iGEM 2021 — Team Jianhua

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Jianhua/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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Jianhua/Engineering

iGEM 2021 - Jianhua

![](https://static.igem.org/mediawiki/2021/9/91/T--Jianhua--greenJianhua.png)

### Engineering

### Parts Submitted

Basic Parts:

| Part Name | Type |
| --- | --- |
| [BBa\_K4081992](http://parts.igem.org/Part%3ABBa_K4081992) | FKP |
| [BBa\_K4081996](http://parts.igem.org/Part%3ABBa_K4081996) | Lac12 |
| [BBa\_K4081998](http://parts.igem.org/Part%3ABBa_K4081998) | FucT2 |
| [BBa\_K4081995](http://parts.igem.org/Part%3ABBa_K4081995) | TEF1 promoter |
| [BBa\_K4081994](http://parts.igem.org/Part%3ABBa_K4081994) | GAP promoter |
| [BBa\_K4081990](http://parts.igem.org/Part%3ABBa_K4081990) | ADH1 promoter |
| [BBa\_K4081888](http://parts.igem.org/Part%3ABBa_K4081888) | CDT2 |

Composite Parts:

| Part Name | Type |
| --- | --- |
| [BBa\_K4081085](http://parts.igem.org/Part%3ABBa_K4081085) | pGAP-FKP-tADH1 |
| [BBa\_K4081238](http://parts.igem.org/Part%3ABBa_K4081238) | pTEF1-Lac12-tADH1 |
| [BBa\_K4081333](http://parts.igem.org/Part%3ABBa_K4081333) | pADH1-FucT2-tADH1 |

### abstract

This year, we successfully co-express three proteins(FKP, LAC12, FucT2) in Saccharomyces cerevisiae BY4741.
These three genes will work together to synthesize 2’-FL.

### Construct recombinant plasmids

1.We PCR GAP promoter([BBa\_K4081994](http://parts.igem.org/Part%3ABBa_K4081994)) from vector
PML104.
Get TEF1 promoter ([BBa\_K4081995](http://parts.igem.org/Part%3ABBa_K4081995)) from the genome of
Saccharomyces cerevisiae BY4741.
Get ADH1 promoter([BBa\_K4081990](http://parts.igem.org/Part%3ABBa_K4081990)) from vector pAUR123.

![](https://static.igem.org/mediawiki/2021/7/79/T--Jianhua--Eng_1.png)

*Figure 1. Gel electrophoresis results of PCR promoter.
Lane 1: Marker;
Lane 2: GAP promoter(700bp);
Lane 3: TEF1 promoter(577bp);
Lane 4: ADH1 promoter(461bp).*

2.The result of PCR showed that we successfully construct recombinant plasmid.

![](https://static.igem.org/mediawiki/2021/d/da/T--Jianhua--Eng_2.png)

*Figure 2. Restriction digest test result proves success.*

3.To get the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene AurR from the plasmid with
homology arms of BY4741,
we perform PCR twice, and then connect the two fragments with ligase.
The gel electrophoresis results of PCR shows we successfully get the target fragment.

![](https://static.igem.org/mediawiki/2021/b/b4/T--Jianhua--Eng_3.png)

*Figure 3. PCR the target fragment: the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene
“AurR”.
Line1: Marker; Line2-6: target fragment(11022bp).*

### Co-express three proteins(FKP, LAC12, FucT2)

1.Transform the target fragment into BY4741 by lithium acetate conversion method to integrate genes into the
genome of BY4741 for expression.
Screen for transformants by AbA-YPD selection medium.

![](https://static.igem.org/mediawiki/2021/9/98/T--Jianhua--Eng_4.png)

*Figure 4. Transformants on AbA-YPD selection medium*

2.The result of PCR showed that we successfully integrated the target fragment into the yeast genome.

![](https://static.igem.org/mediawiki/2021/e/e0/T--Jianhua--Eng_5.png)

*Figure 5. Gel electrophoresis results of PCR.
Lane 1: Marker; Lane 2: PCR product of the upstream of integration site (1797bp);
Lane 3: PCR production of the downstream of integration site (1005bp).*

3.The result of SDS-PAGE showed that we successfully co-express three proteins
FKP([BBa\_K4081992](http://parts.igem.org/Part%3ABBa_K4081992)),
LAC12([BBa\_K4081996](http://parts.igem.org/Part%3ABBa_K4081996)) and
FucT2([BBa\_K4081998](http://parts.igem.org/Part%3ABBa_K4081998)).

![](https://static.igem.org/mediawiki/2021/a/a5/T--Jianhua--Eng_6.png)

*Figure 6. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD),
LAC12(65KD), FucT2(35KD).*

To conclude, we demonstrated the engineering success of our modules through experiments.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Jianhua/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

* [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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Jianhua/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)