iGEM 2022 — Team uestc-biotech

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![](https://static.igem.wiki/teams/4335/wiki/notebook.png)

# Timeline

(please click the time table to explore more)

**June, 2022**

**2022-06-01~2022-06-07**

Select the team members for the experiment, wiki, human
practice,
modeling, and artwork respectively and form a team of 15 members.
We analyzed iGEM competition and shared our understanding of it collectively.
After communicating with our PI, we finally selected our topic this year----Chlipid: a highly productive
biofuel factory.

**2022-06-08~2022-06-14**

Our team members looked up and read the literature
extensively,
and
finally settled the culture, transformation, gene editing, and gene expression methods of *Chlamydomonas
reinhardtii*, as well as the according design and improvement of the Crispr/cas9 system.
Our team members did cas9 plasmid and sequence summary, gene transformation system construction, lipid gene
and the summary of protocols.

**2022-06-15~2022-06-30**

 We Started to experiment with different culture methods,
metabolic
pathway studies and CRISPR system selection for *Chlamydomonas reinhardtii*.
We designed 6 Composite Parts, they are HSP70A Promoter+3×Flag+NLS+SpCas9+NLS+Rbcs2 Term、U6 promotor+insert
site+SpScaffold+polyT Term、Rbcs2 Promotor+APHVII+Rbcs2 Term、Rbcs2 Promotor+mCherry+Rbcs2 Term、Rbcs2
Promotor+Staygold+Rbcs2 Term.
We sent the sequences we needed to Tsingke Biotechnology Co., Ltd for synthesis, and got our synthesized
sequence fragments at the end of June.

click to close

---

**July, 2022**

**2022-07-01~2022-07-07**

We designed and improved the following experiments:
*Chlamydomonas reinhardtii* culture experiment, paddle plate conservation and conservation recovery
amplification experiment, TEA electrophoresis experiment, electro-transformation and glass bead
transformation experiment, OD measurement by Thermo Fisher ELISA, and plasmid extraction protocol.
We reviewed the literature to identify genes and pathways in the metabolic pathway of *Chlamydomonas reinhardtii* that can significantly affect its lipid production.
We did liquid and solid cultures of *Chlamydomonas reinhardtii* and finalized six different stress conditions
to be performed on *Chlamydomonas reinhardtii*: N stress, Fe stress, salt stress, N+S stress, N+P stress, and S+P stress.
We found that our cultivation of *Chlamydomonas reinhardtii* was contaminated by stray bacteria, so we
disinfected our laboratory and light incubator.

**2022-07-08~2022-07-14**

 We have designed and improved the protocol for lipid
extraction.
We reviewed the literature and identified seven genes to be knocked out by the Crispr-cas9 system to promote
lipid production in *Chlamydomonas reinhardtii*: LACS1, LACS2, DTH1, CHT7, PEPC1, ACX2, PLA2, and ICL1.
We designed protocols for six stress conditions of *Chlamydomonas reinhardtii*.
Under the guidance of our advisor Xu Tang we started to build the CRISPR-Cas9 system plasmid.

**2022-07-15~2022-07-21**

 We designed experiments to measure the OD values and cell
numbers at 750 nm of *Chlamydomonas reinhardtii* at different gradient dilutions under normal culture
conditions, and worked with modeling members to establish the growth curve of *Chlamydomonas reinhardtii*
under normal cultivation.
We obtained the plasmid PZHY989 (with fluorescent protein YFP), which was kindly donated by the university
laboratory, and used it to start experimenting with glass bead transformation and electrotransformation
methods, while performing a series of experiments exploring concentration gradient screening pressure, glass
bead size, and electrotransformation parameters.
We tried enzymatic digestion and gel recovery experiments. After running the gel, we found that the striped
loop plasmid was not cut by the enzyme and the concentration of the plasmid after gel recovery was too low,
so we looked up the literature and designed different experiments, trying to solve the problem, but the
result is rather than satisfactory.

**2022-07-22~2022-07-31**

We continued the experiments of enzyme digestion and
gel
recovery, made continuous improvement to the protocol, and finalized our solution of enzyme digestion
overnight and gel recovery in the absence of gel. Finally, we obtained good results.

We Started experimenting with Nile Red staining and kept improving the protocol while observing the
staining results under fluorescence microscope.

We did pre-experiments with six groups of stress conditions. Besides, a method was devised for freezing
and preserving the seeds of *Chlamydomonas reinhardtii*.

We improved the treatment conditions in the electrotransformation protocol we learned from others, made
a second attempt, and set up different concentration gradients of screening pressure for thaumatin so as to
find the most suitable conditions for selecting algae.

We constructed the plamid which involves CRISPR-Cas9 system successfully and started to design sgRNAs.

click to close

---

**August, 2022**

**2022-08-01~2022-08-07**

We found false positives of our algae after
electrotransformation. After communicating with our PI, we decided to use a 96-well plate for the secondary
selection of our algae.

Based on the results of the plate after the second electrotransformation, we determined the screening
pressure of 25 μg/mL as the screening pressure after electrotransformation, which laid the foundation for
the later electrotransformation experiments.

The modified method of plasmid mini extraction reduces the loss of reagents.

The most suitable Nile Red staining method was explored from the variables of microwave heating time,
whether DMSO was added or not, and the staining solvent.

We finished the design of all the sgRNAs and sent them to the company for synthesis and started to
insert our sgRNAs into the insert site of our system.

**2022-08-08~2022-08-14**

We summarized the experience of electrotransformation
and designed a protocol for secondary screening and subsequent observation of fluorescence based on the
grown single algal colonies.

Due to the low conversion rate and a low number of positive clones in the early stage resulted from
electrotransformation, we retrieved the factors which influence the electrotransformation efficiency and
designed two more sets of electrotransformation protocols which were remained to be tried.

We did the in-depth drawing of the growth curve for algae of WT and algae under Fe and N stress
conditions, Nile red staining, and quantitative analysis of fluorescent images experiments.

We did the first round of fragment insertion. And we found that only a few succeeded, and the control
groups of DH5α transformation were often contaminated with spurious bacteria.

**2022-08-15~2022-08-21**

Based on the experimental results, we decided to
change the antibiotic used for antibacterial purposes from Amp to Tim. The concentration of usage was 250
μg/mL. These gestures led to the reduction of the miscellaneous bacteria.

In our constructed plasmid with empty vectors (pTX2038, pTX2039, etc.) as well as plasmid vectors that
have been combined with different gRNAs, different electrotransformation protocols were tried while
performing formal electrotransformation experiments with these materials in the hope of obtaining optimal
processing conditions.

We learned to use a fluorescence spectrophotometer and performed quantitative analysis of Nile Red
staining for a new set of stresses.

We consulted our PI and Advisor for the advice on the second round of fragment insertion, after which
process we increased the cycle number of Golden Gate assembly. Although we focused on an aseptic operation
to avoid contamination by spurious bacteria, the insertion success rate was still not high.

**2022-08-22~2022-08-31**

We purchased the Electroporation Buffer (ME-Suc): MAX
Efficiency™ Transformation Reagent (therfisher science, #A24229). Subsequently, the electrotransformation
experiments were performed according to its instructions. And the positive results were obtained, so we
decided to continue using this protocol for subsequent electrotransformation experiments.

We did a quantification of Nile Red staining results by using a fluorophotometer.

click to close

---

**September, 2022**

**2022-09-01~2022-09-07**

Positive clones were obtained by
electrotransformation and target bands were successfully verified by algal colony PCR.

In the following stage, we continuously did electrotransformation experiments.

The glass bead transformation experiment was continued and single algal colonies were found to grow in
the blank group during the glass bead process.

We did the third round of plasmid construction, this time we were more careful. Finally our success rate
was much higher though certain failure still exists.

**2022-09-08~2022-09-14**

We continuously did electrotransformation
experiments.

The glass bead conversion experiments were continued and the screening pressure of glass bead conversion
was re-executed.

**2022-09-15~2022-09-21**

Continuously doing electrotransformation experiments

**2022-09-22~2022-09-30**

Continuously doing electrotransformation experiments.

click to close

---

**October, 2022**

**2022-10-01~2022-10-07**

We organized the data and results of the stress
conditions.

We selected appropriate pictures for our various experiments.

We organized the results of transcriptome analysis and then wrote our wikipage.

**2022-10-08~2022-10-12**

We organized and analyzed our previous results, and
then wrote our wiki.

click to close

# Labnote

[Download the
file](https://static.igem.wiki/teams/4335/wiki/protocol.pdf)

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{% extends "laytest.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4335/wiki/engineering.png)

#

* [Prologue](#title_1)
* [Choosing a chassis](#title_2)
* [Constructing a genome editing system for algae](#title_3)

+ [Introduction and stimulation of CRISPR/Cas9 system](#stitle_1)
+ [The first wet lab attempt at CRISPR/Cas9 system](#stitle_2)
+ [Perfection of CRISPR/Cas9 system](#stitle_3)

* [Putting the system into practical use](#title_4)
* [Reference](#title_5)

![Back to top](https://static.igem.wiki/teams/4335/wiki/backtotop.png "Back to Top")

Prologue

---

Engineering, as commonly suspected, is to design and build certain products to meet certain needs.
Engineering a biological system is not much different. First, we shall identify our needs. The question of
*What and Why should we build* hit us way before the project started. We tried to locate a
significant topic
that can be solved by building a biological system. This year, after witnessing the deterioration of the
world situation, unusual climates appear much more frequently, and continuous fights for resources in many
areas, we chose to focus on energy issues and related climate issues.

As we have introduced in the project description, we plan to design a biological system, either based on
biological traits already present or create one from scratch, that can produce biofuels using microalgae
as chassis. Microalgae accumulate TAGs, an important storage lipid, to maintain energy homeostasis and
carbon storage. Attempts to use genome engineering to increase TAGs accumulation have been done for years,
however, there hasn't been much success due to the complexity of algal metabolic pathways.

Choosing a chassis

---

Though the aim seems hard to achieve, we are soon devoted to designing our own biological system. To start
with, we need to decide on the specific microalgae as the chassis. We consulted our PIs, who proposed we
target *Chlamydomonas reinhardtii*, the classic chassis among microalgae since it's the first time
for us
and the PIs to engineer microalgae. However, *Chlamydomonas reinhardtii* has multiple strains. After
comparing data from the Chlamydomonas Resource Center of the University of Minnesota and the Freshwater
Algae Culture Collection at the Institute of Hydrobiology of China, we chose CC-125 (the basic “137c” wild
type strain) and CC-503 (a very clean wall-deficient mutant) as candidate strains. For more than a month,
we cultured both strains in standard TAP media and drew growth curves describing the relationship between
OD750 (reflects cell number) and time. To better analyze algae cultivation efficiency, we introduced the
following formula by conducting a series of experiments and stimulations to construct the correspondence
between cell number and OD value.

We found that CC-503 has a larger population size and a longer lifespan than CC-125, not to mention that
CC-503 is wall-deficient which make it easier to be transformed. We then decided *Chlamydomonas
reinhardtii*
strain CC-503 as the final chassis.

Constructing a genome editing system for algae

---

## Introduction and stimulation of CRISPR/Cas9 system

Chosen the chassis, the next step is to select genetic methods and engineer a matching system. Based on a
review named *Innovations in improving lipid production* : Algal chemical genetics by Nishikant Wase,
we
noticed that many attempts were done with overexpression of positive regulation genes in TAGs or fatty
acids synthesis pathways but few have tried to knock down or knock out positive regulation genes in
glucose synthesis pathways or TAGs degradation pathways. Combined with our careful investigation and
previous experience of the team, we supposed this is a good year for us to dive again into CRISPR system
to see what can we achieve. Among the large Cas protein family, we selected the S. pyogenes (Sp) Cas9
endonuclease as our gene-editing tool.

The moment we chose to use CRISPR/Ca9 system, we knew there was a problem that can not be neglected,
off-target. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA
(sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial
complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively and
rely on binary classification schemes to identify strong off-targets. Therefore, we had to construct a
model that could provide a quantitative assessment of the off-target effects of different sgRNAs to guide
and offer better choices on gRNAs so that we could know precisely which gRNAs to synthesize that could
make the most out of our transformation process and guarantee gene editing efficiency. Our modelling group
made a marvellous contribution to this matter.

 They constructed a kinetic model based on thermodynamics
which can give more discriminative off-target scores for better differentiation of different gRNAs
compared to MIT and CFD scores. And the model we used can overcome the binary limitations of the previous
physics-based binary off-target predicting model and considers off-target effects with both high
possibility and low possibility. And an idea of accurately calculating the cleavage efficiency of a
certain sgRNA based on the Moran process is given by us.

## The first wet lab attempt at CRISPR/Cas9 system

### 1. Plasmids design

Although we did suspect building a knock-out system targeting algae will be difficult, the available
results were still surprisingly insufficient. We selected a CRISPR/Cas9 plasmid designed to target
photoreceptor genes in *Chlamydomonas
reinhardtii*, according to Andre Greiner, as the foundation to
engineer our own parts. The origin design of the plasmid seems to perfectly fit our needs but we want to
insure all the parts are capable to fulfil our aim of knocking out different genes located in algal
nuclear genome in vivo. Therefore, we performed codon optimization, kept 2 different promoters for gRNAs,
and replaced reporter genes with mCherry and Staygold (a newly discovered highly photostable green
fluorescent protein). pTX2038 and pTX2040 are designed.

![](https://static.igem.wiki/teams/4335/wiki/egn1p.png)
![](https://static.igem.wiki/teams/4335/wiki/egn1-1p.png)

**Figure 1.** Structure of pTX2038 and pTX2040 plasmids.

### 2. Testing the designs

We chose the commonly used electronic transformation to transform pTX2038 and pTX2040 without cloning
specific gRNAs but remaining gRNAs inserting site into algae.

![](https://static.igem.wiki/teams/4335/wiki/egn2p.png)

**Figure 2.** Selection and genetic editing efficiency of thaumatin-resistant colonies after
transformation.
**(A)** Growth of  *Chlamydomonas reinhardtii* in the plates after electrotransformation (7 days).
**Negative
control** : no plasmid was added. All dishes shown in the figure contain TAP medium supplemented with
25
µg/mL of Hyg, except for the positive control of the wild-type *Chlamydomonas reinhardtii* strain.

**(B)** Statistics of positive clones after transformation and the frequency of transformation.

![](https://static.igem.wiki/teams/4335/wiki/egn3p.png)

**Figure 3.** Gel run of samples from colony PCR.
**(A)** Sequence comparison of Cas9, HgR, and mCherry fragments in pTX2038.

**(B)** Sequence alignment of Cas9, HgR, and StayGold fragments in pTX2040. **M**: 2000bp DNA
marker; **NC**: wildtype;  **PC**: Linear plasmid. 1-3 indicate the different transformants
selected.

The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold
reporter gene  **(Figure 3A and 3B)** fragments were all consistent with the DNA bands of the positive
control
group as well as being matched with the position of DNA Marker, which indicates that the fragmented PCR
products transferred into the vector were in a high concentration and normal expression state. In sum, the
effectiveness of transformation could be testified.

To further demonstrate the correct insertion of the vector at the cellular expression level, we performed
microscopy whose centers were the autofluorescent genes contained in the construct vectors: mCherry and
StayGold. And autofluorescence in red of Chlorophyll was used as a negative control.

![](https://static.igem.wiki/teams/4335/wiki/eng4p.jpeg)

**Figure 4.** Images of WT *Chlamydomonas reinhardtii* and positive clones after transformation at
20×
magnification. **WT**: wild type; **DIC**: bright field. 60ms exposure time;  **Chlorophyll**:
EX: Form 625nm to 650nm,
10ms exposure time;  **mCherry**: EX: Form 515nm to 555nm, 300ms exposure time; **StayGold**: EX:
Form 465nm to
495nm, 300ms exposure time.

Compared with the wildtype, the cells of *Chlamydomonas reinhardtii* with fluorescent reporter gene showed
different degrees of fluorescence and normal autofluorescence expression of chlorophyll, which further
proved that the vector could be expressed normally when transferred into *Chlamydomonas reinhardtii* (**Figure4**).

## Perfection of CRISPR/Cas9 system

We learned from this first attempt that transforming CRISPR plasmids into algae is not a smooth process. Our team tried a protocol that describes a method of transforming  *Chlamydomonas reinhardtii* nuclear genome through electroporation. This scheme requires at least 3 days of work, which usually leads to small colonies within 1-2 weeks, but no single colony is successfully screened after many attempts. We struggled for months to get the result of the plasmids transformation. We began to question our design on the methods.

In order to improve the efficiency of electric conversion, we increased the amount of linear plasmid, improved the parameters of electroporation, and used electroporation buffer (ME Suc): MAX efficiency ™ Convert the reagent (Therfisher, # A24229) to make further attempts.

For the plasmids, we noticed that although we have successfully proved that Cas9 protein and the reporters were transformed into algae, we only had concrete test results on reporters. Cas9 protein hasn't had the chance to show its ability! We found that psy1 (phytoene synthase-1 gene, disruption of psy1 produces white colonies that are easy to detect and count) is a valuable and mind-blowing reporter gene which is an ideal in vivo target gene for us to attempt our first transformation towards  *Chlamydomonas reinhardtii*.

We used the tool called CRISPOR on the sgRNA design website, together with the off-target prediction model we constructed, to perform sgRNA design for psy1. The following are the test results on psy1 knock-out.

![](https://static.igem.wiki/teams/4335/wiki/egn5p.png)

**Figure 5.** The phytoene synthase gene, PSY1, was chosen as a target gene. sgRNA transcription driven by Chlamydomonas U6 promoters (CrU6) was assayed.

Comparison between wildtype *Chlamydomonas reinharditii* and transformed positive clone of PSY1 gene in bright field(**Figure 5**).

Here, we provide a scheme to verify the successful introduction and proper operation of the CRISPR/Cas system by targeting the endogenous PSY1 gene for vector construction and transformation. Moreover, by microscopic bright-field comparison of the wild type with the PSY1 mutant, if the field of view turns white, the system could prove to be feasible.

4. Putting the system into practical use

---

According to previous research, scientists have found that algae accumulate more TAGs when they are cultured in deprived conditions such as nutrition deficiency, temperature or light treatment and many researchers have conducted transcriptome analysis towards those deprived treated algae. The most use one is nitrogen deprivation, which play an important role in regulating gene expression and metabolic pathway. Iron is also an essential micronutrient for all organisms and plays an important role in the growth and metabolism of *Chlamydomonas reinhardtii*. However, research on iron stress in *Chlamydomonas reinhardtii* is relatively blank. Therefore, we chose conventional stress - nitrogen stress and novel stress - iron stress among various stresses to observe their effects on the growth and lipid production of *Chlamydomonas reinhardtii*. And we managed to identify several differential expressed genes (**Table 1**).

**Table 1.** Potential genes to promote lipid production in the four metabolic pathways.

![](https://static.igem.wiki/teams/4335/wiki/engt1p.png)

Now we managed to find some target genes by our own approach but we couldn't neglect the fruits of previous generations so we browsed more than 50 relative papers to observe more important enzymes in algal metabolic pathways that may have huge impacts on TAPs accumulation. From previous research, we selected 7 genes from 5 different pathways as possible target genes and from our transcriptome analysis results we predicted 12 genes as possible targets (**Figure 6**). We shall knock out each of them using our engineering system and then test these mutants' growth conditions and TAGs accumulations.

![](https://static.igem.wiki/teams/4335/wiki/egn6p.png)

**Figure 6.** The selected genes. The

**blue**

ones are reported genes and the

**green**

ones are the genes obtained by using transcriptome analysis.

We planned to continue drawing growth curves as done before, and use Nile red dying, lipid extraction and GC-MS to quantitively and qualitatively analyze TAG and fatty acids accumulation so that we could create engineered algae to produce a large amount of biofuel raw material in a fragmentation tank like a factory.

5. Reference

---

1. Eslami-Mossallam, B. et al. A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity. Nature communications 13, 1367, (2022).
2. Greiner, A. et al. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. The Plant cell 29, 2498-2518 (2017).
3. Wase, N., Black, P. & DiRusso, C. Innovations in improving lipid production: Algal chemical genetics. Progress in lipid research 71, 101-123 (2018).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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

![](https://static.igem.wiki/teams/4335/wiki/results.png)

#

* [Overview](#title_1)
* [Confirmation of optimal growth conditions of Chlamydomonas reinhardtii](#title_2)

+ [Basic growth curve](#stitle_1)
+ [Optimization of the aseptic culture system](#stitle_2)

* [Identification of key genes promoting lipid production in Chlamydomonas reinhardtii](#title_3)

+ [Culture and analysis of Chlamydomonas reinhardtii under different stress treatments](#stitle_3)
+ [Transcriptome sequencing analysis](#stitle_4)

* [The strategy of using genetic engineering](#title_4)

+ [The design and construction of CRISPR/Cas9 system](#stitle_5)
+ [Transformation](#stitle_6)
+ [The strategy of efficient lipid production for Chlamydomonas reinhardtii](#stitle_7)

* [Outlook](#title_5)
* [References](#title_6)

![Back to top](https://static.igem.wiki/teams/4335/wiki/backtotop.png "Back to Top")

Overview

---

Our results consist of three main parts: Firstly, confirmation of optimal growth conditions of *Chlamydomonas reinhardtii*. Secondly, observation and transcriptome analysis of *Chlamydomonas reinhardtii* under different stress conditions. Thirdly, genetic engineering of *Chlamydomonas reinhardtii* by using CRISPR/Cas9 system.

1 Confirmation of optimal growth conditions of *Chlamydomonas reinhardtii*

---

## 1.1 Basic growth curve

To confirm the **culture pattern** of *Chlamydomonas reinhardtii* in our lab, we measured the absorbance values of *Chlamydomonas reinhardtii* at 750nm every 12h to obtain the growth curve of *Chlamydomonas reinhardtii*. Then, to further confirm the relationship between absorbance values at 750nm and cell number, we used a hemocytometer plate to count *Chlamydomonas reinhardtii* with different absorbance values and obtained the **corresponding formula** between absorbance values and cell numbers.

![](https://static.igem.wiki/teams/4335/wiki/result-1.png)

**Figure 1.**Growth curve and fitted curve of *Chlamydomonas reinhardtii*.
**(A)**Growth curve of *Chlamydomonas reinhardtii*.
 **(B)**Fitted curve of *Chlamydomonas reinhardtii*.

Before 36h, *Chlamydomonas reinhardtii* grew rapidly (**Figure 1A**). After that, the growth of *Chlamydomonas reinhardtii* reached a plateau. Also, there was a correlation between the absorbance value at 750nm and cell number in *Chlamydomonas reinhardtii* (**Figure 1B**).

## 1.2 Optimization of the aseptic culture system

Keeping **sterile** is fundamental to microalgae biotechnology as well as basic scientific research. However, **contamination by bacteria and/or fungi** in microalgae cultures is often a triky problem.

Amp is the most commonly used antibiotic for bacterial inhibition when culturing *Chlamydomonas reinhardtii*. However, in the process of cultivation, even if we used Amp with a concentration three times higher than that used in the paper, the pollution of *Chlamydomonas reinhardtii* culture still remains, which seriously affects the accuracy of experimental data.

To solve this problem, we selected two other antibiotics commonly used in culturing plants: Cef and Tim, and carried out exexperimental research.

![](https://static.igem.wiki/teams/4335/wiki/result-2.png)

**Figure 2.**Growth and contamination of *Chlamydomonas reinhardtii* after 3 days of culture in TAP medium which contained **different graded concentrations of Cef, Tim and Amp**.
 **(A)**Absorbance values of *Chlamydomonas reinhardtii* at 750nm.
 **(B)**Contamination of *Chlamydomonas reinhardtii* after centrifugation.

The experimental results showed that the use of TAP medium containing Tim had little effect on the growth of *Chlamydomonas reinhardtii* (**Figure 2B**), and the use of Tim at a concentration of 250μg/mL resulted in the least contamination (**Figure 2A**). Therefore, we decided to use this condition to replace previously used 300μg/mL Amp to cultivate *Chlamydomonas reinhardtii* so as to reduce the contamination of miscellaneous bacteria under normal culture conditions.

It is worth noting that **Tim** was applied to cultivate *Chlamydomonas reinhardtii* for the first time. Here, we provide a **reference** for other teams that take *Chlamydomonas reinhardtii* as the chassis organism to **select the conditions for cultivation**, which enables them to **obtain more accurate experimental data**.

2 Identification of key genes promoting lipid production in *Chlamydomonas reinhardtii*

---

## 2.1 Culture and analysis of *Chlamydomonas reinhardtii* under different stress treatments

### 2.1.1 Selection of stress treatments

**Nutrition deficiency**, temperature and light are important factors to regulate lipid production in *Chlamydomonas reinhardtii*, among which nutrition **deficiency caused by stress treatment** has a significant effect on **promoting lipid production**.

**Nitrogen** and its compounds play an important role in regulating gene expression and metabolic pathways in *Chlamydomonas reinhardtii*. Nitrogen stress can significantly promote lipid production, and it is **the most studied and effective stress treatment ever**. **Iron** is an essential micronutrient for all organisms and plays an important role in the growth and metabolism of *Chlamydomonas reinhardtii*. Studies showed that severe Iron stress can lead to the accumulation of different types of lipids in algal cells, especially **triacylglycerol (TAGs)**, under which condition the initial growth of *Chlamydomonas reinhardtii* will not be strongly inhibited. Although Iron stress is relatively poorly studied in *Chlamydomonas reinhardtii*, it is **still a very promising treatment** for promoting its lipid production.

Therefore, we chose **conventional** Nitrogen stress and **novel** Iron stress among various stress treatments to observe their effects on growth and lipid production of *Chlamydomonas reinhardtii*.

### 2.1.2 Effects on growth and lipid production of *Chlamydomonas reinhardtii* under different stress treatments

We cultured *Chlamydomonas reinhardtii* in TAP under Nitrogen and Iron stress respectively for 48h and **performed three biological replicates to ensure data accuracy**.

To explore the effects on growth of *Chlamydomonas reinhardtii* under different stress treatments, we measured the absorbance value at 750nm on a regular basis during the culture process and used the obtained basic growth curve for comparison.

To explore the effects on lipid production of *Chlamydomonas reinhardtii* under different stress treatments, we used fluorescence microscopy to qualitatively detect the lipid content and used Image J to quantitatively analyze the lipid production status. For the staining method, we chose **Nile red staining**, which is the most commonly used method for rapid detection of lipid content in microalgae.

![](https://static.igem.wiki/teams/4335/wiki/result-3.jpg)

**Figure 3.** Growth and lipid production of *Chlamydomonas reinhardtii* under different stress treatments.
**(A)** Fluorescence microscopy image. G light (492nm-577nm) excitation, 80ms exposure time, 400 sensitivity.
 **(B)** Growth curves of the control group(TAP), Nitrogen stress group, and Iron stress group.
 **(C)** Average fluorescence intensity analysis.

In the early stage, there was no significant difference between Nitrogen stress group and the control group, but gradually the growth rate of Nitrogen stress group got lower than that of the control group, indicating that Nitrogen stress treatment had a certain inhibitory effect on the growth of *Chlamydomonas reinhardtii*. However, the growth rate of Iron stress group was constantly higher than that of the control group, indicating that Iron stress treatment did not limit the growth of *Chlamydomonas reinhardtii* (**Figure 3B**).

The average lipid content of the control group did not change with time. At 8h, the average lipid content of Nitrogen stress group and Iron stress group was 142.3% and 194.8% of that of the control group respectively. At 24h, the average lipid content under Nitrogen stress group and Iron stress group was 259.7% and 232.7% of that of the control group respectively. To conclude, in the process of culture, Iron stress treatment had a small increase in the average lipid production, while Nitrogen stress treatment showed a significantly better result than the other two groups (**Figure 3C**). The fluorescence microscopy images showed consistent findings (**Figure 3A**).

## 2.2 Transcriptome sequencing analysis

The environment lacking nutrients or essential mineral factors would cause algal cells to **undergo a series of physiological reactions**, which **involve the differential expression of a series of genes**. To **minimize environmental differences**, we chose to compare Nitrogen stress group and Iron stress group to identify genes that may affect lipid production. Previous experiments had shown that the growth of Nitrogen stress group and Iron stress group began to differ at 8h. In addition, the lipid production of Nitrogen stress group and Iron stress group was significantly different at 24h. Therefore, we sampled *Chlamydomonas reinhardtii* cultured for 8h and 24h respectively under both Nitrogen stress treatment and the Iron stress treatment for **transcriptome sequencing**.

### 2.2.1 Sequencing data statistics

The raw data from **Illumina HiSeq sequencing** was filtered to obtain the initial data of four samples, 28.31Gb clean reads in total. The percentage of Q30 bases in each sample was more than 93.14%, indicating that the error probability of base recognition was low (**Table 1**).

**Table 1.** Sequencing data statistics.

![](https://static.igem.wiki/teams/4335/wiki/result-table-1.png)

We obtained the total gene expression of each sample ([Click](https://static.igem.wiki/teams/4335/wiki/all-fpkm-density.pdf) here know more). FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) is often used as a measure of transcript or gene expression level.

### 2.2.2 Differential gene expression analysis

Gene expression is **time-specific** and **space-specific**. We performed **differential expression analysis of transcripts** under four different conditions and obtained differential gene expression results in pairs (**Table 2**). [Click](https://static.igem.wiki/teams/4335/wiki/all-deg-final.pdf) here know more.

**Table 2.** The calculation of differential expression genes.

![](https://static.igem.wiki/teams/4335/wiki/result-table-2.png)

Between the comparison of group N8h\_vs\_Fe8h and group N24h\_vs\_Fe24h, about 72% of all 5398 transcripts were differentially expressed (criteria: Fold Change≥2, FDR＜0.05), which is consistent with previous studies showing that Nitrogen or Fe stress treatment induces extensive transcriptional changes related to lipid production. Considering that the total lipid production under Nitrogen stress treatment and Fe stress treatment had obvious differences over time, we made an in-depth analysis of the reasons for the difference in the total lipid production between these two groups.

### 2.2.3 Enrichment analysis of differential gene expression

**KEGG pathway-based analysis** (**Figure 4**) revealed that differential expressed genes were mainly enriched in "**Fatty acid biosynthesis**", "**Fatty acid metabolism**", "**Nitrogen metabolism**" and "**Valine, leucine and isoleucine degradation**". Consequently, we did specific analysis of these four pathways.

![](https://static.igem.wiki/teams/4335/wiki/result-4.png)

**Figure 4.** KEGG enrichment map of differential expressed genes.
 **(A)** N8h\_vs\_Fe8h KEGG enrichment map of differentially expressed genes.
 **(B)** N24h\_vs\_Fe24h KEGG enrichment map of differentially expressed genes.

### 2.2.4 Cluster analysis of differential genes by thermography

**Hierarchical cluster analysis** was conducted on the differentially expressed genes screened from the above pathways, and the genes of the same metabolic pathway were clustered to form thermography of the clustering results of differentially expressed genes. Consequently, the target genes that significantly increased the lipid content of *Chlamydomonas reinhardtii* were selected.

![](https://static.igem.wiki/teams/4335/wiki/result-5.png)

**Figure 5.** Fatty acid biosynthesis clustering thermography.

Based on the analysis of KEGG database, 25 genes were annotated into the fatty acid pathways for synthesis, of which 13 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 3 genes with the greatest differential degree (**Figure 5**).

![](https://static.igem.wiki/teams/4335/wiki/result-6.png)

**Figure 6.** Fatty acid metabolism clustering thermography

Based on the analysis of KEGG database, 40 genes were annotated into metabolism pathway of fatty acid, of which 18 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 2 genes with the greatest differential degree (**Figure 6**).

![](https://static.igem.wiki/teams/4335/wiki/result-7.png)

**Figure 7.** Nitrogen metabolism clustering thermography.

Based on the analysis of KEGG database, 18 genes were annotated into the nitrogen metabolism pathway, of which 9 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 4 genes with the greatest differential degree (**Figure 7**).

![](https://static.igem.wiki/teams/4335/wiki/result-8.png)

**Figure 8.** Valine,leucine and isoleucine degradation clustering thermography

Based on the analysis of KEGG database, 20 genes were annotated to metabolic pathways valine,leucine and isoleucine degradation, among which 113 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 2 genes with the greatest differential degree (**Figure 8**).

**Table 3.** Potential genes to promote lipid production in the four metabolic pathways.

![](https://static.igem.wiki/teams/4335/wiki/result-table-3.png)

To sum up, based on the amount of expression, we summarized the differential expression of four pathways, and selected **12 genes** with high/low expression (**Table 3**), which can be used as the sites for subsequent regulation genes to provide target sites for gene editing of lipid production in *Chlamydomonas reinhardtii* and the resources and information of genes for basic research and application research.

3 The strategy of using genetic engineering

---

## 3.1 The design and construction of CRISPR/Cas9 system

Based on transcriptome sequencing results and previously reported genes, we aim to improve lipid production in *Chlamydomonas reinhardtii* under non-stress conditions by using **synthetic biology** and **gene editing methods**.

We designed the **CRISPR/Cas9 system** for editing  *Chlamydomonas reinhardtii* (**Figure 9**). In this system, we selected **mCherry**, which is the best available reporter gene in the plant field, and **StayGold**, a bright green fluorescent protein that has recently been shown to be highly photostable as our reporter genes and we optimized this two elements according to the codon preference of *Chlamydomonas reinhardtii*. At the same time, each vector carried an optional marker box, C. reinhardtii503 which could be granted resistance to **Hyg**. In the selection of promoters, we selected RBCS2 and HSP70A. For terminators, polyT Term and RBCS2 Term were selected.

We used **Golden Gate Assembly** and **Gibson Assembly** to construct each element into pTX2038 and pTX2040 vectors, and amplified and expressed them in *E.coli DH-5α* to obtain the basic skeleton vector. To verify the successful construction of the vector, PCR amplification was performed.

![](https://static.igem.wiki/teams/4335/wiki/result-9.jpg)

**Figure 9.** Structure of pTX2038 and pTX2040 plasmids and single colony PCR verification.
 **(A)** The structure of pTX2038.The pTX2038 contains three main components: Cas9, mCherry and HgR.
 **(B)** The structure of pTX2040.The pTX2040 contains three main components: Cas9, StayGold and HgR.
 **(C)** Cas9, StayGold, HgR sequence fragments of pTX2038 and Cas9, StayGold, HgR sequence fragments of pTX2040 were successfully amplified by single colony PCR. **M**: 2000bp DNA Marker; **Cas9, HgR, mCherry, StayGold**: fragments of corresponding elements.

The brightness of the primer fragment amplified PCR product matched with the DNA Marker marker position (**Figure 9C**), which initially proved that the sgRNA of the target gene was inserted into the vector. Subsequently, we performed **Sanger forward sequencing** and sequence comparison for each insert site to further verify the successful vector construction.

## 3.2 Transformation

### 3.2.1 The determination of optimal screening concentration

According to the specificity of the codon of *Chlamydomonas reinharditii*, our team optimized and designed a **new part** for the **ChlamyHgR** ([Part:BBa\_K4335003](http://parts.igem.org/Part%3ABBa_K4335003)) based on the existing part, and the new part acted as the selection standard for transforming positive clones. However, the inhibition effect of hygromycin varies with species. Therefore, we designed and performed experiments to determine the minimum inhibitory concentration of Hyg on the growth of *Chlamydomonas reinhardtii* in solid medium.

![](https://static.igem.wiki/teams/4335/wiki/result-10.png)

**Figure 10.** The wild type *Chlamydomonas reinharditii* CC-503 (2 x 108 cells) cells were placed on TAP solid medium supplemented with different concentrations of Hyg (10, 20 and 25µg/mL), and the growth of Chlamydomonas reinhardtii on the solid plate was continuously observed. The pictures displayed correspond to a 2-week old culture. **The experiment was repeated in three copies**.

After two weeks of incubation, we observed an **inverse relationship** between the number of colonies growing in the culture dish and the concentration of Hyg (**Figure 10**). Only in the plates with a concentration of **25µg/mL Hyg** we consistently observed inhibition of cell growth. Therefore, we decided to use this concentration in TAP media to carry out the experiments described below.

### 3.2.2 The procedure of transformation

We chose to use the method of **electrotransformation** to transform *Chlamydomonas reinhardtii*. In order to improve the efficiency of electrictransformation, we used GeneArt® MAX Efficiency® Transformation Reagent for Algae (**Therfisher, # A24229**) to carry out the experiment.

1-2 x 108 cells of algal solution and 2-4μg of linear plasmids were used in each electrotransformation. The linear pTX2038 and pTX2040 vectors were respectively electroporated into *Chlamydomonas reinhardtii* strains at **635V, 31μF, and 800Ω**. The cells were allowed to recover for 14-16 hours under the condition of continuous light and vibration treatment. Single colonies were separated on solid plates containing 25μg/mL Hygromycin. For detailed information, you can refer to our **workflow**(**Figure11**).

![](https://static.igem.wiki/teams/4335/wiki/result-11.png)

**Figure 11.** Workflow diagram summarizing steps for electrotransformation.

After 7 days, the resistant colonies which grew vigorously were obtained in the experimental group (**Figure 12A**), while no algae grew in the negative control group. The number of monoclone which had been successfully transferred into pTX2038 and pTX2040 in the plate was counted respectively and their transformation efficiencies was also calculated respectively (**Figure 12B**), after which process the editing efficiency of 1.9-2.1 x 10-6 was achieved, and **a relatively effective transformation system** of *Chlamydomonas reinhardtii* was established.

![](https://static.igem.wiki/teams/4335/wiki/result-12.png)

**Figure 12.** Selection and genetic editing efficiency of thaumatin-resistant colonies after transformation.
 **(A)** Growth of *Chlamydomonas reinhardtii* in the plates after electrotransformation (7 days). **Negative control**: no plasmid was added. All dishes shown in the figure contain TAP medium supplemented with 25µg/mL of Hyg, except for the positive control of the wild-type Chlamydomonas reinhardtii strain.
 **(B)** Statistics of positive clones after transformation ang the frequency of transformation.

### 3.2.3 Molecular test

To verify whether the transfer of the vector into 503 cells of *Chlamydomonas reinhardtii* at the **molecular level** is successful or not, we used the **colony PCR method** to ensure full integration of the vector.

![](https://static.igem.wiki/teams/4335/wiki/result-13.png)

**Figure 13.** Gel run of samples from colony PCR.
 **(A)** Sequence comparison of Cas9, HgR, and mCherry fragments in pTX2038.
 **(B)** Sequence alignment of Cas9, HgR, and StayGold fragments in pTX2040. **M**: 2000bp DNA marker; **NC**: wildtype; **PC**: Linear plasmid. **1-3 indicate different transformants selected**.

The monoalgal colonies of pTX2038 and pTX2040 have been transferred. The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold reporter gene (**Figure 13**) fragments were all consistent with the DNA bands of the positive control group as well as matched with the position of DNA Marker, which indicates that the fragmented PCR products transferred into the vector were in a high concentration and normal expression state. In sum, the effectiveness of transformation could be testified.

### 3.2.4 Fluorescent validation

To further demonstrate the correct insertion of the vector at the **cellular expression level**, we performed microscopy whose centers were the autofluorescent genes contained in the construct vectors: **mCherry** and **StayGold**. And autofluorescence in red of Chlorophyll was used as a negative control. It is worth noting that although both chlorophyll autofluorescence and mCherry will appear red after fluorescence excitation at certain wavelengths, they do not affect the observation because the wavelength range of their excitation of fluorescence does not overlap.

![](https://static.igem.wiki/teams/4335/wiki/result-14.jpg)

**Figure 14.** Images of WT *Chlamydomonas reinhardtii* and positive clones after transformation at 20x magnification. **WT**: wild type; **DIC**: bright field. 60ms exposure time; **Chlorophyll**: EX:Form 625nm to 650nm, 10ms exposure time; **mCherry**: EX:Form 515nm to 555nm, 300ms exposure time; **StayGold**: EX:Form 465nm to 495nm, 300ms exposure time.

Compared with the wildtype, the cells of *Chlamydomonas reinhardtii* with fluorescent reporter gene showed different degrees of fluorescence and normal autofluorescence expression of chlorophyll, which further proved that the vector could be expressed normally when transferred into *Chlamydomonas reinhardtii* (**Figure 14**).

## 3.3 The strategy of efficient lipid production for *Chlamydomonas reinhardtii*

### 3.3.1 Target gene selection

To promote lipid accumulation in *Chlamydomonas reinhardtii*, we based on existing reports to focus on blocking the decomposition of fatty acid, promoting the secretion of fatty acid out of the cellular, reducing the TCA cycle, reducing pathways of carbon flowing for biomass synthesis, blocking the initial stage of LD degradation, promoting the generation of the acyl glyceride, and regulating cell cycle to select a series of genes (**Figure 15**): **ACX2** (NCBI; Gene ID:5727823), **LACS1** (NCBI; Gene ID:5728486), **LACS2** (NCBI; Gene ID:5716500), **PEPC1** (NCBI; Gene ID:66056659), **ICL1** (NCBI; Gene ID:5720950, **DTH1** (NCBI; Gene ID:5722030, **PLA2** (NCBI; Gene ID:5725622, **CHT7** (NCBI;Gene ID:5722949).

Meanwhile, by transcriptome analysis, we selected a series of genes with the greatest degree of variation (**Figure 15**): **3-ACPR** (Gene ID:gene-CHLRE\_04g216950v5), **ACC** (Gene ID:gene-CHLRE\_08g359350v5), **BCCP** (Gene ID:gene-CHLRE\_17g715250v5), **Acox** (Gene ID:gene-CHLRE\_11g467350v5), **ω-6FAD** (Gene ID:gene-CHLRE\_17g711150v5), **CYP450 family** (Gene ID:gene-CHLRE\_01g007950v5), **CA** (Gene ID:gene-CHLRE\_04g223100v5), **NRT** (Gene ID:gene-CHLRE\_09g396000v5), **NiRs** (Gene ID:gene-CHLRE\_09g410750v5), **DLD** (Gene ID:gene-CHLRE\_01g016514v5), **KADHs** (Gene ID:gene-CHLRE\_06g311050v5), **MCC** (Gene ID:gene-CHLRE\_03g181200v5).

![](https://static.igem.wiki/teams/4335/wiki/result-15.png)

**Figure 15.** The selected genes. The

**blue**

ones are reported genes and the

**green**

ones are the genes obtained by using transcriptome analysis.

### 3.3.2 sgRNA design

We used the tool called **CRISPOR** on the sgRNA design website. At the same time, combined with the off-target prediction [**model**](https://2022.igem.wiki/uestc-biotech/model), the exons which have higher rankings were selected to design sgRNA, and the sequences with higher comprehensive scores were selected to design three sgRNA for each gene (**Figure 16**). For details about the primers we designed, please refer to the attachment [prime](https://static.igem.wiki/teams/4335/wiki/prime.pdf).

![](https://static.igem.wiki/teams/4335/wiki/result-16.jpg)

**Figure 16.** Schematic diagram of respective sgRNA positions of some target genes (PLA2, ICL1, CHT7).

### 3.3.3 Vector construction

We used **Golden Gate Assembly to insert the sgRNA fragments** of each target gene into the pTX2038 and pTX2040 vectors, respectively, and amplified the expression in ***E. coli DH-5α* to obtain a CRISPR/Cas9 system** that could knock out the target sequence. In order to **verify** the successful construction of the vector, **PCR amplification** was performed by the forward of target gene and downstream primers iGEM007 (pTX2038) or iGEM008 (pTX2040). Please refer to the attachment [prime](https://static.igem.wiki/teams/4335/wiki/prime.pdf) for primer sequences.

![](https://static.igem.wiki/teams/4335/wiki/result-17.jpg)

**Figure 17.** Gel run of samples from colony PCR. Primer length ~660 bp. **M**: 2000 bp DNA Marker; **38**: pTX2038 vector; **40**: pTX2040 vector; **sgRNA1-3**: different sgRNAs are designed.

The brightness of amplified primer fragments matched the position of DNA Marker (**Figure 17**), which initially **proved** that the **sgRNA** of the target gene **was inserted into the vector**. Subsequently, **Sanger forward sequencing** and **sequence alignment** were performed for each insert site to **further verify** the successful construction of the vector.

### 3.3.4 Creation and validation of PSY1 mutants

To demonstrate the integrity of the introduced vector and the proper functioning of the CRISPR/Cas9 system, we chose to use the phytoene synthase gene PSY1 as the target gene, the knockout of which will result in the cell state of *Chlamydomonas reinhardtii* being white under microscopic bright-field conditions. We successfully aspirated parts of algal solutions amplified in 96-well plates for filming and observation in the bright field of the microscope, and used wild type as a negative control (**Figure 18**).

![](https://static.igem.wiki/teams/4335/wiki/result-18.png)

**Figure 18.** Comparison between wildtype *Chlamydomonas reinharditii* and transformed positive clones in bright field.

Here, we provide a scheme to verify the successful introduction and proper operation of the CRISPR/Cas9 system by targeting the endogenous PSY1 gene for vector construction and transformation, and by microscopic bright-field comparison of the wild type with the PSY1 mutant, which proves that the system is feasible if the field of view turns white.

4 Outlook

---

Based on the results, we confirmed the effective culture system and stress conditions. Meanwhile, we completed the microalgae culture under stress conditions and transcriptome analysis of *Chlamydomonas reinhardtii*. We also discovered a series of genes with a large degree of difference. In addition, we successfully constructed a CRISPR/Cas9 knockout system and a relatively mature transformation system for *Chlamydomonas reinhardtii* . The feasibility of which was verified by using PSY1 as a reporter gene.

In the future, the following work is projected to be done.

1. Molecular biological validation of these genes' functions in the process of lipid anabolic biology was performed for genes with a large degree of variation obtained from transcriptome analysis.

2. Complete the construction of all candidate gene mutants and evaluate their effects on the lipid production capacity of *Chlamydomonas reinhardtii*.

3. Improve the overall design of the applicable system and technology, and develop an integrated strategy for multi-gene.

References

---

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