the tubes. To fix this problem chlamy was cultured in Erlenmeyer flasks, which prevented pellet formation and allowed for more light to reach the cells. In addition, the media was also changed to 5000 px Allen media so that nitrate would be the sole source of nitrogen available. We also changed the strain of chlamy transformed with the De-Slimer to Pstu 4, as it had higher levels of luciferase activity than Pstu 5 did.
For this experiment, we used Allen media which doesn't have very much phosphorus in it. Due to this the transformed and the untransformed cells performed about the same with a phosphorus concentration uptake of about 1.1-1.3 mg/L
Again, we calculated T-Tests for each of the different strains done in triplicate which didn’t show significant differences across the entire experiment.
Despite using Allen media so that nitrate was the only source of nitrogen, we did not see any significant change in media nitrate concentration throughout the experiment.
One trend we noticed was that the chlamy transformed with Psr1 only outperformed untransformed chlamy in TAP media, which had the highest phosphate concentrations of any of the media we used for the assays, as shown in this bar graph. We speculate that at lower levels of phosphate, untransformed chlamy may turn on expression of its own Psr1 gene, causing it to uptake as much phosphate as our transformed chlamy.
