iGEM 2022 — Team latvia-riga

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# NoteBook

Timeline

```
March - Ideation, team sellection, fundraising
April - Problem selection, fundraising, team application
May - Research/experiment planing, DNA design, fundraising
June - First DNA construction, GGA, the yeast arrives, discussion with
Makerere_Uganda 2022, start of the collaboration with Uppsala 2022 team
July - Summer camps, InterLab study 2022, growth media optimisation,
collaboration with ESTONIA-TUIT 2022
August - InterLab study 2022, Baltic Jamboree 2022, yeast transformation,
growth media optimisation, collaboration with Technion-Israel 2022
September - Carotenoid content analysis, yeast transformation, composit
GGA, ELTE 2022 Mock Jamboree, Researcher's Night 2022
October - yeast transformation, wiki building, the GRAND Jamboree
```

GGA for TUs

```
1. Take 10μL of promoter, terminator and gene of choice (10ng/uL).
2. Add 2μL of 10x ligase buffer.
3. Add 1μL of T4 ligase.
4. Add 1μL of BsaI restrictase.
5. Add 6μL of nuclease free water.
6. Incubate 5min at 37°C.
7. Incubate 5min at 10°C.
8. Cycle 10-20 times.
9. Incubate 5min at 60°C.
```

```
# PCR for TUs

1. Add 5μL 10x reaction buffer.
2. Add 1μL dNTP mix.
3. Take 10μL of each assembled TU.
4. Add 1μl of the respective forward and reverse primers for each TU.
Each TU needs the respective primer pair which will extend the fragments and
 add SapI sites with unique fusion sites.
5. Add 1μL Taq polymerase.
6. Add 31 μL water.
7. Anneal at 58°C for 5min, 20 cycles.

# R. toruloides transformation

1.Inoculate yeast in 10 mL of YPD medium and incubate overnight
with a stirring speed of 160 rpm at 30°C.
2. Dilute the culture to an OD600 of ~0.2 in 10 mL of YPD and incubate again until an OD600 of ~0.8.
3. Harvest the culture in a sterile 50 mL centrifuge tube at 3000 g for 10 min.
4. Resuspend the cell pellet in 25 mL of sterile water and centrifuge again.
5. Resuspend the cells in 1 mL 100 mM lithium acetate (LiAc) (pH 7.5)
and transfer 500 μl of the suspension to a 1.5 mL microfuge tube.
6. Centrifuge the cells at 3000 g for 30s and remove LiAc.
7. In the following order, add 240 μL of polyethylene glycol (PEG) 4000 (50% w/v),
 36 μL of 1M LiAc (pH 7.5), 24 μL of sterile water.
8. Transfer 10 μl of pre boiled salmon sperm DNA (10 mg/mL, at 100°C for 10 min) to the tube.
9. Add 50 μL of transforming DNA (0.1–10 μg,
removed from the bacterial backbone by either restriction enzymes or by PCR amplification).
10. Mix the cell pellet and the added reagents by vigorous vortexing.
11. Incubate at 30°C for 30 min.
12. Add 34 μL of dimethyl sulfoxide (DMSO)
13. Heat shock at 42°C for 15 min.
14. Centrifuge the tube at 3000 g for 30 s and remove the supernatant.
15. Resuspend the pellet in 200 uL of water and spread on an agar YPD plate, grow at 30°C overnight.
16. The next day replica plate onto multiple solid
YPD plates containing hygromycin/G418 and leave to grow at 30°C for two days.

# Ura3 mutant selection

1. Grow R. toruloides aerobically grown at 30°C  in SD medium supplemented with uracil for 18h.
2. Prepare 5FOA solution in DMSO (100mg/mL).
3. Spread 150uL of 5FOA solution on a 15mL agar SD plate supplemented with uracil.
4. Spread 200uL of the culture (OD=0.05) on SD+5FOA uracil containing agar plates.
5. Incubate at 30°C for 2 days.
6. Replica plate onto SD plates with and without uracil to isolate potential ura3 mutants.

# Carotenoid extraction

1. Weigh harvested and washed cells were weighed (wet cell biomass).
2. Add 5 mL of DMSO to each sample.
3. Vortex the samples carefully to make sure that the cell pellet has been resuspended completely.
4. Add 5 mL of acetone, 5 mL of hexane and 5 mL of 20% NaCl solution in deionized water and
vortex after each addition.
5. Centrifuge (2500 g, 5 min).
6. Collect the upper layer of the extract.

# UV-Vis measurements

All measurements were carried out with the PerkinElmer Lambda 35 UV-Vis spectrometer.
1. Pour 3 mL of each extract in a 10 mm quartz cuvette.
2. Fill the reference cuvette with hexane.
3. Record the spectra from 350 to 600 nm.
4. Dissolve the beta-carotene standard in hexane at 1.3·10-5 M.

# HPLC analysis

1. Dissolve the commercial beta-carotene standard in a mixture of
tetrahydrofuran (THF) and acetonitrile (50:50, v/v).
2. Prepare calibration samples in concentrations ranging from 16 to 250 mg/mL.
3. Prepare a C18 column with the UV detector operating at 450 nm.
4. Maintain the column temperature at 25 °C. Use a mixture of acetonitrile-THF
(60:40, v/v) as the mobile phase with a flowrate of 1 mL/min.
5. Create a calibration curve based on the area of beta-carotene peaks at
different sample concentrations.
6. Remove the solvent from the yeast beta-carotene extracts using a rotary
evaporator.
7. Redissolve the extract powder in 1 mL of acetonitrile-THF mixture (50:50, v/v).
8. Perform the analysis as described in steps 3 – 4.
9. Using the standard calibration curve, determine the beta-carotene
concentrations in the extracts.
```

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# Engineering

# In our project we followed the design-build-test-learn cycle of synthetic biology.

Design

Before anything can be done in the lab, a clear goal is needed. Our goal being crocin production, we investigated the pathways for crocin production. We found out that *Rhodotorula toruloides* only needs 3 added reactions for crocin synthesis to be possible. Using a genome-scale metabolic model this was confirmed (see [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D))
After the decision to use *R. toruloides* as our crocin producing organism, the necessary transcriptional unit sequences were created and codon optimised.

Build

The build cycle included the creation of transcriptional units and transformation of *R. toruloides*.
The transcriptional unit (TU) creation involved a DBTL cycle itself, because our methods of Golden Gate assembly (GGA) and PCR amplification needed to be optimised for high GC content sequences. After many cycles of testing possible solutions for the TU creation, the correct primers and PCR conditions were found.
Before transforming *R. toruloides* with the final construct, which involves 3 protein coding genes and an antibiotic resistance gene, the antibiotic resistance gene needed to be tested. This is because, if our selection marker doesn’t work, selection of transformants would be impossible.

Test

After transforming *R. toruloides* with antibiotic resistance, the possible transformants were tested for antibiotic resistance. By using both G418 and hygromycin as the antibiotics, we achieved some colony growth only with hygromycin and its resistance as the marker.
Although we saw growth on hygromycin, later testing couldn’t prove that the colonies were *R. toruloides* and suggested that an infection with a hygromycin resistant bacteria occurred.

Learn

2 possible things could lead to us not getting transformants - either our part sequences weren’t optimal for *R. toruloides* or our transformation protocol didn’t work as expected. This could be further investigated by using another transformation method - for example Agrobacterium mediated transformation which has been shown to work with this yeast.
The next DTBL cycle would involve part creation for *Agrobacterium* mediated transformation, transformation of the yeast with *Agrobacterium*, and testing of the transformants for antibiotic resistance. If this cycle again would yield no successful transformants, a deeper look into codon optimization and part creation would be required.

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# Experiments

# Codon Optimization

Each organism has codons, which it uses more or less frequently. Because of this, even if different transcribed RNA sequences result in the same translated amino-acid sequence, expression of the protein can vary based on the codons that are used. Therefore, codon optimization is needed for optimal gene expression. In our case, we needed to reverse engineer the proteins that allow for crocin synthesis in Crocus sativus into DNA sequences, which use the optimal codons for our unconventional yeast *Rhodotorula toruloides*.

The most frequently used codons of *R. toruloides* result in a DNA sequence that is high in G-C content [1]. The G-C content of the *R. toruloides* genome is 62% [2]. Companies which provide DNA synthesis, however, can not synthesise large DNA fragments with a high G-C content. Although these companies, such as IDT and Twist Bioscience, offer online sequence optimization, they don’t offer codon optimization for our unconventional yeast *R. toruloides*. Therefore, we used DNA Chisel [3], a python library that allows for the use of custom codon usage tables.

With no added constraints for the codon optimisation task, the optimised sequence had a high GC content and highly repetitive regions - traits that don’t allow for custom DNA synthesis. Different constraints, such as maximum GC content and maximum amount of repetitive sequences, were added. This resulted in a balancing act where optimal codon usage for our yeast was balanced with the criteria needed to synthesise the DNA. Although the sequences

---

REFERENCES

```
[1]https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=5537&aa=1&style=N
[2]https://www.ncbi.nlm.nih.gov/genome/?term=Rhodotorula%20toruloides[Organism]&cmd=DetailsSearch
[3]https://github.com/Edinburgh-Genome-Foundry/DnaChisel
```

---

# DNA construct creation

To achieve crocin synthesis in *R. toruloides* 3 genes from Crocus sativus need to be added. Additionally, an antibiotic resistance gene is needed to select successful transformants.

#### Level 0 parts

---

7 new parts for use in *R. toruloides* were created. These parts are:- 1. Codon optimized zeaxanthin 7,8(7',8')-cleavage dioxygenase from Crocus sativus.
- 2. Codon optimized crocetin glucosyltransferase 2 from Crocus sativus.
- 3.Terminator from *R. toruloides* (NP11) mRNA-heat shock 70kDa protein gene
- 4. Codon optimized kanamycin resistance gene from Escherichia coli
- 5. Promoter from *R. toruloides* (NP11) glucose-6-phosphate isomerase (PGI) gene
- 6. Codon optimized beta-carotene hydroxylase from Crocus sativus.
- 7. Codon optimized hygromycin resistance gene from Escherichia coli

To create these parts, restriction sites used in BioBrick and RFC1000 assembly were removed. RFC1000 prefix and suffix for each part were added to the ends. Flanking the RFC1000 prefix and suffix, each part also has a BioBrick prefix and suffix, which allows for easy PCR amplification for all parts using the same primers and also allows the use of the conventional Biobrick assembly method.

![](https://static.igem.wiki/teams/4398/wiki/exp/experimentslevel0part.png)

#### Level 1 Part Assembly

---

We used the iGEM RFC1000 standard for the assembly of our DNA constructs. This method uses the type IIs restriction enzyme BsaI to digest and T4 ligase to ligate the level 0 parts. Each level 1 part includes a promoter, transcribed sequence and a terminator. We used the same promoter PPGI for all level 1 parts. The PPGI promoter is the strongest constitutive promoter reported for *R. toruloides* NP11 [1].

Before the Golden Gate Assembly (GGA) was performed each level 0 part was amplified by PCR using Taq polymerase [2]. After the PCR amplification, the required parts for GGA (promoter, transcribed sequence and terminator for each transcriptional unit (TU) ) were mixed together (7 ul each) and purified using Thermo Scientific GeneJET PCR purification kit. The GGA was performed with the purified mix for each transcriptional unit. For each transcriptional unit 10 ul the purified mix, 2 ul 10x T4 ligase buffer, 0.5ul of BsaI, 0.5 ul of T4 ligase and 7ul of water were added in a PCR tube. The GGA mix was cycled between 5 min 37 C and 5 min 16 C for 10 cycles, followed by 5 min enzyme inactivation at 60 C.

![](https://static.igem.wiki/teams/4398/wiki/exp/experimentstu1-4.png)

#### Level 2 Assembly

---

Before level 2 assembly each transcriptional unit was amplified by PCR with primers that contained overhangs with SapI restriction recognition sites and fusion sites according to the iGEM RFC1000 standard.
For each transcriptional unit 10ul of the GGA product was used in a 50ul PCR reaction [2]. The PCR reaction was performed according to [3] protocol.
![](https://static.igem.wiki/teams/4398/wiki/exp/experimentstupcr.png)

After PCR amplification gel electrophoresis was performed and the DNA fragments near 3kb were purified. Level 2 assembly was performed by mixing the purified transcriptional units 1-4 (4 ul each), adding 2 ul 10x T4 ligase buffer, 0.5 ul SapI and 0.5 ul T5 ligase. The GGA mix was cycled between 5 min 37°C and 5 min 16°C for 10 cycles, followed by 5 min enzyme inactivation at 60°C.

![](https://static.igem.wiki/teams/4398/wiki/exp/51deb976-e6a6-443d-b2b1-842f3b4a0e0c.jpeg)

---

```
     REFERENCES
     [1] Wang, Y., Lin, X., Zhang, S., Sun, W., Ma, S., and Zhao, Z. K. (2016)
     Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides.
      Yeast, 33: 99– 106. doi: 10.1002/yea.3145.
     [2] https://international.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273
```

---

# Transformation, colony PCR and qPCR

*R. toruloides* does not have a described 2-micron plasmid ORI sequence. One of the described methods for transformation uses Agrobacterium tumefaciens, which has been proven to be capable of infecting fungi [1]. The Agrobacterium tumefaciens-mediated transformation (ATMT) has been successfully used to incorporate foreign DNA into the genome of *R. toruloides* [2]. Since ATMT is a time consuming method that presents multiple difficulties we chose a protocol described by Nora et al. to incorporate the linear DNA construct into *R. toruloides* [3]. This method relies on the non-homologous end joining mechanism where the foreign DNA is inserted in random locations of the yeast genome.

We tested if the DNA has successfully been integrated by adapting a previously described yeast colony PCR protocol [4].

---

```
  REFRENCES:
  [1] Hooykaas PJJ, van Heusden GPH, Niu X, Reza Roushan M, Soltani J, Zhang X, van der Zaal BJ. Agrobacterium-Mediated
  Transformation of Yeast and Fungi. Curr Top Microbiol Immunol. 2018;418:349-374. doi: 10.1007/82_2018_90. PMID: 29770864.
  [2] Park YK, Nicaud JM, Ledesma-Amaro R. The Engineering Potential of Rhodosporidium toruloides as a Workhorse for
  Biotechnological Applications. Trends Biotechnol. 2018 Mar;36(3):304-317. doi: 10.1016/j.tibtech.2017.10.013. Epub 2017 Nov 10.
  PMID: 29132754.
  [3] Nora, L.C., Wehrs, M., Kim, J. et al. A toolset of constitutive promoters for metabolic engineering of Rhodosporidium
  toruloides. Microb Cell Fact 18, 117 (2019). https://doi.org/10.1186/s12934-019-1167-0
  [4] Lõoke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 2011
  May;50(5):325-8. doi: 10.2144/000113672. PMID: 21548894; PMCID: PMC3182553.
```

---

# Uracil auxotrophic strain creation

Using auxotrophy-complementing genes as selection markers eliminates the need for antibiotic based positive clone identification. A strain unable to synthesise a compound necessary for its growth is auxotrophic and can survive only if it can be taken from the environment. The auxotrophy-complementing genes therefore can be used to create a prototroph that is able to survive in minimal media without the previously necessary amino acid, nucleotide or other organic compound. Widespread antibiotic pollution promoting the resistance to them is the main reason auxotrophy genetic markers should be preferred.

*R. toruloides* has been transformed with resistance genes for hygromycin, bleomycin and zeocin which are some of the effective antibiotics for the wild type yeast [1]. In our opinion auxotrophic selection methods should be the first to be implemented for novel yeasts. The creation of auxotrophic strains would allow for future genetic engineering that forgoes environmentally harmful and often exotic and expensive antibiotics. Therefore we set to create a uracil auxotrophic *R. toruloides* strain and design a complementing gene to be used as a selection marker for our and future iGEM and research projects.

URA3 gene encodes orotidine 5-phosphate decarboxylase which is necessary for the synthesis of uracil - a nucleotide essential for cell survival. The Ura3p also converts 5-Fluoroorotic acid to 5-fluorouracil which is a suicide inhibitor that causes cell death. Knowing that URA3 strains are killed off by FOA5 and ura3 strains are unable to grow without supplemented uracil both positive and negative selection experiments can be designed.

FOA5 toxicity to wild type strains can be used to screen for useful mutants that are uracil auxotrophic, since those would not be affected by FOA5 presence. We are using a protocol developed by [2] to isolate a uracil auxotrophic strain of *R. toruloides* which could be later confirmed by sequencing.

---

```
     REFERENCES
     [1] Park YK, Nicaud JM, Ledesma-Amaro R. The Engineering Potential of Rhodosporidium toruloides as a Workhorse for
     Biotechnological Applications. Trends Biotechnol. 2018 Mar;36(3):304-317. doi: 10.1016/j.tibtech.2017.10.013.
     Epub 2017 Nov10. PMID: 29132754.
     [2] Wang F, Yue L, Wang L, Madzak C, Li J, Wang X, Chi Z. Genetic modification of the marine-derived yeast Yarrowia
     lipolytica with high-protein content using a GPI-anchor-fusion expression system. Biotechnol Prog.
      2009 Sep-Oct;25(5):1297-303. doi: 10.1002/btpr.235. PMID: 19743190.
```

---

# Medium optimization for higher beta-carotene yields

Amounts of *R. toruloides* carotenoid production can be highly variable. There are many factors that affect carotenoid yields, such as composition of growth medium, temperature and lighting conditions. Furthermore, it is mentioned in literature that additives such as asparagine can boost carotenoid production. In order to achieve higher yields of our final product (crocin), it was important to make as much beta-carotene as possible since carotene is the starting point of our biosynthesis cycle.

For our experiments we prepared four different media. Medium 1: defined medium with salts (10 g/L glucose; 0.52 g/L KH2PO4; 0.52 g/L MgSO4; 4 g/L NH4NO3; 10 g/L asparagine); medium 2: SD minimal medium (20 g/L glucose; 6.7 g/L yeast nitrogen base); medium 3: SD minimal medium with added arginine (10 g/L); Medium 4: YPD medium with salts (40 g/L glucose, 7 g/L KH2PO4, 2 g/L (NH4)2SO4, 2 g/L Na2SO4, 1.5 g/L MgSO4·7H2O, 1.5 g/L yeast extract). All media were prepared with deionized water and sterilised by autoclaving at 121°C. Tetracycline (10 mg/L) was added to sterilised and cooled solutions. For each experiment, 50 mL of medium was transferred to sterile 250 mL flask and inoculum was added from an SD night culture for the final OD to be around 0.2.

We chose four different growth conditions for each medium: 30°C with light; 30°C without light; 20°C with light; 20°C without light. Prepared cultures were put in incubators and grown for 120 hours while shaking at 160 rpm. Optical density at 600 nm was measured after 120 hours. The final OD for most of the culture reached 20. Cells were harvested with centrifugation (7000g, 2 min) and washed with distilled water twice. The carotenoids were extracted from wet cell biomass and quantified via  [UV-Vis Spectroscopy.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

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# Results

# DNA construct creation

We successfully created 7 new basic (level 0) and 5 new composite (level 1) parts. (see [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D))

# Basic parts

Basic parts were ordered from IDT and Twist Bioscience. These fragments were amplified with PCR using primers that are complementary to the Biobrick prefix and suffix.

Basic parts1. 1. Codon optimized zeaxanthin 7,8(7',8')-cleavage dioxygenase from *Crocus sativus*.- 2. Codon optimized crocetin glucosyltransferase 2 from *Crocus sativus*.- 3. Terminator from *Rhodotorula toruloides* (NP11) mRNA-heat shock 70kDa protein gene.- 4. Codon optimized kanamycin resistance gene from *Escherichia coli*.- 5. Promoter from *Rhodotorula toruloides* (NP11) glucose-6-phosphate isomerase (PGI) gene.- 6. Codon optimized beta-carotene hydroxylase from *Crocus sativus*.- 7. Codon optimized hygromycin resistance gene from *Escherichia coli*.

![](https://static.igem.wiki/teams/4398/wiki/level-1-parts.png)

# Composite parts

The composite parts were made using Golden Gate Assembly and then amplified using PCR with primers that add the necessary overhangs for level 2 assembbly

Transcriptional units1. TU1: PPGI + GLT2 + terminator- TU2: PPGI + zcd + terminator- TU3: PPGI + beta-carotene hydroxylase + terminator- TU4: PPGI + kanamycin resistance + terminator- TU5: PPGI + hygromycin resistance + terminator (not seen in the picture)

![](https://static.igem.wiki/teams/4398/wiki/results-level-2.png)

# Medium optimization

![](https://static.igem.wiki/teams/4398/wiki/kolbas.jpg)

UV-Vis measurements were used to compare total carotenoid content in *R. toruloides* extracts grown in different conditions. Absorption patterns were quite different, but some things could be noticed. First, growth at 20°C yields higher amounts of carotene than in 30°C in all cases. Second, extra lighting can boost carotenoid yields, but it can be noticed that change of light conditions does not affect all samples equally. Dramatical yield drop is detected in media 1 and 3, whereas changes in medium 2 between light and no-light (n) samples are almost negligible. Third, medium 4 gives lowest carotenoid yields in all conditions by a margin. As we were more interested in beta-carotene in our samples, we also ran an experiment for beta-carotene standard. The standard gave us two characteristic beta-carotene peaks at 452 and 483 nm. It can be seen that some samples present those two characteristic carotene peaks, especially samples from M2 medium, however the peaks are shifted bathochromically (460 and 490 nm, respectively). This shift can be easily explained: beta-carotene standard spectrum was recorded in dry hexane solution, whereas our extracts definitely had some water in them as well as residual acetone and DMSO from the extraction process, therefore making the solvent more polar and causing a bathochromic shift. Our very approximate estimates from UV-Vis spectroscopy suggest that at best growth conditions (M2, 20°C, extra light) *R. toruloides* produce beta-carotene at 1.5 mg/L concentration.

![](https://static.igem.wiki/teams/4398/wiki/uv-chart.png)- UV-Vis absorption patterns of the *R. toruloides* extracts.

  However, UV-Vis spectroscopy is more suitable for total carotenoid quantification than single carotenoid species detection. TLC analysis gave us more knowledge about types of carotenoids found in our extracts. It became clear that medium M2 was the best for beta-carotene production. Surprisingly to us, it turned out that M2 samples grown in 30°C actually had more beta-carotene than those grown in 20°C. Other mediums barely produced any beta-carotene, with M3 and M4 mediums at 20°C being second- and third-best carotene producers. From literature studies we know that the mildly polar compound giving the orange band in TLC plate is torularhodin. As can be identified, torularhodin is responsible for major part of absorption in M2 20°C samples. We could not identify the third major component on the extract with the highest polarity that did not move from the start line in hexane and in other TLC running solutions. This compound most likely has UV-Vis absorption peak at 520 nm as it is more characteristic to samples that contain less beta-carotene and torularhodin.

  ![](https://static.igem.wiki/teams/4398/wiki/tlc.png)- TLC of *R. toruloides* extracts

    ![](https://static.igem.wiki/teams/4398/wiki/calibration.png)

    ![](https://static.igem.wiki/teams/4398/wiki/samples.jpeg)

    - Beta-carotene calibration curve for HPLC
      ![](https://static.igem.wiki/teams/4398/wiki/standardhplc.png)

      Beta-carotene elution time was determined to be at 4.57 min.

      HPLC analysis was performed on five extracts.

      - Beta-carotene yields from *R. toruloides* grown in different mediums
        ![](https://static.igem.wiki/teams/4398/wiki/res0.jpg)

        The greatest yields of beta-carotene were achieved for *R. toruloides* grown in medium 2.
        It was also interesting to observe how the amounts of different carotenes - torularhodin and beta-carotene - varied among the different samples. For example, extracts from *R. toruloides* grown in M2 medium at 30°C had more beta-carotene than torularhodin, while the opposite was true for extracts from cells grown in the same M2 medium at 20°C. It seems like *R. toruloides* produces more beta-carotene at lower temperatures.

        ![](https://static.igem.wiki/teams/4398/wiki/20deg.png)
        ![](https://static.igem.wiki/teams/4398/wiki/30deg.png)

# Uracil auxotrophic strain creation

*R. toruloides* seems to be semi-resistant to 5-FOA. The first generation of yeasts formed thick matts of colonies on the 5-FOA plates. After successional replica plating onto new 5-FOA plates, as well as SD plates with and without uracil, multiple possible URA3 mutants were isolated. Each colony was plated onto an SD plate with and without uracil to confirm the auxotrophy. None of the isolated mutants displayed inability to grow on SD without uracil, meaning they were not uracil auxotrophic.

Previous research suggests that not all 5-FOA resistant *R. toruloides* colonies in experiments like this are deletion mutants of URA3. Furthermore, the number of ‘false’ URA3 mutants can be high in proportion to all 5-FOA resistant colonies [1]. It has to be noted that this sort of random mutation induction causes random gene disruption. The albino phenotype of many mutants can be attributed to the deletion of CAR2 - gene encoding carotene cyclase without which beta-carotene is not formed in the cells [1]. For precise URA3 deletion CRISPR-Cas9 aided method can be used [2].

![](https://static.igem.wiki/teams/4398/wiki/trauki.jpg)- Successional SD 5-FOA, SD-uracil and SD+uracil plates of *R. toruloides* (bottom to top)

# Transformations

We tried to transform our yeast with the DNA constructs we had created. Because *R. toruloides* prefers non-homologous recombination instead of homologous recombination, we tried exploiting this property and insert linear DNA fragments with no homology arms, hoping that it would insert in a place that doesn’t impact other physiological functions (see [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)).

Before inserting other genes, it is important to know that our selection marker is working properly, in this case - our antibiotic resistance genes. We performed transformations with both TU4 (kanamycin resistance) and TU5 (hygromycin resistance). We performed the transformations, plate them on YPD plates for overnight growth and then the next day transfer the colonies over with a cloth on YPD plates containing the respective antibiotic.

For our TU4 transformations we observed no growth on plates containing kanamycin over 2 transformation attempts.

For our TU5 transformations in the second attempt we observed white colonies growing on plates containing hygromycin. Worried by the colonies not turning orange as the wild-type, we put the cells under a microscope. We observed that these were slow growing bacteria and that we have had contamination during our transformation process.

![](https://static.igem.wiki/teams/4398/wiki/tu5-transform.png)- "Transformants" from the 2nd TU5 transformation
  ![](https://static.igem.wiki/teams/4398/wiki/microscope.png)- "Transformants" under 1000x magnification
    ![](https://static.igem.wiki/teams/4398/wiki/wild-type-microscope.png)- *R. toruloides* wild-type under 1000x magnification

---

```
REFERENCES:
  [1] Koh, C.M.J., Liu, Y., Moehninsi et al. Molecular characterization of KU70 and KU80 homologues and exploitation of a
  KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides.
  BMC Microbiol 14, 50 (2014). https://doi.org/10.1186/1471-2180-14-50
  [2] Otoupal PB, Ito M, Arkin AP, Magnuson JK, Gladden JM, Skerker JM.
  Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides.
  mSphere. 2019 Mar 20;4(2):e00099-19. doi: 10.1128/mSphere.00099-19. PMID: 30894433; PMCID: PMC6429044.
```

---

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