iGEM 2020 — Team Jiangnan_China

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# Team:Jiangnan China/Notebook

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* Project
  + [Inspiration and Description](https://2020.igem.org/Team%3AJiangnan_China/Description)
  + [Proof of Concept](https://2020.igem.org/Team%3AJiangnan_China/Proof_Of_Concept)
  + [Implementation](https://2020.igem.org/Team%3AJiangnan_China/Implementation)
* Dry Lab
  + [Model](https://2020.igem.org/Team%3AJiangnan_China/Model)
* Wet Lab
  + [Parts](https://2020.igem.org/Team%3AJiangnan_China/Parts)
  + [Engineering Success](https://2020.igem.org/Team%3AJiangnan_China/Engineering)
  + [Results](https://2020.igem.org/Team%3AJiangnan_China/Results)
* Human Practices
  + [Integrated Human Practices](https://2020.igem.org/Team%3AJiangnan_China/Human_Practices)
  + [Collaboration](https://2020.igem.org/Team%3AJiangnan_China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AJiangnan_China/Partnership)
* [Science Communication](https://2020.igem.org/Team%3AJiangnan_China/Education)
* Team
  + [Team members](https://2020.igem.org/Team%3AJiangnan_China/Team)
  + [Attribution](https://2020.igem.org/Team%3AJiangnan_China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AJiangnan_China/Notebook)
* Future
  + [Contribution](https://2020.igem.org/Team%3AJiangnan_China/Contribution)
  + [Prospect](https://2020.igem.org/Team%3AJiangnan_China/Prospects)

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* MAY
* JUN
* JUL
* AUG
* SEP
* OCT

1. Week1 MAY 1-MAY 2

Codon optimization of green fluorescent protein (GFP) gene sequence was carried out.

2. Week2 MAY 3-MAY 9

Promoter Ppgki was used to express the gene gfp. The recombinant plasmid with the gene gfp was integrated into Starmerella Bombicola. Fluorescence intensity of the recombinant strains were measured using laser scanning confocal microscope. According to the results, it was verified that GFP could be successfully expressed in S. Bombicola, which could be used as a reporter gene for promoter screening.

3. Week3 MAY 10-MAY 16

After sequence analysis of S. Bombicola whole genome, 13 promoter sequences were obtained by PCR using specific designed primers.

4. Week4 MAY 17-MAY 23

The fusion structures “promoter-GFP-terminator” (Ptef1, Ptef2, Pgpd, Peno, Ptpi and Ptdh1 ) were assembled by fusion PCR and inserted into plasmid pPHP to obtain the recombinant plasmids, which were respectively linearized and then electroporated to the wild-type S. Bombicola to obtain the recombinant strains.

5. Week5 MAY 24-MAY 30

Pfao, Ppgi, Padh1, Pcyp52m1, Pmob and Padh1 were fused with GFP by fusion PCR and inserted into the starting plasmid pPHP to obtain the recombinant plasmids, which were respectively linearized and then electroporated to the wild-type S. Bombicola to obtain the recombinant strains.

6. Week6 MAY 31

Promoters of UGTA1 and UGTB were fused with the gene gfp by fusion PCR and inserted into the starting vector pPHP to obtain the recombinant plasmids, which were linearized and then electroporated to the wild-type S. Bombicola to obtain the recombinant strains, respectively.

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# Team:Jiangnan China/Engineering

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* Project
  + [Inspiration and Description](https://2020.igem.org/Team%3AJiangnan_China/Description)
  + [Proof of Concept](https://2020.igem.org/Team%3AJiangnan_China/Proof_Of_Concept)
  + [Implementation](https://2020.igem.org/Team%3AJiangnan_China/Implementation)
* Dry Lab
  + [Model](https://2020.igem.org/Team%3AJiangnan_China/Model)
* Wet Lab
  + [Parts](https://2020.igem.org/Team%3AJiangnan_China/Parts)
  + [Engineering Success](https://2020.igem.org/Team%3AJiangnan_China/Engineering)
  + [Results](https://2020.igem.org/Team%3AJiangnan_China/Results)
* Human Practices
  + [Integrated Human Practices](https://2020.igem.org/Team%3AJiangnan_China/Human_Practices)
  + [Collaboration](https://2020.igem.org/Team%3AJiangnan_China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AJiangnan_China/Partnership)
* [Science Communication](https://2020.igem.org/Team%3AJiangnan_China/Education)
* Team
  + [Team members](https://2020.igem.org/Team%3AJiangnan_China/Team)
  + [Attribution](https://2020.igem.org/Team%3AJiangnan_China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AJiangnan_China/Notebook)
* Future
  + [Contribution](https://2020.igem.org/Team%3AJiangnan_China/Contribution)
  + [Prospect](https://2020.igem.org/Team%3AJiangnan_China/Prospects)

![](https://static.igem.org/mediawiki/2020/1/1e/T--Jiangnan_China--Title-Engineering_Success.gif)

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The core of our project is to construct a recombinant strain of *S. Bombicola* to produce the sophorolipid with our expectation. Step by step, we faced a lot of problems and failures. With careful consideration and effective improvement, finally we achieved engineering success.

↓ Please click these pictures ↓

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)](https://2020.igem.org/Team%3AJiangnan_China/Engineering#Promoters)

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Promoters Screening

Background:

We know that involvement of strong promoters in carrier construction could enhance the expression level of the corresponding genes, while weak promoters correspond to inefficient expression.[1] So, how to acquire the promoters with different transcriptional level? How to know the expression intensity of different promoters?

![](https://static.igem.org/mediawiki/2020/4/49/T--Jiangnan_China--en-1.png)

Research:

Our research focused on finding an appropriate reporter gene to express in *S. Bombicola*. Then we can define a Promoters Screening Method to measure the translation intensity of different promoters. We found that the green fluorescent protein (GFP) was usually used for a template protein to determine promoter translation intensity and was convenient to observe and test,[2] so it may be a good choice.

Imagine

The green fluorescent protein (GFP) gene can be expressed in S. bombocola and be used as reporter gene to measure the translation level of different promoters.

Design

Design the GFP expression cassettes. One promoter was used to express GFP and one terminator was used to terminate its expression.

Bulid

After the comparison and analysis of S. bombicola’ s genome. Contrast analysis was carried out with DNAMAN to obtain about 1500 bp upstream of each gene initiation codon as its functional region of promoter. By designing specific primers, the genome of *S. Bombicola* ATCC 22214 was used as the template. Gene sequences of 14 promoters were obtained from Polymerase Chain Reaction (PCR).

Amplify the GFP gene and terminator Tsyn7 using PCR.

The fusion fragment “promoter-GFP-terminator” was assembled by fusion PCR and inserted into the plasmids pPHP to obtain the recombinant plasmids. These plasmids were respectively linearized.

Click the picture to go to the Parts Pages

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Fig1. GFP expression cassette

Test

The recombinant yeast was cultured on YPD plate added hygromycin B. After 48 hours of incubation, the existed colonies would be conducted fluorescence observation and transcription level analysis. However, none of the transformants showed fluorescence and expressed mRNA.

Learn

This result might be related to low conversion efficiency. But one more important reason is that S. bombicola is eukaryote, it is complex for heterologous gene to express in it.

Improve

We needed to adopt codon optimization[4] to improve the expression level of GFP. As a result, the GFP gene was successfully heterogeneously expressed in S. bombicola.

![](https://static.igem.org/mediawiki/2020/d/d4/T--Jiangnan_China--en-3.png)

Research:

Some researches were done to find the most frequently used codon in S. bombicola.[5]

Imagine:

The green fluorescent protein (GFP) gene can be expressed in S. bombocola and be used as reporter gene to measure the translation level of different promoters.

Design

The optimum codons were used to optimize the GFP gene. Then we used one acquired promoter to express GFP in S. bombicola. If the GFP was successfully expressed in S. bombicola, then other promoters were also going to be used to express GFP, respectively.

Bulid

The 14 promoters obtained from the S. bombicola were amplified using PCR.

Obtain the GFP gene and terminator Tsyn7 by PCR.

The fusion fragment “promoter-GFP-terminator” was assembled by fusion PCR and inserted into the plasmid pPHP to obtain the recombinant plasmids. These plasmids were respectively linearized.

Test

After incubation for 48 hours, 14 kinds of transformants strains were acquired. Then through the analysis of fluorescence and transcription level, we successfully defined the intensity of different promoters.

![](https://static.igem.org/mediawiki/2020/7/73/T--Jiangnan_China--Engineering_Success-Fig.2.png)

Fig2. FITC-Mean of promoters

![](https://static.igem.org/mediawiki/2020/5/54/T--Jiangnan_China--Engineering_Success-Fig.3.png)

Fig3. RT-qPCR of promoters

Learn

The 14 promoters obtained from S. bombicola have shown different intensity. The promoter Ptef1 was the strongest promoter.

![](https://static.igem.org/mediawiki/2020/3/3e/T--Jiangnan_China--footer.png)

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# Team:Jiangnan China/Experiments

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Jiangnan China/Results

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* Project
  + [Inspiration and Description](https://2020.igem.org/Team%3AJiangnan_China/Description)
  + [Proof of Concept](https://2020.igem.org/Team%3AJiangnan_China/Proof_Of_Concept)
  + [Implementation](https://2020.igem.org/Team%3AJiangnan_China/Implementation)
* Dry Lab
  + [Model](https://2020.igem.org/Team%3AJiangnan_China/Model)
* Wet Lab
  + [Parts](https://2020.igem.org/Team%3AJiangnan_China/Parts)
  + [Engineering Success](https://2020.igem.org/Team%3AJiangnan_China/Engineering)
  + [Results](https://2020.igem.org/Team%3AJiangnan_China/Results)
* Human Practices
  + [Integrated Human Practices](https://2020.igem.org/Team%3AJiangnan_China/Human_Practices)
  + [Collaboration](https://2020.igem.org/Team%3AJiangnan_China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AJiangnan_China/Partnership)
* [Science Communication](https://2020.igem.org/Team%3AJiangnan_China/Education)
* Team
  + [Team members](https://2020.igem.org/Team%3AJiangnan_China/Team)
  + [Attribution](https://2020.igem.org/Team%3AJiangnan_China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AJiangnan_China/Notebook)
* Future
  + [Contribution](https://2020.igem.org/Team%3AJiangnan_China/Contribution)
  + [Prospect](https://2020.igem.org/Team%3AJiangnan_China/Prospects)

![](https://static.igem.org/mediawiki/2020/c/c9/T--Jiangnan_China--result-logo.gif)

Cyanobacteria degradation

We have learned that the cyanobacteria can be degraded efficiently by sophorolipid from some papers. So the cyanobacteria degradation experiment was carried out to verify its effect.

Pretest study: Determination of the appropriate content of cyanobacteria

The risen cyanobacteria were collected and were acting as the algae mother liquor, which had a concentration of 0.936 g/mL. Three groups were divided by diluting the mother liquor twice, 10 times and 20 times, respectively. Add equal amount of sophorolipids powder to the test beakers and mix up to the final content of 20 mg/L. Then observe the degradation effect of different groups.

Result1:

After 5 minutes, the cyanobacteria in group 3 have been degraded almost totally. There was also obvious phenomenon in group 1 and group 2. After 2 hours, most of the cyanobacteria have been degraded.

Summary: To achieve the adequate reaction time, the group 1 is the best choice.

![](https://static.igem.org/mediawiki/2020/6/64/T--Jiangnan_China--results-1.PNG)

Fig.1. Degradation effect of cyanobacteria after 5 minutes

Degradation process: Find a preliminary method for determining the degradation efficiency

Dilute the mother liquor 10 times and add them to two centrifuge tubes. Add 2 mL sophorolipid with a concentration of 15 mg/mL to one centrifuge tube as the test group, while the control group added equal deionized water. Shake well and then observe the degradation process. After 15 min, these two tubes were centrifuged at 7000 RPM for 2min, and then observe the phenomenon.

Result2:

The cyanobacteria have been degraded totally after adding sophorolipid with a high concentration. After centrifuging, the dead cyanobacteria settled down into precipitate, while the live cyanobacteria floated on the top.

[

]( https://static.igem.org/mediawiki/2020/3/3e/T--Jiangnan_China--results-viedio.mp4)

Video1. Cyanobacteria degradation process

Summary: Observing the precipitation and rising cyanobacteria after centrifugation can be a preliminary method for determining the degradation efficiency.

Best ratio: Test the best ratio that can maximize the degradation of cyanobacteria.

Dilute the mother liquor twice and add them to eight centrifuge tubes. Then we mixed two kinds of pure sophorolipids (both acid and lactone with the concentration of 0.991 mg/mL) to obtain mixed sophorolipids with different ratios shown in the table below. 10 μL of these combinations were added to the corresponding tubes, respectively. The degradation efficiency was measured by preliminary centrifugation observation and testing the chlorophyll content of living cells.

Result3:

Observe the amount of precipitate and rising cyanobacteria. In Fig.2, Lactone : Acid ratio of 10 : 0, 8 : 2 and 6:4 seem to have the most precipitate and least rising cyanobacteria.

![](https://static.igem.org/mediawiki/2020/8/86/T--Jiangnan_China--result-centent1-img3.png)

Fig.2. Preliminary concentration observation

Result4:

Chlorophyll in living cells was extracted with acetone. Test the absorbance of different groups at the λ=663 and λ=645 and note them as E663 and E645. Then the relative chlorophyll content can be calculated by the formula “10.115×E663-1.877×E645”.

![](https://static.igem.org/mediawiki/2020/e/e4/T--Jiangnan_China--result-centent1-table.png)

Tab.1. The relative chlorophyll content

Summary: The lactone : acid SL ratio of 8:2 has the highest efficiency on cyanobacteria degradation.

![](https://static.igem.org/mediawiki/2020/a/a7/T--Jiangnan_China--result-centent1-img5.png)

Fig.3. The degradation efficiency of cyanobacteria

Promoter Screening

Our team intends to use strong promoter to express Cas9 protein and sgRNA and use different promoters to regulate different ratios of acid and lactone sophorolipids via regulating the expression level of key gene. Therefore, we need to identify the intensities of different promoters screened before constructing the recombinant strains producing sophorolipids in this work.

Construction of GFP expression plasmid: Expression of GFP in Starmerella bombicola.

We constructed GFP expression cassette and transformed it into S. bombicola to express GFP. After incubation, observe the fluorescence (Fig 4).

![](https://static.igem.org/mediawiki/2020/f/f4/T--Jiangnan_China--result-centent1-img6.png)

![](https://static.igem.org/mediawiki/2020/1/16/T--Jiangnan_China--result-centent1-img7.png)

![](https://static.igem.org/mediawiki/2020/8/8d/T--Jiangnan_China--result-centent1-img8.png)

Fig.4. Analysis of GFP expression in S. bombicola

Result5:

GFP can be successfully expressed in Starmerella bombicola.

Promoter screening: Determination of the promoter intensity

We obtained 14 different promoters from S. bombicola and identified their functions. These promoters were used to direct the expression of GFP protein in S. bombicola. We found that the effect of these promoters on GFP expression were different, and the intensity of these promoters was also different.

Result6:

From the Fig.5. below, we can know the intensity of these 14 promoters.

![](https://static.igem.org/mediawiki/2020/2/28/T--Jiangnan_China--result-centent1-img10.png)

![](https://static.igem.org/mediawiki/2020/8/86/T--Jiangnan_China--result-centent1-img9.png)

Fig.5.Analysis promoters’ intensity by fluorescence observation and transcription level analysis

Summary: Ptef1 is the strongest promoter that can be used to express sgRNA and Cas9 protein.

CRISPR/Cas9 System

As an efficient gene editing system, CRISPR/Cas9 system was used to insert the promoters screened into specific sites of S. bombicola genome to regulate the ratio of acid and lactone sophorolipids in this work.

**NLS nuclear localization signal peptide:**Guide Cas9 through the nucleus.

Since the molecular weight of Cas9 nuclease is high, Cas9 nuclease cannot freely pass through the nuclear pores. We need to add a nuclear localization sequence (NLS) to guide it through the nucleus. Under the guidance of sgRNA, Cas9 nuclease can cut the specific sites of genome to form the double strains breaks (DSBs), and then the target gene could be engineered.

We linked the optimized NLS sequence to the 3' end of the gene gfp to construct an expression plasmid, and then transformed it into S. bombicola. The positive colonies were screened by detecting the green fluorescence intensity of GFP (Fig.6). We found that the luminescent region was concentrated in the nucleus. According to the results, it was concluded that the signal peptide can be used for the subsequent construction of the CRISPR/Cas9 system in S. bombicola.

![](https://static.igem.org/mediawiki/2020/2/2c/T--Jiangnan_China--result-centent3-img1.png)

Fig.6 The effect of the signal peptide on the subsequent construction of the CRISPR/Cas9 system in S. bombicola.

Without signal peptide (Left) and with signal peptide (Right)

Result7:

The optimized NLS can successfully guide Cas9 nuclease through the nucleus to cut the specific sites.

Determination of gene editing efficiency: Verifying the feasibility of CRISPR/Cas9 gene editing system

We carried out the verification experiments of single, double and triple gene-editing efficiency. We firstly integrated the Cas9 expression cassette into the genome as a cutting tool for gene editing, and then used the colonies containing Cas9 nuclease to verify the subsequent editing efficiency.

√ Single gene-editing efficiency

We constructed the recombinant fragment PXA1-hphex-PXA1-sgPXA1-13ex and transformed it into S. bombicola, which achieved the knockout of PXA1 gene and inserted the hygromycin resistant gene as a selection marker. The transformants were spread on YPD plus hygromycin plates. Then the positive transformants were conducted genome extraction for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the Pxa1 site.

The single gene-editing efficiency was 100%.

![](https://static.igem.org/mediawiki/2020/b/b4/T--Jiangnan_China--result-centent3-img2.png)

Fig.7 Gel electrophoresis analysis of the positive transformants

√ Double gene-editing efficiency

Secondly, we inserted GFP expression cassette into the S. bombicola genome, and obtained the positive colonies by analysing green fluorescence intensity. The positive colonies can be used as the starting strains to verify the double-gene editing efficiency. We constructed the expression fragment PXA1-hphex-PXA1-sgPXA1-13ex-linker-sg-GFP-GFP-donor, which realized the knockout of GFP genes and inserted the hygromycin resistant gene. The colonies were screened on the solid medium with hygromycin (Fig.8, Left), which also lost the function of expressing GFP (Fig.8, Right). It was verified that the double gene-editing of CRISPR/Cas9 system in S. bombicola was successful in this work.

The double gene-editing efficiency was 99%.

![](https://static.igem.org/mediawiki/2020/d/d4/T--Jiangnan_China--results-Fig8.png)

Fig. 8. Double-gene editing efficiency of CRISPR/Cas9 system in S. bombicola

√ Triple gene-editing efficiency

We also identified a gene related to synthesis of leucine, and used it as the target gene to verify the editing efficiency of CRISPR/Cas9 system in S. bombicola. It was verified that the triple-gene editing efficiency of CRISPR/Cas9 system in S. bombicola was high using the leucine synthesis gene, PXA1, and gfp (Fig9).

The triple gene-editing efficiency was 30%.

![](https://static.igem.org/mediawiki/2020/b/b8/T--Jiangnan_China--result-centent3-img5.png)

![](https://static.igem.org/mediawiki/2020/6/6e/T--Jiangnan_China--result-centent3-img6.png)

Fig.9. Triple-gene editing efficiency of CRISPR/Cas9 system in S. bombicola

Medium with leucine (Left) and minimal medium without leucine (Right)

Result8:

Single gene-editing efficiency can up to 100%, double one can up to 99% and triple one can up to 30%.

Summary: Successfully construct CRISPR/Cas9 system with high gene-editing efficiency in S. bombicola.

Production of Sophorolipids

Pure acid type and lactone type sophorolipids

The strong promoters were used to express UGTB, and as a result, the production of sophorolipids had increased. Starting with the high production-level strains, we knocked out SBLE to produce pure acid-type sophorolipid and over-expressed SBLE with strong promoters to produce pure lactone-type sophorolipid.

Result9:

The yield had boosted a lot by over-expressing UGTB. The pure acid-type and lactone-type sophorolipid had also been produced (Fig.10).

![](https://static.igem.org/mediawiki/2020/d/d7/T--Jiangnan_China--1820.png)

Fig.10. Sophorolipids yield

![](https://static.igem.org/mediawiki/2020/3/3e/T--Jiangnan_China--footer.png)

![](https://static.igem.org/mediawiki/2020/5/53/T--Jiangnan_China--Allfooter.png)