iGEM 2024 — Team hkust

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}2024 HKUST iGEM DHA Express{% endblock %}
{% block page\_content %}

![Experiment](https://static.igem.wiki/teams/5159/module-logo/experiment.png)
![](https://static.igem.wiki/teams/5159/learn-more.png)

# Notebook

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| Week 1 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w1.pdf) |

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| --- |
| Week 2 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w2.pdf) |

|  |
| --- |
| Week 3 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w3.pdf) |

|  |
| --- |
| Week 4 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w4.pdf) |

|  |
| --- |
| Week 5 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w5.pdf) |

|  |
| --- |
| Week 6 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w6.pdf) |

|  |
| --- |
| Week 7 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w7.pdf) |

|  |
| --- |
| Week 8 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w8.pdf) |

|  |
| --- |
| Week 9 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w9.pdf) |

|  |
| --- |
| Week 10 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w10.pdf) |

|  |
| --- |
| Week 11 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w11.pdf) |

|  |
| --- |
| Week 12 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w12.pdf) |

|  |
| --- |
| Week 13 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w13.pdf) |

|  |
| --- |
| Week 14 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w14.pdf) |

|  |
| --- |
| Week 15 |
| [Wet lab & Circuit Design](https://static.igem.wiki/teams/5159/notebook/igem-notebook-w15.pdf) |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}2024 HKUST iGEM DHA Express{% endblock %}
{% block page\_content %}

[• Cycle 0](#firstContent)
[• Cycle 1](#Cycle1)
[• Cycle 2](#Cycle2)
[• Cycle 3.1](#Cycle3.1)
[• Cycle 3.2](#Cycle3.2)
[• Cycle 4](#Cycle4)
[• Engineering success in E. coli](#Engineering success in E. coli)
[• Transformation of Yarrowia
lipolytica](#Transformation of Yarrowia lipolytica)
[• References](#references)
![](https://static.igem.wiki/teams/5159/claw-1.png)

![Project](https://static.igem.wiki/teams/5159/igem-project-img.png)
![](https://static.igem.wiki/teams/5159/learn-more.png)

# Cycle 0

## Design

Growth rate, cell density and lipid accumulation are key factors determining the yield of final
products when considering microbial industrial production. So to better understand the strain and
set the basis for further engineering, we plan to first test and optimize cultivation conditions
(including supporting media) for *Yarrowia lipolytica* growth and lipid accumulation.

The standard and very basic media for cultivating the yeast is YPD and YNB, but there are lots more
variables to be explored. Carbon and nitrogen sources are the 2 main components that determine the
growth of the strain, and the C/N ratio has a significant impact on the lipid accumulation
[[1]](#references)
. There
are also papers reporting that phosphate limitation is crucial to DHA accumulation [[2]](#references). Therefore, we
divided the experiments in cycle 0 mainly into 2 parts by its purpose, and under each part the
following factors are investigated:

## Part 1. Optimization for Growth

* Different carbon source (glucose, glycerol, glucose+glycerol)
* Conc. of carbon source

## Part 2. Optimization for lipid accumulation: Effect of nitrogen source depletion

Our chassis Yarrowia lipolytica is known for being able to grow on various carbon sources,
among which glucose and glycerol are the most common ones. In our case carbon source also
serves as the substrate for DHA synthesis. It contributes to one of the main costs in industrial
fermentation. This was noticed through our interviews with experts in the current bioproduction
industry,
 [Dr. Young-Kyuong Park](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
working with *Yarrowia lipolytica* and
[Dr. Zhu Yuanmin](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
working
with
DHA-producing microalgae *Schizochytrium sp.*. Various researches tried to use a cheaper
alternative
carbon source
for bioproduction including crude glycerol or food waste extract[[4][5]](#references). Therefore we also designed
experiments
to verify the feasibility of doing so and its impact on the growth of our chassis.

Besides, the scale of culture is another key factor to be considered, and will be further
discussed in our
[Engineering Cycle 4](#Cycle4)
together with some other key factors such as
temperature, pH and oxygen supply.

## Build

To further understand how different substrates affect the metabolism and growth of *Yarrowia
lipolytica*,
our dry lab established a Genome-scale Metabolic Model (See our
[Dry Lab](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
page for more
detail),
which helps us identify the crucial factors, make predictions and design our experiments
correspondingly.

Our target strain *Yarrowia lipolytica* Po1f is kindly provided by
[Dr. Du Fei from Nanjing Normal
University](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D),
who’s group is working on a similar topic with Yarrowia lipolytica. For other media preparation,
please
refer to the
[Experiment page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

## Test

The change of OD600 over time was measured by taking the sample every 1 hour, which gives
us
important
information about
the growth rate and final biomass, as a linear coloration between cell dry mass (CDM) per unit
volume
and OD600 was suggested
by various researches[[6]](#references) and past iGEM teams (
[2023 KUleuven - Model](https://2023.igem.wiki/kuleuven/model)
). As a result, the maximum
growth rate during the
exponential phase and the time taken to reach the stationary phase under different conditions were
also
obtained.

The results also assemble the prediction made by our models.

For lipid accumulation, we used the lipid staining method to visualize the lipid accumulation in the
cells under different
growth phases and conditions using a microscope.

A GC-MS analysis was also conducted to characterize the lipid profile in the original strain, at the
same
time providing a
quantified result for reference along with the lipid-staining method, and serving as the control for
GC-MS analysis in later cycles.
For more details about the experiment and results, please refer to our
[Experiment page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
.

## Learn

By analyzing the data derived, we found that glycerol will lead to a higher growth rate of
*Yarrowia
lipolytica*,
which is important to the efficient industrial production. Also nitrogen limiting
conditions will lead to
a higher lipid accumulation by some number. However, these might not be the optimal conditions for
growth, suggesting
a separation of growth and production stages under different conditions, which lead to our
[Engineering Cycle 3.1](#Cycle3.1)
and will be discussed later in detail.

More importantly, in cycle 0, we found out that the lipid accumulation in the native strain, though
optimized, is
still far from the reported cases for industrial production. Therefore, we would like to further
increase the lipid
accumulation in *Yarrowia lipolytica* before introducing the DHA synthesis pathway, which
redirects
the
existing fatty
acid to DHA. Here comes our engineering cycle 1.

# Cycle 1

## Design

In order to improve lipid accumulation in *Yarrowia lipolytica*, we refer to various researches
and
found
that preventing
fatty acid degradation is one of the more effective strategies. Fatty acid including DHA is degraded
through the
beta-oxidation pathway in *Yarrowia lipolytica*, which happens in an organelle called
peroxisome where
most enzymes for
beta-oxidation are localized. Knowing that, we consulted
[Prof. R. A. Rachubinski](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
, expert in
peroxisome biogenesis, and
further confirmed that termination of the peroxisome biogenesis lead to the total disruption of
beta-oxidation, and
effectively promotes fatty acid accumulation in the cell in form of triacylglycerol (TAG), which was
suggested to be a
more effective strategy than increasing the precursors.

Through the discussion and relevant research, we know that the PEX proteins are the key proteins in
peroxisome biogenesis,
which help form the peroxisomal membrane ring complex and transport other important proteins during
peroxisome formation.
Knockout of PEX genes will lead to disruption of peroxisome biogenesis, especially PEX10, which was
proved to promote lipid
accumulation to the largest extent among other existing strategies and not affecting the normal cell
growth. So in our engineering
cycle 1, we decided to construct a PEX10 knockout strain to improve lipid accumulation.

We designed a construct with the 5’ and 3’ homologous arms of PEX10 gene flanking a LEU2 selection
marker, containing a LEU2
native promoter, LEU2 coding sequence and a terminator, which enables part of the Pex10 to be
replaced
by the LEU2 expression
cassette through homologous recombination.

However, for selection after future transformations with the same LEU2 marker gene, we need to
release
the LEU2 gene from the genome.
For this part we consulted
[Prof. Jee Loon Foo](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
from NUS and he kindly provided us a solution using
the
Cre-loxP system and gave us
the plasmid containing the Cre recombinase, which can recognize the region flanked by the loxP sites
and
enable its release.
Therefore we also added the loxP sites flanking our previous LEU2 expression cassette.

Before constructing the KO cassette, we would like to first perform a proof-of-concept by chemically
impairing the peroxisomal function to see whether the logic under our proposed strategy is correct.
This is planned to be achieved by catalase inhibitor 3-amino-1,2,4-triazole treatment and lipid
staining. For details please refer to our
[Proof-of-concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)
page.

## Build

According to our design, we ordered the gBlocks and assembled them with Gibson assembly at once in
*E.
coli*, verified, linearized
with restriction digestion, then transformed into *Yarrowia lipolytica* with lithium acetate
method.
For
more details about the cloning process,
please refer to the
[Cloning](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27cloning%27%29%20%7D%7D)
page.

## Test

Thanks to Prof. Rachubinski, we obtained a peroxisomal-targeting antibody, anti-SKL, to detect the
presence of peroxisome before and after the
knockout through observing if the immune-staining marker clusters (have peroxisomes) or spread
uniformly
in the cytosol (no peroxisome). At the
same time we will do lipid staining to observe and compare the lipid accumulation in the strains.
After
the
knockout of PEX10, the peroxisome is expected to almost disappears, and the lipid accumulation would
beis increased by a large extent.

To investigate the impact of PEX10 knockout on the lipid profile, we also plan to do a GC-MS
analysis of the PEX10-KO strain and compared the result with the original one, investigating
whether PEX10 knockout will have any influence on the lipid profile and its implication on DHA
production.

## Learn

In this cycle we performed a proof-of-concept test indicating the effectiveness of the strategy, and
further moved on to constructing a PEX10-KO strain, which is expected to show a better ability to
accumulate lipids up to 50-60% percent of DCW under an optimized condition. This level of
accumulation can be further raised by combining with other strategies such as knocking down the MFE2
gene, another key gene catalyzing the beta-oxidation in *Yarrowia lipolytica*. We are also
planning to
test it out after the successful knockout of PEX10, which can serve as another proof-of-concept, and
similar approaches will be directly applied to the knockout of other genes.

Up to this point, we finished optimizing the lipid accumulation, but since *Yarrowia lipolytica*
normally do not produce DHA, we have to introduce a new
pathway and redirect the synthesis of fatty acid to DHA to get a DHA-accumulating Yarrowia
lipolytica
strain. Here comes our next cycle to introduce the
PUFA synthase pathway for DHA production.

# Cycle 2

## Design

Thanks for feedbacks from
[Prof. Ye Chao](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
of Nanking Normal University and
[Prof. Henry Lam](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
in
HKUST, we first established a genome-scale metabolic model (GEM)
to predict the change in flux and identify potential targets for metabolic engineering after
introducing
the DHA synthesis pathway. We also build a DHA yield
model using ODE analysis with Matlab and some AI-aided parameter prediction tools, which can predict
the
DHA production curve over time. Please refer to our
[Dry Lab](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
page for more details. These models indicate that introducing the PUFA synthase
pathway
into the cell can indeed lead to predicted DHA production, and
the correlation between the expression level and DHA yield, which guide us through the design of
this
cycle and further optimization strategies in the next cycle.

In order to express the PUFA synthase described on the
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
page in *Yarrowia
lipolytica*,
we designed to first put each subunits under the same strong constitutive
promoter pTEFin (
[BBa\_K5159005](https://parts.igem.org/Part%3ABBa_K5159005)
) and before the common *Yarrowia lipolytica* terminator
LIP2t
(
[BBa\_K5159009](https://parts.igem.org/Part%3ABBa_K5159009)
) from the registry, after consulting
[Prof. Yong Lai](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
specialized
in yeast synthetic biology in HKUST, and experienced researcher of *Yarrowia lipolytica*,
[Dr.
Young-Kyuong Park](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
. In order to detect the expression, we also added a His-tag
after each subunit and with a linker. Though we are using the same tag, expression of different
subunits
can still be differentiated according to their diverse size.
We also made sure that the addition of the His-tags will not change the proteins’ conformation and
affect the assembly by predicting the structures with software
, after discussion with
[Prof. David Banfield](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
, another expert in
yeast
study in HKUST. All the coding sequences are codon-optimized for *Yarrowia lipolytica*,
and the open reading frames are divided into shorter fragments to facilitate the gBlock synthesis.

Expressing such a large enzyme with a total gene length of 20kb is not a common practice, therefore
we
again reached out to
[Dr. Du Fei](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
who has experience in a similar project.
From her feedback we decided to transform linearized fragments into *Yarrowia lipolytica*, and
make
use of the non-homologous end-joining mechanism, which has a higher chance in *Yarrowia
lipolytica*,
to randomly integrate the gene into the yeast's genome.

## Build

Given the length of the construct and the time limit, also thanks to the suggestion from our PI, we
finally decided not to get all subunits expressed in *Yarrowia lipolytica*, but rather complete
the
successful
expression of subunit C, PPTase and detection of activity for subunit C as a proof-of concept that
all subunits, with the
same promoter and terminator, can be successfully expressed in *Yarrowia lipolytica* and
cluster
together to
achieve its function of synthesizing DHA. To support this assumption, our
Dry Lab
is also planning to
simulate
the assembly of the cluster in cytosol in the future.

To increase the cloning efficiency, we first constructed a new plasmid backbone containing the TEFin
promoter and the LIP2 terminator with spacer, then inserted our ORF\_C into
the new backbone and finally added the LEU2 selection marker cassette to the plasmid by digestion
and
ligation. The same assembly and transformation approach (except the addition of LEU2 selection
marker)
was employed as the previous cycle. Refer to the
[Cloning](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27cloning%27%29%20%7D%7D)
page for more details.

## Test

The successful expression of each protein subunit, with the His-tag added, will be detected by
Western
Blot,
and the expression level is quantified through image analysis. Given the fact that individual
subunits
of
PUFA synthase, if expressed correctly, still contain certain functioning domains that can catalyze
the
reaction, for example, dehydration, during the fatty acid synthesis on its own, a change in fatty
acid
profile
after the expression is expected. Therefore, we also plan to further verify the activity of the
subunit C with
GC-MS to compare the change in fatty acid profile before and after expressing the subunit.

For experimental details, please refer to our
[Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
page.

## Learn

After the proof-of-concept expression of a functioning subunit in *Yarrowia lipolytica* and
measuring the expression level, we can make a relatively more accurate prediction of the DHA yield
using
our
genome-scale metabolic model and mathematical yield model, which can be further improved according
to
the models. This leads to the next cycle, which is the optimizations for increasing DHA yield.

# Cycle 3.1

## Design

During our discussion with
[Prof. David Banfield](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
, we realized that constitutive expression of
such
a large enzyme may not be good for the yeast, and a controlled expression is preferred.
While according to our engineering cycle 0, there are different optimal conditions for cell growth
and
lipid accumulation, so our design divided the whole process into two stages: **growth stage**
and **production stage**. We first explored the genome-scale metabolic model (see
[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
page) to predict if
this strategy will help increase the yield by comparing the case to constitutive expression,
and show a favorable result.

In order to achieve the control between stages, we need to use an inducible promoter to control the
gene
expression. However, we found that there are only few inducible promoters characterized
in *Yarrowia lipolytica*, though it is already a common strain for industrial production, which
was
also mentioned during our interview with
[Dr. Young-Kyoung Park](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
, an expert in
*Yarrowia lipolytica* research, to be a major challenge. Given this situation, in this cycle we
changed our focus to characterizing more inducible promoters in *Yarrowia lipolytica*.

From the result of cycle 0, we found out that nitrogen-limiting conditions are very beneficial for
lipid
accumulation, in our case DHA accumulation. So we choose nitrogen-limitation to be our
inducing condition and identified several native promoters induced by nitrogen starvation, cleaving
1500
bp upstream of the coding gene. Among them two promoters pCIT1 and pIDP2 are chosen for testing.
We use GFP under the promoters to test its activity and sensitivity to the nitrogen level by
fluorescence measurement, and compare them to the standard TEF strong constitutive promoter to
quantify
the promoter strength.

Knowing that the native inducible promoters do not usually have a high enough expression level for
production cases, we would also aim to improve the promoters by increasing its strength. We add
Upstream
Activating Sequence (UAS), in our case most-studied UAS1B from upstream of the native XPR promoter,
to
amplify the inducible promoters, which can significantly increase their strength with increasing
copy
numbers added.

We know the UAS functions in a distance-dependent manner, which means they need to be closer to the
core
promoter region in order to work well. However currently the promoters are all simply a cut of 1500
or
1000 base pairs in front of the native gene, and not well characterized where exactly the promoter
region is. So after attempting to predict the conserved elements and known regulatory sequences, we
decided to
sequentially truncate the promoter from the 5’ end and try to identify the actual promoter region.
At
the same time we also try to add an intron sequence from pTEFin after the promoter, which is another
reported strategy to increase its strength.

![A graph showing the improved promoter construct by adding UAS and intron sequences](https://static.igem.wiki/teams/5159/eng-suc/wiki-graphs-zip-13-copy-2.webp)

A graph showing the improved promoter construct by adding UAS and intron
sequences

## Build

Using the same approaches mentioned above, we planned to construct 9 plasmid by inserting different
promoter or promoter modifications into a GFP-Lip2t-pSB1C3 new backbone, and 3 of them are
successfully
constructed and tested:

* pTEF (
  [BBa\_K5159025](https://parts.igem.org/Part%3ABBa_K5159025))
* pCIT1
  (
  [BBa\_K5159026](https://parts.igem.org/Part%3ABBa_K5159026)
  )
* pIDP2 (
  [BBa\_K5159027](https://parts.igem.org/Part%3ABBa_K5159027)
  )

Please refer to
[Engineering success in *E. coli*](#Engineering success in E. coli)
section for more details. The following promoter
constructs are also designed for characterizing and improving the original pCIT1, to be constructed
soon with a same approach: 1UAS1B+pCIT1, 2UAS1B+pCIT1, 4UAS1B+pCIT1, pCIT1+TEF intron, pCIT1-810 bp,
and pCIT1-500 bp or pCIT-1000 bp, depending on the testing results of pCIT1-810 bp.

## Test

We acquired YNB without nitrogen source as the control medium and plan to test the promoter by GFP in
different media with a serial nitrogen source concentration. Measurements are repeated to get more
data points near the threshold concentration and the change of fluorescence over time was obtained.
The fluorescence values are normalized with OD600 and compared to the GFP fluorescence
under pTEF.
Influence of copy numbers integrated
into the genome was minimized statistically by comparing the average value of multiple fluorescent
colonies.

## Learn

In this cycle, we identified an inducible promoter that can be used for the stage control strategy in
*Yarrowia lipolytica*. With the promoter characterized, people can also apply the strategy to
many
other biomanufacturing cases in *Yarrowia lipolytica* where a controlled expression and lipid
accumulation are preferred.

With interchanges between 2 different conditions, the stage control strategy is highly associated
with
the overall design of the bioproduction process and hardwares involved, which lead to our
[Engineering
Cycle 4](#Cycle4)
, the bioreactor design.

# Cycle 3.2

## Design

With our
[Dry Lab](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
models, we perform sensitivity analysis to identify the metabolic substrates
and
pathways that affect DHA yield the most, and NADPH was found to be one of the key limiting factors.
During fatty acid synthesis, NADPH is sorely used and oxidized to NADP+ to provide the reduction
power.
Extensive fatty acid synthesis leads to the depletion of NADPH without enough replenishment.
Thus we identified an enzyme that can change NADP+ back to NADPH. It is achieved by enabling cell
metabolism to prefer using NADP+ instead of normally NAD+ during glycolysis to provide the oxidation
power.

Then we introduced the new metabolic pathway with GapC enzyme into our
[Genome-scale Metabolic
Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
and predicted the engineering outcome, which shows an increased DHA flux and turns out
to
be the most
significant improvement predicted among other potential engineering strategies. Prediction with the
[Yield Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
by adding the equation representing this pathway further verified our
optimization
strategy.
Therefore, we designed to introduce the codon-optimized GapC gene into our *Yarrowia
lipolytica*
with a constitutive TEF promoter and LIP2 terminator.

# Cycle 4

## Design

As a biomanufacturing project, the final goal is to reach an industrial-scale production of DHA with
our
engineered strain. According to cycle 3.1, a two-stage fermentation approach was adopted,
so a corresponding reactor system was proposed and modeled, which was shown on our
[Hardware
page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)
.

One of the biggest problems here is that lab-scale results cannot be directly used to calculate the
final
yield, cost and productivity, and the scale-up effect must be considered.

Our initial plan was to design and prototype the hardware for two-stage DHA production with our
strain,
however, after consulting Mr. Dhruv and Tasin, founders of
[AlGreen](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
and
[Prof. Marshal Liu](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
in HKUST
expertise
in industrial biomanufacturing, we understand that such hardware is already mature and in use in
existing facilities, which can be directly applied to our case, while it is unrealistic to redesign
an
production scale hardware without prior experience in the industry. Interviews with
[Prof. Henry Lam](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
and
[Dr. Zhu Yuanmin](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
further confirmed
this
idea.
Therefore, we decided to instead focus on modeling and testing the scale-up effect of our engineered
chassis.

## Build

Please refer to the [Hardware
page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) for more details.

## Test

Thanks to the suggestion and helps from [Prof. Marshal Liu](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) and technician Mr. Bruce
Chan, we
managed to conduct fermentation experiments with the 1 L bioreactor in the food lab under Chemical
and Biological Engineering (CBE) department, HKUST, to test the growth and lipid accumulation of our
strain under a preliminary scale-up scenario. We cultivated our engineered strain in the 1L-reactor
and obtained the growth curve with the same method described in
Cycle
1.

## Learn

From the data derived, we modified the parameters in our scale-up model and thus predicted the
conditions
required during the industrial scale operation at some ton or liter scale. Based on the results,
we further built a cost model to analyze the cost-effectiveness of our system.

# Engineering success in *E. coli*

We first successfully constructed the plasmids for expression of PPT and ORF\_C under pTEFin via SLiCE
assembly
in *E. coli* (Please refer to the [Cloning](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27cloning%27%29%20%7D%7D) page for details). The construct is
ready
to be
expressed in
*Yarrowia
lipolytica*.

![Graph 1. Gel electrophoresis result showing the correct band size after colony PCR corresponding to the pTEFin-PPT-LIP2t construct](https://static.igem.wiki/teams/5159/eng-suc-fin/new-eng-suc-img-1.png)

Graph 1. Gel electrophoresis result showing the correct band size after colony
PCR
corresponding to the pTEFin-PPT-LIP2t construct

![Graph 2. Digestion check results with restriction enzymes (BamHI+StyI) indicating the correctness of pTEFin-PPT-LIP2t constructs obtained](https://static.igem.wiki/teams/5159/eng-suc-fin/new-eng-suc-img-2.png)

Graph 2. Digestion check results with restriction enzymes (BamHI+StyI) indicating the correctness of
pTEFin-PPT-LIP2t
constructs obtained

Expression cassettes for testing the promoter pCIT1, pIDP2 and pTEF with green fluorescence protein
(GFP)
were
also successfully constructed in E. coli, and ready to be transformed into *Yarrowia
lipolytica*.

![Graph 3. Gel electrophoresis results showing the correct band size after colony PCR corresponding to the pCIT1-GFP-LIP2t construct. Please refer to our notebook for results showing success of the other 2 constructs.](https://static.igem.wiki/teams/5159/eng-suc-fin/new-eng-suc-img-3.png)

Graph 3. Gel electrophoresis results showing the correct band size after
colony
PCR
corresponding to the pCIT1-GFP-LIP2t construct. Please refer to our notebook for results showing
success
of the
other 2 constructs.

![Graph 4. Digestion check results with restriction enzymes ApaI and StyI indicating the correctness of pCIT1-GFP-LIP2t constructs obtained. Please refer to our notebook for results showing success of the other 2 constructs.](https://static.igem.wiki/teams/5159/eng-suc-fin/new-eng-suc-img-4.png)

Graph 4. Digestion check results with restriction enzymes ApaI and StyI
indicating
the
correctness of pCIT1-GFP-LIP2t constructs obtained. Please refer to our notebook for results showing
success of
the other 2 constructs.

# Transformation of *Yarrowia lipolytica*

We then demonstrated a cloning of these genes in *E. coli* and further towards the expression
and
production of
DHA in *Yarrowia lipolytica*. We are constructing all the fragments and transforming them into
*Yarrowia
lipolytica*.

So far we showed a proof of those transformations of *Yarrowia lipolytica* with the LEU2 gene
and a
successful
transformation was shown. We are still in progress transforming and testing the expression of our
constructs in
*Yarrowia lipolytica*.

![](https://static.igem.wiki/teams/5159/eng-suc/eng-suc-img-5a.jpg)

![](https://static.igem.wiki/teams/5159/eng-suc/whatsapp-image-2024-10-02-at-19-38-03.jpeg)

Graph 5. (Left) *Yarrowia lipolytica* colonies observed on the selective
leu-
YNB
plate
after transformed with the linear LEU2 marker; (Right) No colony was observed on the selective leu-
YNB
plate
with untransformed *Yarrowia lipolytica*.

# References

Reference:

[1] Young-Kyoung Park, Rodrigo Ledesma-Amaro (2023). What makes Yarrowia lipolytica well suited for
industry? Trends in Biotechnology. Volume 41, Issue 2, 242-254,
<https://doi.org/10.1016/j.tibtech.2022.07.006>.

[2] Gemperlein, K., Dietrich, D., Kohlstedt, M. et al (2019). Polyunsaturated fatty acid production
by
Yarrowia lipolytica employing designed myxobacterial PUFA synthases. Nat Commun 10, 4055.
<https://doi.org/10.1038/s41467-019-12025-8>.

[3] Sofija Jovanovic Gasovic, Demian Dietrich, Lars Gläser, Peng Cao, Michael Kohlstedt, Christoph
Wittmann (2023). Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid
catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at
the
gram scale, Metabolic Engineering, Volume 80, 45-65. <https://doi.org/10.1016/j.ymben.2023.09.003>.

[4] Ishibashi, Y., Goda, H., Hamaguchi, R. et al (2021). PUFA synthase-independent DHA synthesis
pathway
in Parietichytrium sp. and its modification to produce EPA and n-3DPA. Commun Biol 4, 1378.
<https://doi.org/10.1038/s42003-021-02857-w>.

[5] Guo P, Dong L, Wang F, Chen L and Zhang W (2022), Deciphering and engineering the
polyunsaturated
fatty acid synthase pathway from eukaryotic microorganisms. Front. Bioeng. Biotechnol. 10:1052785.
<https://doi.org/10.3389/fbioe.2022.1052785>.

[6] Hauvermale, A., Kuner, J., Rosenzweig, B. et al (2006). Fatty acid production in Schizochytrium
sp.:
Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids 41,
739–747. <https://doi.org/10.1007/s11745-006-5025-6>.

[7] J.G. Metz et al (2009). Biochemical characterization of polyunsaturated fatty acid synthesis in
Schizochytrium: Release of the products as free fatty acids. Plant Physiology and Biochemistry, 47,
472–478. <https://doi.org/10.1016/j.plaphy.2009.02.002>.

[8] Yu-Lei Jia, Fei Du, Fang-Tong Nong, Jin Li, Peng-Wei Huang, Wang Ma, Yang Gu, and Xiao-Man Sun
(2023). Function of the Polyketide Synthase Domains of Schizochytrium sp. on Fatty Acid Synthesis in
Yarrowia lipolytica. Journal of Agricultural and Food Chemistry, 71 (5), 2446-2454.
<https://doi.org/10.1021/acs.jafc.2c08383>.

[9] Qin J, Kurt E, L Bassi T, Sa L and Xie D (2023). Biotechnological production of omega-3 fatty
acids:
current status and future perspectives. Front. Microbiol. 14:1280296.
<https://doi.org/10.3389/fmicb.2023.1280296>.

[10] Xi Xie (2019). Functional analysis of ketoacyl synthase and dehydratase domains from a PUFA
synthase of Thraustochytrium in Escherichia coli and Arabidopsis thaliana. Thesis Submitted to the
College of Graduate and Postdoctoral Studies in Partial Fulfilment of the Requirements for the
Degree of
Doctor of Philosophy in the Department of Food and Bioproduct Sciences University of Saskatchewan,
Saskatoon, Saskatchewan, Canada.
<https://harvest.usask.ca/server/api/core/bitstreams/5ee0b3d7-f4d3-4980-9476-6876a967238d/content>.

[11] Wang, S., Lan, C., Wang, Z. et al (2020). PUFA-synthase-specific PPTase enhanced the
polyunsaturated fatty acid biosynthesis via the polyketide synthase pathway in Aurantiochytrium.
Biotechnol Biofuels 13, 152. <https://doi.org/10.1186/s13068-020-01793-x>.

[12] Hayashi, S., Satoh, Y., Ujihara, T. et al (2016). Enhanced production of polyunsaturated fatty
acids by enzyme engineering of tandem acyl carrier proteins. Sci Rep 6, 35441.
<https://doi.org/10.1038/srep35441>.

[13] Trujillo U, Va ́zquez-Rosa E, Oyola-Robles D, Stagg L.J, Vassallo D.A, et al (2013). Solution
Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase
Reveals
Beads-on-a-String Configuration. PLoS ONE 8(2): e57859. <https://doi.org/10.1371/journal.pone.0057859>.

[14] M. Larroude, T. Rossignol, J.-M. Nicaud, R. Ledesma-Amaro (2018). Synthetic biology tools for
engineering Yarrowia lipolytica. Biotechnology Advances, Volume 36, Issue 8, 2150-2164.
<https://doi.org/10.1016/j.biotechadv.2018.10.004>.

[15] Lizhen Cao, Mingxue Yin, Tian-Qiong Shi, Lu Lin, Rodrigo Ledesma-Amaro, Xiao-Jun Ji (2022).
Engineering Yarrowia lipolytica to produce nutritional fatty acids: Current status and future
perspectives. Synthetic and Systems Biotechnology, Volume 7, Issue 4, 1024-1033.
<https://doi.org/10.1016/j.synbio.2022.06.002>.

[16] Lynn Wong, Jake Engel, Erqing Jin, Benjamin Holdridge, Peng Xu (2017). YaliBricks, a versatile
genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metabolic
Engineering Communications, Volume 5, 68-77. <https://doi.org/10.1016/j.meteno.2017.09.001>.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}2024 HKUST iGEM DHA Express{% endblock %}
{% block page\_content %}

[• Protocols](#firstContent)
[• Lipid Profile](#Lipid extraction and lipid profile)
[• Lipid Staining](#Lipid Staining)
[• DHA Measurement](#DHA Measurement)
[• Growth Curve](#Growth Curve)
[• Western Blot](#Western Blot)
![](https://static.igem.wiki/teams/5159/claw-1.png)

![Experiment](https://static.igem.wiki/teams/5159/module-logo/experiment.png)
![](https://static.igem.wiki/teams/5159/learn-more.png)

# Protocols

|  |
| --- |
| General Protocol |
| [General Protocol](https://static.igem.wiki/teams/5159/experiment/experiment-pdf/igem2024-dha-express-general-protocols-1.pdf) |

|  |
| --- |
| Experiment Protocol |
| [Experiment Protocol](https://static.igem.wiki/teams/5159/experiment/experiment-pdf/igem2024-dha-express-experiment-protocols-1.pdf) |

# Lipid Profile

## Aim

The aim of this experiment is to assess the impact of the introduced genetic circuit of PUFA synthase on the
lipid profile,
especially the DHA accumulation, in engineered  *Y. lipolytica* using Gas Chromatography-Mass Spectrometry
(GC-MS). The lipid profile of the
non-engineered *Y. lipolytica* will be used as a control for measuring the changes in the lipid profile in
the engineered *Y. lipolytica*.

## Experimental Design

Lipid extraction:

\* All centrifugation was performed using Eppendorf Centrifuge 5702 R

* Inoculate a colony of *Y. lipolytica* from a YPD plate in 15 mL of YPD medium for 1 day (for 24-h
  culture) or 2 days (for 48-h culture) before
  the experiment date.
* Harvest 15 mL *Y. lipolytica* culture by centrifugation at 4,000 x g for 10 min at room temperature.
  Discard the supernatant.
* Wash the cells with double-distilled H2O or TE buffer twice.
* Add 4 mL chloroform, 8 mL methanol, and 3.2 mL of 1% NaCl to the pellet, vortex after every addition.
* Agitate on a tube rotator at 30 rpm overnight.
* Add 4 mL chloroform and 4 mL of 1% NaCl, invert 30 times.
* Centrifuge at 4,000 x g for 5 min, and collect the bottom layer.
* Blow dry with a gentle stream of nitrogen.
* (After gravimetric analysis,) Dissolve the lipid in 1 mL of methanol and chloroform mixture (2:1, v/v) to
  prevent DHA
  oxidation.
* Samples are stored at or below 4℃ until ready for extraction to minimize oxidation and degradation of
  samples.

Gravimetric analysis:

* Put an empty beaker in a 90℃ oven overnight and record the weight.
* Put the same empty beaker in the 90℃ oven overnight again and record the weight.
* If the difference between the weights is ±0.0005, acceptable. Take the average of the two values.
* Insert the entire sample in a beaker and blow-dry with nitrogen gas until it is almost dry.
* Put the beaker in the 90℃ oven overnight and record the weight.
* Put the same beaker in the 90℃ oven overnight again and record the weight.
* If the difference between the weights is ±0.0005, acceptable. Take the average of the two values.
* Calculate the difference between the average values from the empty beaker trial and the sample-containing
  beaker trial. This difference will be the amount of the lipid in the sample.

+ Only proceed to subsequent steps if the difference in weight is significant.

FAME (Fatty Acid Methyl Ester):

* 2 mg of the lipid sample are reconstituted in 1 mL of 0.5 M methanolic NaOH and hydrolyzed at 80℃ for 40
  min.
* Add 1 mL of fresh 10% BF3 (dissolved in methanol). Transesterification is performed at 80℃ for 40
  min.

+ Keep the temperature the same all the time.

* After transesterification, 2 mL of double-distilled H2O and 1 mL of hexane are added to the
  sample to quench the reaction.

Gas Chromatography-Mass Spectrometry (GC-MS):

* Sample preparation for testing GC-MS

+ Prepare the vials containing the transesterified and quenched lipid samples.
+ Prepare the vials containing the standard fatty acids.

* Lipid samples are measured with GC-MS (Thermo Scientific ISQ with TRACE 1300 GC-MS) to confirm peaks.

## Results

GC-MS for non-engineered *Y. lipolytica*:

* For the experiment, three fatty acid standards that are known to be abundant in
  *Y. lipolytica* were prepared, namely palmitic acid(C16:0), oleic acid(C18:1), and linoleic acid (C18:2).
  The GC-MS result of the oleic
  acid standard was omitted from the report since it was not significantly detected due to random error. Since
  all conditions for the three standards were the same, this error
  was unexpected and unable to be explained. Further replication trials for the oleic acid standard would be
  conducted. If the results from the additional trials are unclear, an
  oleic acid standard from a different manufacturer would be used.

![Figure 1. GC-MS result of Linoleic acid (C18:2) Spectrum of the transesterified linoleic acid (C18:2) sample in GC-MS. The peak of the linoleic acid standard sample is indicated as a single intense peak. ](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-1-gc-ms-result-of-linoleic-acid-c18-2.png)

Figure 1. GC-MS result of Linoleic acid (C18:2)
Spectrum of the transesterified linoleic acid (C18:2) sample in GC-MS. The peak of the linoleic acid standard
sample is indicated as a single intense peak.

![Figure 2. NIST library of linoleic acid As one of the standards, a linoleic acid sample after FAME (Fatty Acid Methyl Ester) was tested. Mass spectra of the peaks were matched against the NIST library.](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-2-nist-library-of-linoleic-acid.png)

Figure 2. NIST library of linoleic acid
As one of the standards, a linoleic acid sample after FAME (Fatty Acid Methyl Ester) was tested. Mass spectra
of the peaks were matched against the NIST library.

![Figure 3. GC-MS result of Palmitic acid (C16:0)Spectrum of the transesterified palmitic acid (C16:0) sample in GC-MS. The peak of the palmitic acid standard sample is indicated as a single intense peak.](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-3-gc-ms-result-of-palmitic-acid-c16-0.png)

Figure 3. GC-MS result of Palmitic acid (c16:0)Spectrum of the transesterified
palmitic acid (C16:0) sample in GC-MS. The peak of the palmitic acid standard sample is indicated as a single
intense peak.

![Figure 4. NIST library of palmitic acid As another standard, a palmitic acid sample after FAME (Fatty Acid Methyl Ester) was also tested. Mass spectra of the peaks were matched against the NIST library. ](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-4-nist-library-of-palmitic-acid.png)

Figure 4. NIST library of palmitic acid
As another standard, a palmitic acid sample after FAME (Fatty Acid Methyl Ester) was also tested. Mass spectra
of the peaks were matched against the NIST library.

![Figure 5. GC-MS result of lipid from Y.lipolytica cultured for 24h ](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-5-gc-ms-result-of-lipid-from-y-lipolytica-cultured-for-24h.png)

Figure 5. GC-MS result of lipid from *Y. lipolytica* cultured in YPD medium for
24h

![Figure 6. GC-MS result of lipid from Y.lipolytica cultured for 48h ](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-6-gc-ms-result-of-lipid-from-y-lipolytica-cultured-for-48h.png)

Figure 6. GC-MS result of lipid from *Y. lipolytica* cultured in YPD medium for
48h

* The GC-MS chromatograms of two standards: palmitic acid (C16:0) and linoleic acid (C18:2), were recognized
  by comparing their mass spectra to the corresponding references reported in the NIST library (Fig. 1,2,3,4).
  The presence of chlorine in the linoleic acid sample is hypothesized as the result of chloroform usage
  during the lipid extraction process. (Fig. 2)
* In the lipid extract from *Y. lipolytica* samples that were grown in YPD medium for 24 hours and 48
  hours, palmitic acid was detected.
* Based on the result, the major lipid component present in non-engineered *Y. lipolytica* was palmitic
  acid (C16:0), but linoleic acid (18:2) was not detected.
* The major limitation of this experiment is that the profile of the lipid extracted from *Y.lipolytica*
  is unclear.
* Several aspects could potentially be improved:

+ First, there were some random errors during the process of lipid extraction before GC-MS analysis, despite
  the consistent conditions.
  Certain amounts of lipid pellets might have been accidentally removed together while removing the aqueous
  layer of the centrifuged samples.
+ Second, there were some limitations in the culture medium of *Y. lipolytica*, so the lipid
  accumulation in *Y. lipolytica* was not sufficient.

* In order to measure the amount of lipid extracted from *Y. lipolytica*, another experiment of
  gravimetric analysis was
  conducted between lipid extraction and FAME to check if the lipid samples extracted from the non-engineered
  *Y. lipolytica* contained a sufficient amount of lipid.

Lipid amount - gravimetric analysis:

![OD600 measurement and gravimetric analysis result ](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/table-1-od600-measurement-and-gravimetric-analysis-result.png)

Table 1. OD600 measurement and gravimetric analysis result

![Figure 7. Lipid extract yield that is normalized to cell weight. The two-tailed P value was 0.0493, considered to be statistically significant. ](https://static.igem.wiki/teams/5159/experiment/lipid-profile-new/figure-7-lipid-extract-yield-that-is-normalized-to-cell-weight-the-two-tailed-p-value-was-0-0493-considered-to-be-statistically-significant.png)

Figure 7. Lipid extract yield that is normalized to cell weight. The two-tailed P
value was 0.0493, considered to be statistically significant.

* Since unknown bacteria were observed in culture with YNB, ampicillin was added to the YNB medium to prevent
  undesired bacteria growth.

+ Considering the potential impact of ampicillin on the growth of the *Y. lipolytica*, this result
  should be confirmed again with YNB medium without ampicillin.

* Gravimetric analysis was used to measure weight of the lipid extracted from *Y. lipolytica*.
* Result shows that YPD has higher OD600, mean cell weight, and mean lipid weight. However, their
  lipid
  weight normalized by cell weight was lower than that of YNB with ampicillin (Table 1, Fig. 7).
* Hence, it is concluded that YNB is a more advantageous candidate as a growth medium for lipid accumulation
  in *Y. lipolytica*.

## Future Plan

GC-MS for non-engineered *Y. lipolytica* (grown in YNB medium)

* Based on the results to date, we observed that *Y. lipolytica* grown in YPD medium yielded unclear
  results,
  while gravimetric analysis indicated that *Y. lipolytica* grown in YNB medium demonstrated more effective
  lipid
  accumulation.
* Therefore, a repeat experiment using *Y. lipolytica* grown in YNB medium will be conducted, as this may
  enhance
  lipid accumulation and provide a clearer control lipid profile.
* A new GC-MS for oleic acid, one of the standards, will also be conducted.

GC-MS for engineered *Y. lipolytica* (specifically measure the DHA amount)

* Standard: Pure DHA

+ Add a known quantity of DHA

* Blank: Lipid from non-engineered *Y. lipolytica*
* Spiking: Lipid from engineered *Y. lipolytica* + DHA

+ Add a known quantity of DHA

* Target: Lipid from engineered *Y. lipolytica*
* Observation of the recovery from the spike

+ Compare the peak area of the spiked DHA to a standard.

- Calculate the recovery rate using the formula:
- Recovery rate (%) = (Amount from spiking - Amount from target)/(Amount spiked) \* 100%

+ A recovery rate of 90 to 110% is considered an excellent result.
+ If recovery rate falls out of that range, other factors are affecting the result.

- < 90%:

* Recovery less than 90% could be due to the degradation of DHA by β-oxidation enzymes (e.g.,
  acyl-CoA oxidase) (Liu et al., 2021).

+ Heat-inactivate the enzymes at above 70℃.

* DHA might have been degraded due to oxidation by air.

+ Avoid storage of the samples in the open area to minimize exposure to the air.

- > 110%:

* Recovery over 110% suggests interference by contaminants. Additional DHA can be introduced by
  contaminations from chemical processing of the samples, or external sources such as manufacturing
  facilities or laboratory environments.

+ Expected results: compared to non-engineered *Y. lipolytica*, engineered *Y. lipolytica* has
  higher DHA
  amount (approximately 30%).

## References

Applegate, B. L. (2007). “Extraction, Derivatization, and Analysis of Fatty Acid Methyl Ester (FAME) in
Tissue Homogenates and Blubber by ASE and Gas Chromatography.” Short description of FAME Analysis. Applied
Science, Engineering, and Technology Laboratory, University of Alaska Anchorage.
Gemperlein, K., Dietrich, D., Kohlstedt, M., Zipf, G., Bernauer, H. S., Wittmann, C., Wenzel, S. C., & Müller,
R. (2019). Polyunsaturated fatty acid production by *Yarrowia lipolytica* employing designed
myxobacterial PUFA
synthases. Nature Communications, 10(1), 4055.
<https://doi.org/10.1038/s41467-019-12025-8>
Liu, H., Song, Y., Fan, X., Wang, C., Lu, X., & Tian, Y. (2021). Yarrowia lipolytica as an oleaginous platform
for the production of value-added fatty acid-based bioproducts.
Frontiers in Microbiology, 11. <https://doi.org/10.3389/fmicb.2020.608662>

# Lipid Staining

## Aim

The aim of this experiment is to examine the influence of media type and different nitrogen source levels, and
a peroxisome inhibitor (3-Amino-1,2,4-triazole(3-AT)) in lipid accumulation by using Nile blue and Nile Red
staining assay.

## Experimental Design:

Previous research shows that yeasts cultured in YNB (YNB-defined synthetic media) have higher lipid
accumulation levels in cells than in YPD media (Gasovic et al, 2023). Additionally, nitrogen limitation can
increase lipid
accumulation levels. However, it only compares the most commonly used YNB media (with 5g/L ammonium sulfate as
the nitrogen source) and YNB completely without the nitrogen source (Pomraning et al, 2016). Since nitrogen
source levels may also
affect the growth of the yeast, we decided to investigate the lipid accumulation levels under different
nitrogen source levels and media types.

Some studies show that inhibiting peroxisome in *Y. lipolytica* can increase lipid accumulation (Xue et
al, 2013).
3-amino-1,2,4-triazole (3-AT) has been proven to inhibit peroxisome by inhibiting the peroxisomal catalase (Ueda
et al, 2003). We
decided to add 3-AT to YPD media to investigate the lipid accumulation levels under the addition of peroxisome
inhibitor.

**Nile Blue Staining**

* *Y. lipolytica* culture in different media
  + YPD
  + YNB (with 5g/L ammonium sulfate)
* 96-Well Plate Fluorescence Assay
  + Excitation wavelength: 500 nm
  + Emission wavelength: 625 nm
  + Plate reader: FlexStation 3 Multi-Mode Microplate Reader (6 flashes per second)
  + Plate Design (Nitrogen-limiting): ![Table 2. Plate design for Nile Blue staining of Y.lipolytica cultured in YPD and YNB (with 5 g/L ammonium sulfate)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/table-2-plate-design-for-nile-blue-staining-of-y-lipolytica-cultured-in-ypd-and-ynb-with-5-g-l-ammonium-sulfate.png)

    Table 2. Plate design for Nile Blue staining of *Y. lipolytica* cultured in
    YPD and
    YNB (with 5 g/L ammonium sulfate)
  + Real plate:![Figure 8. Real plate for Nile Blue staining of Y.lipolytica cultured in YPD and YNB (with 5 g/L ammonium sulfate)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/figure-8-real-plate-for-nile-blue-staining-of-y-lipolytica-cultured-in-ypd-and-ynb-with-5-g-l-ammonium-sulfate.jpeg)

    Figure 8. Real plate for Nile Blue staining of *Y. lipolytica* cultured in
    YPD and
    YNB
    (with 5 g/L ammonium sulfate)

**Nile Red Staining**

* *Y. lipolytica* culture in different media
  + YPD
  + YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)
  + YPD (with 10mM 3-AT, a peroxisome inhibitor)
* 96-Well Plate Fluorescence Assay
  + Excitation wavelength: 485 nm
  + Emission wavelength: 535 nm
  + Plate reader: FlexStation 3 Multi-Mode Microplate Reader (6 flashes per second)
  + Plate Design (nitrogen-limiting): ![Table 3. Plate design for Nile Red staining of yeast cultured in YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/table-4-plate-design-for-nile-red-staining-of-yeast-cultured-in-ynb-with-0-1-2-3-4-5-6-7-8-9-10-g-l-ammonium-sulfate-respectively.png)

    Table 3. Plate design for Nile Red staining of *Y. lipolytica* cultured in
    YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)
  + Real plate (nitrogen-limiting):![Figure 9. Real plate for Nile Red staining of Y. lipolytica cultured in YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/figure-9-real-plate-for-nile-red-staining-of-yeast-cultured-in-ynb-with-0-1-2-3-4-5-6-7-8-9-10-g-l-ammonium-sulfate-respectively.jpeg)

    Figure 9. Real plate for Nile Red staining of *Y. lipolytica* cultured in
    YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)
  + Plate Design (peroxisome inhibitor): ![Table 4. Plate design for Nile Red staining of Y. lipolytica cultured in YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/table-3-plate-design-for-nile-red-staining-of-y-lipolytica-cultured-in-ypd-and-ypd-with-10mm-3-at-a-peroxisome-inhibitor.png)

    Table 4. Plate design for Nile Red staining of *Y. lipolytica* cultured in
    YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)
  + Real plate (peroxisome inhibitor):![Figure 10. Real plate for Nile Red staining of Y. lipolytica cultured in YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/figure-10-real-plate-for-nile-red-staining-of-y-lipolytica-cultured-in-ypd-and-ypd-with-10mm-3-at-a-peroxisome-inhibitor.jpeg)

    Figure 10. Real plate for Nile Red staining of *Y. lipolytica* cultured in
    YPD and
    YPD (with 10mM 3-AT, a peroxisome inhibitor)

## Results:

Expected Results:

* The fluorescence of *Y. lipolytica* culture in YNB media is higher than in YPD media, indicating
  higher lipid accumulation levels.
* The fluorescence of *Y. lipolytica* culture in YNB with lower nitrogen source levels is higher than
  in YNB
  with higher nitrogen source levels, indicating higher lipid accumulation levels.
* The fluorescence of *Y. lipolytica* culture in YPD with 3-AT is higher than in YPD without 3-AT,
  indicating
  higher lipid accumulation levels.

Nile Blue Staining

* The average fluorescence of *Y. lipolytica* cultured in YNB is 1.814, while 0.152 in YPD.
* *Y. lipolytica* cultures in YNB media have higher lipid accumulation levels than in YPD media.

![Figure 11. Nile Blue staining result of Y. lipolytica cultured in YPD and YNB (with 5 g/L ammonium sulfate)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/figure-11-nile-blue-staining-result-of-y-lipolytica-cultured-in-ypd-and-ynb-with-5-g-l-ammonium-sulfate.png)

Figure 11. Nile Blue staining result of *Y. lipolytica* cultured in YPD and YNB
(with 5
g/L ammonium sulfate)

Nile Red Staining of nitrogen limiting

* The differences in fluorescence between *Y. lipolytica* cultures in YNB media with different
  nitrogen source levels are minor and fluctuant, especially in the range of 1 g/L to 10 g/L.
* Based on the results, nitrogen limitation may not lead to significant changes in lipid accumulation levels
  in *Y. lipolytica*.

![Figure 12. Nile Red staining result of Y. lipolytica cultured in YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/figure-12-nile-red-staining-result-of-y-lipolytica-cultured-in-ynb-with-0-1-2-3-4-5-6-7-8-9-10-g-l-ammonium-sulfate-respectively.png)

Figure 12. Nile Red staining result of *Y. lipolytica* cultured in YNB (with 0, 1,
2, 3,
4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)

Nile Red Staining of peroxisome inhibitor

* *Y. lipolytica* cultures in YPD media (with 10mM 3-AT, a peroxisome inhibitor) have higher lipid
  accumulation levels than in YPD media.
* Inhibition of peroxisome in *Y. lipolytica* can lead to increased lipid accumulation levels.

![Figure 13. Nile Red staining result of Y. lipolytica cultured in YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)](https://static.igem.wiki/teams/5159/experiment/lipid-staining-new/figure-13-nile-red-staining-result-of-y-lipolytica-cultured-in-ypd-and-ypd-with-10mm-3-at-a-peroxisome-inhibitor.png)

Figure 13. Nile Red staining result of *Y. lipolytica* cultured in YPD and YPD
(with 10mM
3-AT, a peroxisome inhibitor)

## References

Jovanovic Gasovic, S., Dietrich, D., Gläser, L., Cao, P., Kohlstedt, M., & Wittmann, C. (2023). Multi-omics
view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases
polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale. Metabolic
Engineering, 80, 45–65.
<https://doi.org/10.1016/j.ymben.2023.09.003>
Pomraning, K. R., Kim, Y.-M., Nicora, C. D., Chu, R. K., Bredeweg, E. L., Purvine, S. O., Hu, D., Metz, T. O.,
& Baker, S. E. (2016). Multi-omics analysis reveals regulators of the response to nitrogen limitation in
Yarrowia lipolytica. BMC Genomics, 17(1), 138.
<https://doi.org/10.1186/s12864-016-2471-2>
Rakicka, M., Lazar, Z., Dulermo, T., Fickers, P., & Nicaud, J. M. (2015). Lipid production by the oleaginous
yeast Yarrowia lipolytica using industrial by-products under different culture conditions. Biotechnology for
Biofuels, 8(1), 104.
<https://doi.org/10.1186/s13068-015-0286-z>
Rostron, K. A., & Lawrence, C. L. (2017).
Nile red staining of neutral lipids in yeast. In C. Pellicciari & M. Biggiogera (Eds.), Histochemistry of
Single Molecules (Vol. 1560, pp. 219–229). Springer New York.
https://doi.org/10.1007/978-1-4939-6788-9\_16
Ueda, M., Kinoshita, H., Yoshida, T., Kamasawa, N., Osumi, M., & Tanaka, A. (2003). Effect of catalase-specific
inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo. FEMS Microbiology Letters, 219(1),
93–98.
[https://doi.org/10.1016/S0378-1097(02)01201-6](https://doi.org/10.1016/S0378-1097%2802%2901201-6)

Xue, Z., Sharpe, P. L., Hong, S.-P., Yadav, N. S., Xie, D., Short, D. R., Damude, H. G., Rupert, R. A., Seip, J.
E., Wang, J., Pollak, D. W., Bostick, M. W., Bosak, M. D., Macool, D. J., Hollerbach, D. H., Zhang, H., Arcilla,
D. M., Bledsoe, S. A., Croker, K., … Zhu, Q. (2013). Production of omega-3 eicosapentaenoic acid by metabolic
engineering of Yarrowia lipolytica. Nature Biotechnology, 31(8), 734–740.
<https://doi.org/10.1038/nbt.2622>

## Acknowledgement

We thank Biosciences Central Research Facility of HKUST (Clear Water Bay) to support the work in general
equipment core.

# DHA Measurement

## Aim

The aim of this experiment is to examine the concentration of DHA in engineered *Y. lipolytica* culture.

## Experimental Design

DHA standard curve:

* DHA has a specific absorbance peak at 205nm, which can be detected by spectrophotometers. Those absorbance
  values are linearly correlated to DHA concentration. Hence, a DHA standard curve was measured in order to
  examine the concentration of DHA in yeast culture.
* Various concentrations (1, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500 μg/mL) of the DHA sample were
  prepared, using absolute ethanol as solvent.
* The spectrum of pure DHA samples within 190-300nm was analyzed by spectrophotometer.
* A standard curve was plotted using various concentrations measured at 205nm wavelength.

\* All measurement was performed using BioDrop μLite Spectrophotometer.

## Results

* A linear curve of DHA concentration and 205nm absorbance.
* Equation: y = 0.0044x + 0.0791, R2= 0.9852

![](https://static.igem.wiki/teams/5159/experiment/dha-measurement/figure-14-dha-standard-curve-205nm.jpeg)

Figure 14. DHA Standard Curve (205nm)

## Future Plan

DHA measurement:

* Extract lipid from engineered *Y. lipolytica* culture and dissolve in absolute ethanol.
* The 205nm wavelength of the sample will be measured by a spectrophotometer.

\* All measurement was performed using BioDrop μLite Spectrophotometer.

* The DHA concentration of the sample will be calculated using the equation y = 0.0044x + 0.0791.
* If the absorbance of the sample solution is out of range (1-500 μg/mL), dilute the sample or use less
  absolute ethanol to dissolve the lipid.
* Expected result: The DHA production of engineered *Y. lipolytica* culture is around 3g/L or more.

## Reference

Sahi, A. K., Anjali, Varshney, N., Poddar, S., Vajanthri, K. Y., & Mahto, S. K. (2019). Optimizing a
detection method for estimating polyunsaturated fatty acid in human milk based on colorimetric sensors.
Materials Science for Energy Technologies, 2(3), 624–628.
<https://doi.org/10.1016/j.mset.2019.07.001>

# Growth Curve

## Aim

The aim of this experiment is to examine the growth of *Y. lipolytica* in medium with glucose versus
glycerol as
the carbon source. The recorded data is analysed to estimate the time period of each growth phase, the carrying
capacity, and the theoretical growth rate.

## Experimental Design

*Y. Lipolytica* Growth Curve:

* Yeast Y. lipolytica cell growth in liquid medium can be monitored using OD600nm.
* Cell growth is monitored at certain time points after inoculation, an spectrophotometer is used to measure
  its OD600 value.
* The measurements are plotted against time elapsed after inoculation, and the data is fitted to a logistic
  curve.
* The growth curves are compared to identify the optimal carbon source.

Conditions

General Conditions:

* 10g/L Yeast Extract
* 20g/L Peptone
* 120mL media in 1000mL conical flask
* Shaking Incubator: 30˚C ± 2; 225rpm
* Initial pH: N/A
* Initial inoculum: 2 night Y. lipolytica in YPD, washed twice with PBS
  + Final resuspension in sterile water, add 1mL to each flask
  + Same amount is added to each flask

Variable Conditions:

* 20g/L Glucose (Control)
* 20g/L Glycerol
* 40g/L Glucose
* 40g/L Glycerol

Results & Analysis:

Fit to Logistic Curve* Graph

![](https://static.igem.wiki/teams/5159/final/d737d7d5382aedf44e6402178143c65e.jpg)

Figure 15. Sept 16th Growth Curve

First Derivative :

* Estimated Maximum Growth Rate:

  | Steps | Function |
  | --- | --- |
  | Carbon Source | Maximum Growth Rate |
  | 20g/L Glucose (Control) | 0.2245 |
  | 20g/L Glycerol | 0.2410 |
  | 40g/L Glucose | 0.1840 |
  | 40g/L Glycerol | 0.2725 |
* Graph:
  ![](https://static.igem.wiki/teams/5159/final/714b499830278b1ee89bd87b72cc76a1.jpg)

Figure 16. Sept 16th First Derivative

## Results

From our results, we identified that glycerol has a higher maximum growth rate than glucose for both
concentrations. It was also observed that the cell culture reaches mid exponential phase earlier when using
glycerol as the carbon source, which agrees with our modelling results.

## Future Plan

* Repeating the Experiment
* Testing Waste Carbon Sources:

*Y. lipolytica* is known of its ability to utilize waste carbon sources for growth, such as crude glycerol
and
waste cooking oils. In the future, we hope to evaluate the efficiency of using waste carbon sources as media to
reduce the environmental impact and costs of yeast cultivation, as well as potential future work to further
optimize our production process.

# Western Blot

## Aim

The aim of this experiment is to assess the expression of each subunit of PUFA synthase.

## Experimental Design

Each subunit of PUFA synthase is attached to a His-tag, so we use a His-tag antibody as our primary antibody.
An anti-Rabbit/Mouse IgG antibody is used as our secondary antibody.

Protein Sample Preparation

* Harvest *Y. lipolytica* and lyse the cells.
* Extract proteins from *Y. lipolytica* cells.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

* Set up and run a polyacrylamide gel.

Western Blot

* Use anti-His-tag primary antibody and appropriate secondary antibody to detect the subunit of PUFA
  synthase.

## Expected Results

We will be able to detect the subunit of PUFA synthase in our engineered *Y. lipolytica*.

## Reference

Nevada. (2014). Western Blot to Examine Yeast Strains for Protein Expression. iGEM.
<https://static.igem.org/mediawiki/2014/5/52/UNR_Western_Blot_Protocol_to_detect_GFP_in_yeast.pdf>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
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---

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{% endblock %}