iGEM 2021 — Team ECUST_China

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# Team:ECUST China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:ECUST China/Engineering

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* TEAM
  + [Member](https://2021.igem.org/Team%3AECUST_China/Team)
  + [Collaborations](https://2021.igem.org/Team%3AECUST_China/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AECUST_China/Attributions)
* PROJECT
  + [Description](https://2021.igem.org/Team%3AECUST_China/Description)
  + [Design](https://2021.igem.org/Team%3AECUST_China/Design)
  + [Experiments](https://2021.igem.org/Team%3AECUST_China/Experiments)
  + [Proof Of Concept](https://2021.igem.org/Team%3AECUST_China/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AECUST_China/Contribution)
  + [Safety](https://2021.igem.org/Team%3AECUST_China/Safety)
* RESULT
  + [Engineering](https://2021.igem.org/Team%3AECUST_China/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AECUST_China/Implementation)
* MODEL
  + [Model](https://2021.igem.org/Team%3AECUST_China/Model)
  + [Hardware](https://2021.igem.org/Team%3AECUST_China/Hardware)
* [PARTS](https://2021.igem.org/Team%3AECUST_China/Parts)
* HUMAN PRACTICE
  + [Overview](https://2021.igem.org/Team%3AECUST_China/Overview)
  + [Human
    Practices](https://2021.igem.org/Team%3AECUST_China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AECUST_China/Communication)

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Engineering

“To promote the practice with theory, to revise or supplement theory with practice.”

* [Why we choose Saccharomyces
  cerevisiae S288c?](#engineering-title1)
* [Determination of the synthetic pathway](#engineering-title2)
* [Determination of gene source](#engineering-title3)
* [Determination of plasmids](#engineering-title4)
* [optimization through regulatory parts](#engineering-title5)
* [Homologous recombination](#design-title5)
* [Improvement of the thermal stability](#engineering-title6)

At the first beginning, in order to solve the two main problems that limit the development of
phycocyanin—— the poor stability and high production cost. ECUST\_China come out the idea to
synthesize
phycocyanin through rebuild the metabolism pathway on the host of yeast using synthetic biology. It
can
not only reduce the economic cost and save time but also using biosynthesis process to improve
phycocyanin’s thermostability. What we want to build is a kind of optimal foreign protein expression
system and using synthetic biology to rebuild the metabolic pathway in Saccharomyces cerevisiae
S288c.

Why we choose Saccharomyces cerevisiae S288c?

Refered to the survey results and professional suggestions, we finally choose the traditional model
organism——Saccharomyces cerevisiae among all the yeast species to meet the market’s interest. In the
past three decades, saccharomyces cerevisiae has been rebuilt to express a variety of recombinant
proteins and it has already been approved by the U.S. Food and Drug Administration (FDA) and the
European Drug Administration (EMEA) of it’s safety. Saccharomyces cerevisiae is widely used in the
food
industry and has great advantage for it’s nontoxicity property, along with it’s low demand for
oxygen,
clear genetic background, simple to operate and so on. The yeast we use is Saccharomyces cerevisiae
S288C, which is provided by Professor Cai Menghao's laboratory.

Determination of the synthetic pathway

The initial pathway we built is based on the combination of phycocyanobilin and apoprotein
(phycocyanin α subunit) which is catalyzed by the cleavage synthase. The catalytic pathway is
follows. Heme is catalyzed by the heme oxygenase to produce Biliverdin(BV IXα) with ring opening
pyrrole structure and then Biliverdin is further catalyzed by phycobilin reductase to form
phycocynobilin. The 2-3 molecules of phycocyanobilin will bind to the phycocyanin α subunit
catalyzed by the cleavage synthase and eventually produce phycocyanin. Note that cleavage synthase
has a lot family members including E/F lyase (cpcE/F), S/U lyase (cpcS/U) and t-lyase (cpcT). And
what we choose is cpcE/F to catalyze the binding of phycocyan and its reverse reaction.

In addition, our first experimental design was to use surface display system that anchors phycocyanin
protein to the cell wall and makes the yeast turn blue.But after in-depth investigation, we found
that the final location of phycocyanin α subunit and the color base—— phycocyanobilin are different
in cells. Phycocyanin α subunit will finally go to the cell wall after transmit by endoplasmic
reticulum and golgi apparatus through fuse expression with the GPI anchoring domain. While the color
base—— phycocyanobilin is a kind of small molecule pigment.
For phycocyanobilin is not a kind of protein, so it can not be directed transported through adding a
length of signaling peptide.What’s more, phycocyanobilin’s molecular weight is of 586.7g/mol which
is far from the small molecule (molecular weight of 10 times urea) ,meaning it is nearly impossible
to directly cross the membrane. Thus, the chance of the combination of phycocyanin mainly on the
cytosol and phycocyanin α subunit mainly on the cell wall is greatly reduced.

So we reconstructed the experimental pathway to express different genes in different parts of the
cell, but finally let the phycocyanin α subunit and phycocyanobilin recombine in the cytoplasm. The
expression system of phycocyanin is follows:

Heme oxygenase 1 (Hoxl encoding) and pcyA catalyze the production of biliverdin IX and
phycocyanobilin respectively in mitochondria. Then the product phycocyanobilin is transported out of
the mitochondria to the cytoplasmic matrix.The cleavage synthase (encode by cpcE and cpcF) and
phycocyanin α subunits(encode by cpcA) are generated in the endoplasmic reticulum and then enter the
cytoplasm by default. The 2-3 molecules of phycocyanobilin will bind to the phycocyanin α subuni
catalyzed by the cleavage synthase and eventually produce phycocyanin. In yeast, the original
substances are heme, heme oxygenase 1, and we introduce phycocyanin synthase (cpcE and cpcF
encoded), phycocyanin α subunit (cpcA encoded), and ferritin oxidoreductase (pcyA encoded) to
complete the whole metabolic pathway.
In yeast, the original substances are heme, heme oxygenase 1 and phycocyanin, so we need to extra
introduce phycocyanin lyase (cpcE and cpcF coding), phycocyanin a subunit (cpcA coding) and
ferredoxin oxidoreductase (pcyA coding).

![](https://static.igem.org/mediawiki/2021/1/10/T--ECUST_China--engineeringImg1.png)
![](https://static.igem.org/mediawiki/2021/3/38/T--ECUST_China--engineeringImg2.png)

Fig. Phycocyanin experimental synthesis pathway

Reference:

[1] 衣俊杰, 臧晓南, 张学成, 等. 具荧光活性的节旋藻藻蓝蛋白-亚基在大肠杆菌中的重组表达[J]. 中国海洋大学学报,
2011, 41(5): 59-62.

[2] Reuter W, Nicker-Reuter. Molecular assembly of the
phycobilisomes from the cyanobacteria Magstigocladus laminosus, J Photochem Photobiol B:Biol, 1993,
18:51～66

[3] Zhao K H, Deng M G, Zheng M, etal. Novel activity of a
phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to
phycoviobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin. FEBS
Lett, 2000,469:9～13

Determination of gene source

The phycocyanin gene we initially selected was from Galdieria phlegrea (strain 009) whose Tm equals
85 ± 1 ° C(
<https://doi.org/10.1016/j.ijbiomac.2020.02.045>).

While there are no gene nor protein database depend on the Galdieria phlegrea (strain 009). So we use
the the protein sequencing on the paper to find similar sequence on other species through BLAST on
NCBI. Unexpectedly, we found that the phycocyanin sequence is exactly the same as that from another
red algae Cyanidium caldarium and the genetic relationship between the two red algae is very close.
So we change our mind to to use the sequence from Cyanidium caldarium to build phycocyanin and only
build the α Subunits not the β Subunit.

In addition, we also need E/F lyase to catalyze the binding of phycocyanin to phycocyanin α subuni.
But the cpcE/F sequence we can find on NCBI is very limited and often incomplete. Therefore, the
genes related to cleavage synthase could not be found in the genome of Cyanidium caldarium and there
was no homologou schizoisomerase gene. However, we found cpcE/F in Paulinella chromatophora.
Although it is an amoeba, it has photosynthetic endosymbiosis from cyanobacteria.

After in-depth investigation, we found a kind of phycocyanin from the heat-resistant cyanobacteria
Synechococcus lividus PCC 6715. It is reported to withstand the high temperature of 95 ° C and we
could find the homologous cleavage isomerase gene in its genome along with all the needed genes.
Although it is a prokaryote and we don't know whether it can be successfully expressed in yeast,
with it’s excellence in heat-resistant and clear genetic information we finally choose Synechococcus
lividus PCC 6715 as our gene resources to further explore our project.

Determination of plasmids

We initially wanted to import Saccharomyces cerevisiae YPM plasmid, but
considering that this plasmid is not available in our laboratory we turned to choose pTDH3-dCas9
plasmid that was already available. After construction, it was found that the plasmid was
overwhelmed to carry all our interest genes so the transformation rate was very low.

![](https://static.igem.org/mediawiki/2021/2/2a/T--ECUST_China--engineeringImg3.png)

Fig. pTDH3-dCas9 was transformed into simple granules,
but it was not transformed successfully

Therefore, we finally selected the two plasmid systems using pTDH3-dCas9 and P426-SNR52p-gRNA
plasmids. Initially, considering our pcyA gene was introduced into the endoplasmic reticulum and the
other three genes were introduced into mitochondria. We chose to introduce pcyA into pTDH3-dCas9 and
cpcA, cpcE and cpcF into P426-SNR52p-gRNA plasmid. We found GenScript company to help us synthesize
the genes(pcyA,cpcE,cpcFand cpcE). And then we add the primers and terminators to the genes to
complete the expression vector. (For yeast is a kind of eukaryotic cell, each gene calls for a
dependent primer and terminator). When we added expression boxes to each of the genes synthesized by
the company, we found that the different position of genes on the plasmids had little effect on the
final expression. Therefore, while re-splicing the cpcA expression boxes to pTDH3-dCas9 plasmid.
Because dCas9 would affect the normal expression of our plasmid, we further chose to knock out the
dcas9 gene.
The construction of the plasmids are as follows:

![](https://static.igem.org/mediawiki/2021/4/40/T--ECUST_China--engineeringImg4.jpg)

Fig. plasmid construction of
cpcE-cpcF-p426-snr52p-grna plasmid

![](https://static.igem.org/mediawiki/2021/8/8d/T--ECUST_China--engineeringImg5.png)

Fig. construction of PcyA-CpcA plasmid

optimization through regulatory parts

The phycocyanin αsubunits encoded by cpcA and the lyase encoded by cpcE and cpcF are proteins. In the
yeast's growth period, if there exist large molecules, such as the subunit of phycocyanin. It will
definatly affect the growth of yeast then we can not get the required output of phycocyanin to
support the yeast to get blue. So we use the inducible expression system on the gene of cpcA,cpcE
and cpcF to solve the problem., thus the growth of S. cerevisiae is not affected as much as
possible.

We learned that **GAL1** is a commonly used inducible promoter in S. cerevisiae. The transcription
of GAL
genes involved in galactose metabolism in S. cerevisiae is tightly regulated. **VP16** is a
transcription-activating enzyme that is responsible for the transcription of downstream genes and
can enhance the expression of downstream genes. And **LexA** is a transcriptional regulator in
yeast,
and the protein expressed by it can bind to downstream **LexO**, thus initiating transcription of
downstream genes .

Finally we selected **Lexa-LEXO** as a pair of inductor, and started transcription with GAL1 as
the
inducer promoter also added a VP16 sequence after LexA to increase its expression.

Therefore, there are four expression vectors in the regulation of this module. The first expression
box is PGAL1-Lexa-VP16-CYC1, the second expression box is Lexo-PTDH3-CpcA-CYC1, the third expression
box is Lexo-PTDH3-CpcE-ADH1, and the fourth expression box is Lexo-PTDH3-CpcF-ADH1. At the same
time, we selected the plasmid with 2u which will greatly increase the copy number of plasmids and
finally increase the production of phycocyanin.

While we preserve the constitutive promoter GAP of pcyA for the final product of pcyA is a kind of
small molecule--phycocyanobilin which will not effect the groth of yeast. And the phycocyanobilin
can prepare for the final construction of phycocyan more efficiently for phycocyanin αsubunits call
for 2-3 phycocyanobilin to combine. The pre-synthesis of phycocyanobilin can improve the synthesis
efficiency when the other molecules (encode by cpcE,cpcA and cpcF) was induced without affecting the
growth of S. cerevisiae as much as possible.

The transformed double plasmids were as follows:

![](https://static.igem.org/mediawiki/2021/7/78/T--ECUST_China--engineeringImg6.png)

Fig. CpcE-CpcF-P426-SNR52p-gRNA plasmid
transformation

![](https://static.igem.org/mediawiki/2021/4/45/T--ECUST_China--engineeringImg7.png)

Fig. PcyA-CpcA-pTDH3 plasmid transformation

Homologous recombination

In order to further improve the stability of the yeast recombinant colony and the safety of our products, we knocked out the resistance genes on the two plasmids p426 and pTDH3 and integrated the plasmids without resistance genes into the yeast genome.

HXK2 and PTC1 genes are located on Chromosome VII and Chromosome IV in Saccharomyces cerevisiae S288C respectively. By referring to NCBI, we found that the replacement of these two genes had little influence on yeast growth and phycocyanin synthesis and its properties. We selected a sequence of these two genes (see table) and cloned them into expression box upstream in P426 and pTDH3, respectively. The cloned plasmids were digested with single enzyme and transferred into yeast. The homologous arms at both ends of the plasmid were combined with the corresponding gene sequences of yeast, and the genes on the plasmid were integrated into the yeast genome.

![](https://static.igem.org/mediawiki/2021/9/90/T--ECUST_China--designImg9.png)(Blue marked is the enzyme recognition sequence)

Improvement of the thermal stability

In Saccharomyces cerevisiae, through constructing the biosynthesis pathway of phycocyanin from
Synechococcus lividus, we have achieved the constituent biosynthesis of phycocyanin and phycocyanin
α-subunit. In order to discuss the theoretical possibility of further improve the thermal stability
of our product, site-directed mutagenesis techniques was applied to modify the key amino acid sites.
Comparing amino acid sequence of phycocyanin α-subunit between Synechococcus sp. PCC 6715 and
Synechocystis sp.PCC 6803,we found 40 different sites might related to thermal stability.

**[ Protein sequence ]**

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 10 | 15 | 20 | 25 | 30 | 35 | 40 |
| MKTP**I** | TEA**IA** | **A**AD**T**Q | GRFLS | **N**TELQ | **A**A**D**GR | **FKR**A**V** | A**SME**A |
| A**R**ALT | **N**NAQS | L**ID**GA | AQAVY | **Q**KFPY | TT**TM**Q | G**SQY**A | **STPE**G |
| K**A**KCA | RDIGY | YLR**M**V | TYCLV | AGGTG | P**M**DEY | LIAG**L** | **A**EIN**S** |
| TFDLS | PSWYV | EALKY | IKANH | GLSG**Q** | A**AV**EA | N**A**Y**I**D | YAINA | LS |

Taking Synechococcus amino acid sequence of phycocyanin α-subunit as template. The 40 different sites
were directed mutated respectively by I-Mutant website to predict protein stability changes upon
mutations. The change value of the mutant’s free energy is calculated according the formula of △△G =
△G (mutant) −△G (wild-type strain). For instance, the L (in position 115) was mutated to T because T
was predicted the most stable whose △△G equals -4.84 kcal/mol.

#### GET IN TOUCH

+86 15821261548

IGEM\_ECUST@outlook.com

ID:IGEM\_ECUST

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#### ECUST\_China

East China University of Science and Technology

Shanghai,China

[![](https://static.igem.org/mediawiki/2021/9/97/T--ECUST_China--ECUST_logo.png)](https://www.ecust.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/7/71/T--ECUST_China--bioeng_logo.png)](https://biotech.ecust.edu.cn/)

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# Team:ECUST China/Experiments

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* TEAM
  + [Member](https://2021.igem.org/Team%3AECUST_China/Team)
  + [Collaborations](https://2021.igem.org/Team%3AECUST_China/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AECUST_China/Attributions)
* PROJECT
  + [Description](https://2021.igem.org/Team%3AECUST_China/Description)
  + [Design](https://2021.igem.org/Team%3AECUST_China/Design)
  + [Experiments](https://2021.igem.org/Team%3AECUST_China/Experiments)
  + [Proof Of Concept](https://2021.igem.org/Team%3AECUST_China/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AECUST_China/Contribution)
  + [Safety](https://2021.igem.org/Team%3AECUST_China/Safety)
* RESULT
  + [Engineering](https://2021.igem.org/Team%3AECUST_China/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AECUST_China/Implementation)
* MODEL
  + [Model](https://2021.igem.org/Team%3AECUST_China/Model)
  + [Hardware](https://2021.igem.org/Team%3AECUST_China/Hardware)
* [PARTS](https://2021.igem.org/Team%3AECUST_China/Parts)
* HUMAN PRACTICE
  + [Overview](https://2021.igem.org/Team%3AECUST_China/Overview)
  + [Human
    Practices](https://2021.igem.org/Team%3AECUST_China/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AECUST_China/Communication)

![](https://static.igem.org/mediawiki/2021/8/8b/T--ECUST_China--experimentBanner.png)

Experiments

"Problem inspires us to learn, to practice, to observe."

* [Plasmid build](#experiment-title1)

  [- Get the gene fragment](#experiment-title1-1)

  [- Overlap PCR fragment connection](#experiment-title1-2)
* [Plasmid amplification and sequencing](#experiment-title2)
* [Yeast transformation](#experiment-title3)
* [Verification of phycocyanin](#experiment-title4)
* [Color-rendering experiment](#experiment-title5)

Plasmid build

First of all, according to our experimental design, we need to transfer the total of four segments of
exogenous gene. Because the single plasmid cannot carry all the exogenous gene, we need to build the
double plasmid system, which contributes to insert the gene cpcE and cpcF encoding lyase into P426
SNR52p - gRNA plasmid and insert gene cpcA that encodes alpha subunit of phycocyanin and gene pcyA
that encodes ferredoxin oxidoreductase into pTDH3 plasmid. After consulting with Professor Cai
Menghao, an expert in the study of Saccharomyces cerevisiae in our school. We borrowed plasmids from
Professor Cai Menghao's laboratory and after collaborated with Genscript Company. Genscript company
helped us construct the exogenous gene into two plasmid skeletons and conducted sequence
optimization. However, in order to make the plasmid more suitable for our experimental system, we
constructed the expression box of the plasmid by ourselves. The experimental process is as follows:

Get the gene fragment

To rebuild the plasmid , after getting the first edition of plasmid from company we finished
storage，expansion and extraction of plasmid and so on. At the same time we still need to design
primers, we chose the Primer Premier and Snapegene as Primer design software and by PCR primers
calculator to calculate the parameters such as annealing temperature. After designing the primers,
we obtained the small fragments we needed through PCR for splicing and reconstruction.

![](https://static.igem.org/mediawiki/2021/d/dd/T--ECUST_China--experimentTable1.png)

The target fragments were amplified by PCR using DNA polymerases Pfu, Taq or Prime Star, and the gel
was recovered to measure the concentration of nucleic acid fragments.

PCR segments by gel extraction:

![](https://static.igem.org/mediawiki/2021/2/29/T--ECUST_China--experimentTable2.png)

![](https://static.igem.org/mediawiki/2021/8/8d/T--ECUST_China--experimentImg1.png)**Figure: The fragments of the
constructed plasmid were recovered**

**M: Marker**
**1-4: CYC1(terminator)**
**5-8: cpcE**
**9: GAP
(promoter)**
**10-13: cpcF**
**14-17: cpcA**

![](https://static.igem.org/mediawiki/2021/b/b8/T--ECUST_China--experimentTable3.png)

![](https://static.igem.org/mediawiki/2021/9/90/T--ECUST_China--experimentImg2.png)**Figure: The fragments of the
constructed plasmid were recovered**

**M: Marker**
**1-4: ADH1(Terminator)**

![](https://static.igem.org/mediawiki/2021/a/aa/T--ECUST_China--experimentTable4.png)

![](https://static.igem.org/mediawiki/2021/7/7e/T--ECUST_China--experimentImg3.png)**Figure: The fragments of the
constructed plasmid were recovered**

**M: Marker**
**1-4: LARGE fragment of P426**

Overlap PCR fragment connection

We carry out overlap according to the map of plasmid construction and gel recovery for corresponding
fragments to measure the concentration of nucleic acid fragments. Overlap system (50 µ L) : Pfu
enzyme, two segments to be connected (1:1 mole number), plus ddH2O Supplemented with 46 µ L system,
7-10 cycles were amplified, and then P1 and P4 primers were added to amplify 22-35 cycles.

![](https://static.igem.org/mediawiki/2021/6/69/T--ECUST_China--experimentTable5.png)

![](https://static.igem.org/mediawiki/2021/5/5f/T--ECUST_China--experimentImg4.png)**Figure: link the target gene
fragment**

**M: Marker**
**1-4: GAP+cpcE+CYC1**
**5-7: GAP+cpcF**
**8:
GAP+cpcA+ADH1**

Plasmid amplification and sequencing

After the fragments have been connected, the plasmid was introduced into Escherichia coli (DH5α). 90
μ L of competent bacteria was added into each EP tube, 10 μ L of plasmid was added into Escherichia
coli, evenly beaten, glycerol was added into 20 μ L.Place in ice for 1min and in heat engine at
42.1℃ for 1.5 to 2 minutes (transformation).Since both of our plasmids carry a gene screening marker
for ampicillin resistance, the transformed E. coli is lined on a plate with ampicillin resistance.
Single colonies were selected after 12 h culturing.

![](https://static.igem.org/mediawiki/2021/a/af/T--ECUST_China--experimentImg5.png)**Figure: Growing colony is
containing
E. oil of ptDH3-DCAS9 plasmid**

![](https://static.igem.org/mediawiki/2021/2/2e/T--ECUST_China--experimentImg6.png)**Figure: The growing colony was E.
oil containing plasmid P426-SnR522-GRNA**

Meanwhile, plasmid was extracted from the colony. The colonies were subjected to PCR, with the PCR
system as shown below. Finally, electrophoresis verification, glue recovery and Sanger sequencing
were carried out.

**[Colony PCR system]**

![](https://static.igem.org/mediawiki/2021/1/1f/T--ECUST_China--experimentTable6.png)

![](https://static.igem.org/mediawiki/2021/6/61/T--ECUST_China--experimentImg7.png)

**M : Marker
1-24: cpcA\_pTDH3**

![](https://static.igem.org/mediawiki/2021/0/01/T--ECUST_China--experimentImg8.png)

**M : Marker
1-24: cpcE/F\_p426**

The gel of the 2 and 4 pore of CpcA\_pTDH3 and the gel of 8,15 and 24 pore of the CpcE /F\_p426 PCR
systems (shown on the picture above)were sent for sequencing.

Sequencing result diagram:
Comparison of results determined by the company showed that our plasmid
construction results were consistent with our design expectations.

Yeast transformation

After the early stage of the plasmid construction has been completed, we will further introduce the
dual-plasmid system into saccharomyces cerevisiae BY4741 which is the auxotroph stain of our target
host saccharomyces cerevisiae S288C . The genotype for **BY4741 is [genotype, Mata His3 Δ 1 Leu2 Δ
0
Met15 Δ 0 Ura3 Δ 0]) and there are** Ura gene in plasmid P426 SNR52p - gRNA, Leu gene in
plasmid
pTDH3, from which we can carry out the selection of the successful transformed yeast using
bideficiency medium.

Medium

General yeast medium (YPD or YEPD medium) : yeast extract 10g, protein 20g, glucose 20g, constant
volume to 1L, 2% AGAR powder can be added to become a fixed medium.Sterilization temperature: 115℃.

Saccharomyces cerevisiae screening medium (SD-URA /Leu medium) : YNB medium dry powder 6.7%.Both Met
and His mother solution were prepared 20mg/ mL with 250 µ l of mother solution per 100mL
medium.Sterilization temperature: 115℃.

![](https://static.igem.org/mediawiki/2021/d/d1/T--ECUST_China--experimentImg9.png)

Eletrotransformation

1. Put the micropluser cuvettes in the super clean bench in advance and dry
it for 3-4 hours.
2. Remove the competent cell from -80C refrigerator and melt them in palm. The
80ul competent cells and about 1ug DNA samples were mixed and transferred to a 0.2cm pre-frozen
micropluser cuvettes for ice bath for 5 min.
3. Put the micropluser cuvettes into the chute of
the rotary instrument set in Pic "mode, shock once, then quickly add 1.0m1 pre-cooled 1Mo1 /L
sorbitol solution to the micropluser cuvettes, gently blow and mix, and transfer 500U of bacterial
liquid from the micropluser cuvettes to EP tube with 500 m YPD liquid medium, and culture for about
2h on a shaking bed at 30℃ and 200 r/min.
4. Absorb an appropriate amount of the bacterial liquid
recovered from the electrical transfer in step (3), spread it on the corresponding screening plate,
and incubate it in an inverted dry incubator at 30℃ for about 2 days until the transformants visible
to the naked eye grow.

Verification of phycocyanin

After successfully transforming the double plasmid into yeast, we verified phycocyanin by SDS. After reviewing the data, we found that the molecular weight of phycocyanin was 19.7KD, and we analyze the results according to the bands.

SDS PAGE Protocol:
1. Make the separating gel and stacking gel.
2. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.
3. Prepare the samples.Mix your samples with sample buffer (loading buffer).Heat them in boiling water for 5-10 min.
4. Load prepared samples into wells and make sure not to overflow. Loading protein marker into the first lane. Then cover the top and connect the anodes.
5. Set a volt and run the electrophoresis when everything's done.Generally, about 1 hour for a 120V voltage and a 12% separating gel.
6. Immerse the gel with staining solution,and slowly shake it on horizontal rotator for about 20-30min.
7. Immerse the gel in destaining solution and put it on the same shaker for about 20-30 mins.Change the destaining solution for 3-5 times untill you can see clear bands with almost no blue background.
8. 100ml fixing solution : 50 % Methanol, 10% Acidic Acid, 50 µl Formalaldehyde for one hour to overnight.
9. Wash gel with 50 % ethanol for three times (no less than 20 minutes each).
10.Treat gel for 1min with hypo solution (Sodium Thiosulfate solution 20 mg/100 mL). SAVE 2 ml FOR LATER STEP.Over treating with the hypo solution will result in darker gel in the end.
11. Wash with water for three times (20 seconds each).
12. Treat gel with Silver Nitrite solution (200 mg/100 mL) for 30 minutes.Over treating will result in unwanted artifacts in final stage.
13. Wash with water for three times (20 seconds each).
14. Develop gel in 100 mL developing solution (6 g Sodium Carbonate, 2 mL of the hypo solution, 50 µl Formalaldehyde). This step can take from one minute to 30 minutes.
15.Once developed, stop with 5% Acidic Acid. Once We saw the bands, stopped developing the gel immediately.Store gel in fixing solution.

Color-rendering experiment

We carry out color-rendering experiment to provide basic data for the modeling group, and these data provide support for the construction of color-rendering model.

![](https://static.igem.org/mediawiki/2021/8/86/T--ECUST_China--experimentImg10.png)

Experimental steps:
1. 325 mg/L phycocyanin mother solution 20 mL was prepared
2. Phycocyanin solutions with different concentrations were prepared according to the table below, and 3 centrifuges were prepared for each concentration, which were evenly divided into groups A, B and C

![](https://static.igem.org/mediawiki/2021/a/ad/T--ECUST_China--experimentTable7.png)

3. Three sets of centrifuge tubes A, B and C were immersed in constant temperature water bath at 30℃, 40℃ and 50℃ for 40min respectively
4. Remove the centrifuge tube and shake for 3 s
5. Take 20 μL solution from each centrifuge tube to the plate, add equal volume of deionized water to the first well in each row as blank control, and measure the absorbance value of the solution at 614 nm wavelength with a microplate reader
6. The measured absorbance value minus the absorbance value of the peer deionized water is the final absorbance value of phycyanin solution

Experimental results:

![](https://static.igem.org/mediawiki/2021/d/dc/T--ECUST_China--experimentTable8.png)

#### GET IN TOUCH

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ID:IGEM\_ECUST

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#### ECUST\_China

East China University of Science and Technology

Shanghai,China

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# Team:ECUST China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)