iGEM 2024 — Team uvu-utah-2

![Attribution Image](https://static.igem.wiki/teams/5078/attributions/attribuitions-tube.webp)

# **Lab Notebook** ![Attribution Image](https://static.igem.wiki/teams/5078/attributions/attribuitions-tube.webp)

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## Our Engineering Cycle

## Example 1

### Design

Last year we utilized a green fluorescent tag to check for successful insertion of our plasmid, this was unhelpful due to the green color of
C. reinhardtii so we replaced it with an mCherry tag so that the fluorescence was red, which would be easier to distinguish from auto-fluorescence.

### Build

We inserted the mCherry tag into 4 different strains of C. reinhardtii through electroporation. The mCherry tag was inserted at the C-terminus of our nosZ gene so that if it was expressed it would show expression nitrous oxide reductase.

### Test

We tested for mCherry expression by Western blot and immunofluorescence. Initial test are inconclusive and will be repeated with new controls.

### Learn

A major problem faced while doing the testing is the ability for
C. reinhardtii to auto-fluoresce which makes it difficult for us to distinguish from expression of mCherry or just typical cell function. Next year we will utilize a different tag to test for successful expression of our plasmid.

## Example 2

### Design

Psr1 is a transcription factor that initiates transcription and expression of phosphate intake channel protein. Normally, this gene is only expressed by C. reinhardtii under conditions of phosphate starvation. We theorized that by adding a copy of the gene under the Psad promoter, it will always express regardless of the level of phosphate in the cell, increasing phosphate uptake from the environment.

### Build

Built the pL1 Psr1-nanoLuc plasmid using the C. reinhardtii MoClo cloning kit and Psr1 genes from the Alison Baker Lab using golden gate cloning.

### Test

Confirmed proper construction of PL1 PSR NanoLuc using a restriction enzyme digest on a 1% agarose gel, and verified through sequencing.

![](https://static.igem.wiki/teams/5078/engineering/diagnostic-digest-psr1.webp)

### Learn

Plasmid construction occurred correctly in E. Coli and can be used in future builds to test phosphorus uptake and luciferase expression.

## Example 3

### Design

Knowing that pL1 Psr1-nanoLuc was properly constructed, a Level 2 plasmid was constructed using aadA to combine spectinomycin resistance in order to test incorporation and expression in C. reinhardtii.

### Build

We completed the L2 plasmid using golden gate cloning methods.

### Test

Proper plasmid construction was confirmed using restriction digest on 1% agarose gel and then further confirmed via sequencing.
Properly constructed pL2 aadA Psr1-nanoLuc was used to transform
C. reinhardtii using electroporation. C. reinhardtii was then allowed to grow before testing. We performed a phosphate uptake assay, comparing the rate of uptake of our transformed chlamy compared to untransformed control.

### Design

We designed a plasmid to express nosZ, Ptc amiRNA and Psr1 simultaneoiuisely.

### Build

We assembled the plasmid and confirmed that it was assembled correctly using restriction digest.

### Test

We electroporated the plasmid into chlamy and screened nearly 200 colonies in order to find several that had luciferase activity. However, we found that chlamy transformed with this multi-gene plasmid grew more slowly than control chlamy, and had phosphate uptake levels similar to that of untransformed chlamy.

### Learn

There may be limits to how much exogenous DNA chlamy can incorporate, and still express efficiently.

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# **Plasmid Design & Construction:**

## Nitrogen

![NosZ D. denit L0](https://static.igem.wiki/teams/5078/plasmid-pictures/nosz-d-denit-l0-plasmid-picture.webp)

**NosZ D. denitrificans
Level 0 plasmid**

In our Level 0 plasmid builds, two different bacteria were used as a basis for two versions of nosZ:
nosZ from *[Pseudomonas stutzeri](https://parts.igem.org/Part%3ABBa_K5078000)* and
nosZ from *[Dechloromonas denitrificans](https://parts.igem.org/Part%3ABBa_K5078001)*.
These original hosts of nosZ were chosen due to their part compatibility with “chlamy”.

![NosZ P. stutz L0](https://static.igem.wiki/teams/5078/plasmid-pictures/nosz-p-stutzerii-l0-plasmid-picture.webp)

**NosZ in P. stutzeri
Level 0 plasmid**

![NosZ L0 Digest](https://static.igem.wiki/teams/5078/experiments/digest-of-pstu-and-ddenit-l0.png)

**NosZ L0 Diagnostic Digest**
P. stutzeri and D. denitrificans

By building two versions of nosZ, we could determine which build would work best for reducing nitrous
oxide. These part sequences were found using GenBank and then codon optimized with IDT’s software for
*C. reinhardtii* which prefers a high G, C content in its genome. Once optimized, the correct
restriction sites were added to each end of the sequence to allow for cloning using Golden Gate
technology. We confirmed that these plasmids were correctly built using diagnostic digests.

![NosZ D. denit L1](https://static.igem.wiki/teams/5078/plasmid-pictures/nosz-ddenit-l1-picture.webp)

**NosZ in D. denitrificans
Level 1 plasmid**

Next, the Level 1 plasmid builds were built using parts from the *Chlamydomonas* MoClo kit.
This kit allows for the assembly of parts through the choice of many pieces that align with each
other in a system of genetic constructs.

![NosZ P. stutz L1](https://static.igem.wiki/teams/5078/plasmid-pictures/nosz-pstu-l1-picture.webp)

**NosZ in P. stutzeri
 Level 1 plasmid**

![NosZ L1 Digest](https://static.igem.wiki/teams/5078/experiments/digest-of-pstu-and-ddenit-l1.webp)

**NosZ L1 Diagnostic Digest**
P. stutzeri and D. denitrificans

Each part fits together with unique sticky ends in a designated spot.We used a *C. reinhardtii*
[Psad promoter](https://parts.igem.org/Part%3ABBa_K3002001) from the MoClo kit to allow for
robust gene expression. Following this, the nosZ coding sequence was cloned in-frame with the gene for
[mCherry](https://parts.igem.org/Part%3ABBa_K4770013), a red-fluorescent protein, and this
was followed by a [beta-tubulin terminator](https://parts.igem.org/Part%3ABBa_K3002012). We
confirmed that these plasmids were correctly built using diagnostic digests.

## Phosphorous

![PSR1 L0](https://static.igem.wiki/teams/5078/plasmid-pictures/psr1-l0-plasmid-picture.webp)

**PSR1 Level 0 plasmid**

To increase phosphate uptake from wastewater, we used the MoClo kit to manipulate the expression of two
additional genes. First, we wanted to overexpress the *[Psr1](https://parts.igem.org/Part%3ABBa_K5078003)* gene from *C. reinhardtii* that promotes increased uptake of phosphate. We obtained
the Level 0 part from the Alison Baker Lab at the University of Leeds.

![PSR1 L0](https://static.igem.wiki/teams/5078/experiments/digest-of-psr-l0.webp)

**PSR1 L0
Diagnostic Digest**

![PSR1 L0](https://static.igem.wiki/teams/5078/experiments/digest-of-psr1-l1-picture.webp)

**PSR1 Level 1
Diagnostic Digest**

The Level 1 build started with the promoter from the Psad gene. We attached the PSR1 gene to the
[NanoLuc reporter](https://parts.igem.org/Part%3ABBa_K2619004) gene that can be used in a luminescence
reporting assay. We confirmed that this plasmid was built successfully using a diagnostic digest.

![PSR1 L0](https://static.igem.wiki/teams/5078/plasmid-pictures/psr1-l1-plasmid-picture.webp)

**PSR1 Level 1 plasmid**

![PTC1 amiRNA L0](https://static.igem.wiki/teams/5078/plasmid-pictures/ptc-amirna-l0-picture.webp)

**PTC1 amiRNA
Level 0 plasmid**

Additionally, we designed an artificial [miRNA](amiRNA%20link) to silence expression of the
[PTC1 gene](https://parts.igem.org/Part%3ABBa_K5078002), which codes for a phosphate export protein.

![PTC1 amiRNA Diagnostic Digest](https://static.igem.wiki/teams/5078/experiments/digest-of-pstu-and-ddenit-l1.webp)

**PTC1 amiRNA
Diagnostic Digest**

![PTC1 amiRNA L1 pic](https://static.igem.wiki/teams/5078/experiments/ptc-amirna-l1.webp)

**PTC1 amiRNA
Level 1 plasmid**

For this Level 1 plasmid, we chose to use the [beta-tubulin
promoter](https://parts.igem.org/Part%3ABBa_K3002011), to prevent all genes from relying on the same promoter for the eventual assembly of the Level 2
plasmid. With the PTC1 gene silenced, the algae will be unable to release the phosphate it absorbs and will be
forced to continue phosphate uptake. We confirmed the successful creation of this part by PCR.

We designed all L1 plasmid builds to allow us to assemble an L2 plasmid build that expresses nosZ, PSR1, and the
miRNA to reduce PTC1 expression, along with a gene that allows for antibiotic selection through an additional round
of Golden Gate Cloning.  **We call this plasmid the de-Slimer!**

For a description of our use of the plasmid in *Chlamydomonas reinhardtii*,
please see our [Results page!](https://2024.igem.wiki/uvu-utah-2/results)

Also, please see our most-commonly used Experiment Protocols below.

## Protocols

## Bacteriological Stock Solution Recipes

## Bradford Assay Protocol

## Colony Screening Protocol

## Electroporation

## Designing A gblock For Cloning

## Nutrient Uptake Assays

## Nanoluc Screening

## Pouring Agar Plates

## Golden Gate Assembly

## Tap Agar Plates

## Western Blot

## Passaging Protocol

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## Electroporation of Chlamy

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

![chlamy culture](https://static.igem.wiki/teams/5078/results/chlamy-colony-pic.webp)

## Luciferase assays

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.

![luciferace results](https://static.igem.wiki/teams/5078/results/psr1-luciferase-assay-june10.webp)

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.

## Expression of nosZ-mCherry and Ptc amiRNA

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.

![ptc-rt-qpcr](https://static.igem.wiki/teams/5078/results/ptc-rt-qpcr.webp)

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).

## Phosphate Uptake of Psr1nanoLuc Transformants in TAP media

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.

![exp-1-phosphate-assay](https://static.igem.wiki/teams/5078/results/exp-1-phosphate-assay.webp)

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.

![exp-1-t-test-table](https://static.igem.wiki/teams/5078/results/exp-1-t-test-table.webp)

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.

## Phosphate uptake of Psr1nanoLuc and De-Slimer transformants in mixed media

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.

![exp-2-phosphate-assay](https://static.igem.wiki/teams/5078/results/2-exp-2-phosphate-assay.webp)

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.

![exp-2-t-test-table](https://static.igem.wiki/teams/5078/results/2-t-test-exp-2.webp)

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).

## Nitrate uptake of Psr1nanoLuc and De-Slimer transformants in mixed media

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.

![exp-2-nitrate-assay](https://static.igem.wiki/teams/5078/results/2-exp-2-nitrate-assay.webp)

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.

## Phosphate uptake of Psr1nanoLuc and De-Slimer transformants in Allen media

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.

![exp-3_phosphate_assay](https://static.igem.wiki/teams/5078/results/2-exp-3-phosphate-assay.webp)

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

![exp-3-phosphate-t-test](https://static.igem.wiki/teams/5078/results/t-test-exp-3.webp)

Again, we calculated T-Tests for each of the different strains done in triplicate which didn’t show significant differences across the entire experiment.

## Nitrate uptake of Psr1-nanoLuc and De-Slimer transformants in Allen media

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.

![exp-3-nitrate-assay](https://static.igem.wiki/teams/5078/results/2-exp-3-nitrate-assay.webp)

## Summary

![max phosphate/media](https://static.igem.wiki/teams/5078/results/2-media-max-uptake-comparision.webp)

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.

## Our achievements:

* We successfully assembled 3 simple and 8 new composite parts.
* We successfully electroporated the Level 2 plasmids into chlamy.
* We successfully expressed Psr1-nanoLuc, and found that it caused increased phosphate uptake in TAP media.
* Our Ptc amiRNA part appears to cause decreased expression of Ptc.

## Roadblocks:

* We have not been able to detect expression of nosZ-mCherry.
* Chlamy transformed with De-Slimer do not display increased uptake of nitrate or phosphate compared to untransformed controls.
* Chlamy transformed with De-Slimer appear to grow more slowly than untransformed chlamy.

![slow-growth](https://static.igem.wiki/teams/5078/results/slow-growth.webp)

## Future directions:

* We would like to see if expressing only nitrate or phosphate pathway genes separately improves nutrient rates.
* Instead of looking at nitrate uptake, we would like to see if expression of nosZ causes decreased nitrous oxide production.
* We would like to mimic wastewater treatment plant conditions more closely to see how that effects performance of our chlamy transformed with De-Sliimer.

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