iGEM 2019 — Team GZHS-United

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:GZHS-United/Notebook

[![](https://static.igem.org/mediawiki/2019/e/e8/T--GZHS-United--logo-1.png)](https://2019.igem.org/Team%3AGZHS-United)

* Team
* Project
* Parts
* [Safety](https://2019.igem.org/Team%3AGZHS-United/Safety)
* Human Practices
* Awards

* [Team Members](https://2019.igem.org/Team%3AGZHS-United/Team)
* [Collaborations](https://2019.igem.org/Team%3AGZHS-United/Collaborations)

* [Description](https://2019.igem.org/Team%3AGZHS-United/Description)
* [Design](https://2019.igem.org/Team%3AGZHS-United/Design)
* [Experiments](https://2019.igem.org/Team%3AGZHS-United/Experiments)
* [Notebook](https://2019.igem.org/Team%3AGZHS-United/Notebook)
* [Contribution](https://2019.igem.org/Team%3AGZHS-United/Contribution)
* [Results](https://2019.igem.org/Team%3AGZHS-United/Results)
* [Demonstrate](https://2019.igem.org/Team%3AGZHS-United/Demonstrate)
* [Improve](https://2019.igem.org/Team%3AGZHS-United/Improve)
* [Attributions](https://2019.igem.org/Team%3AGZHS-United/Attributions)

* [Parts Overview](https://2019.igem.org/Team%3AGZHS-United/Parts)
* [Basic Part](https://2019.igem.org/Team%3AGZHS-United/Basic_Part)
* [Composite Part](https://2019.igem.org/Team%3AGZHS-United/Composite_Part)
* [Part Collection](https://2019.igem.org/Team%3AGZHS-United/Part_Collection)

* [Human Practices](https://2019.igem.org/Team%3AGZHS-United/Human_Practices)
* [Education & Engagement](https://2019.igem.org/Team%3AGZHS-United/Public_Engagement)

* [Model](https://2019.igem.org/Team%3AGZHS-United/Model)

## Notebook

#### 2019.7.20

Configure TBE, liquid LB and EDTA

#### 2019.7.21

Gene PCR amplification

#### 2019.7.22

1. The PCR products were subjected to agarose gel electrophoresis, gel recovery and product purification

2. Amplified Arabidopsis APX

#### 2019.7.23

1. Fragment recovery of arabidopsis APX

2. Enzyme cutting target segments

3. Extract plasmid pet28a-rpij

#### 2019.7.24

1. Enzyme digestion vector was tested by electrophoresis and the enzyme digestion product was purified again

2. Enzyme linkage vector and target fragment

#### 2019.7.25

Heat shock transformation of enzyme coupling products, plate culture (no colony was grown, and the analysis should be the wrong enzyme cutting site in the primer)

#### 2019.7.26

Re-amplification of APX(unamplified bands)

#### 2019.7.27

Re-enzyme linked plasmid to target fragment, reheated transformation, coated plate.

#### 2019.7.28

1. No bands were visible on electrophoresis today (colony PCR and PCR purified products were marker). After replacing the dye, streaks were found on electrophoresis. After analysis, it is considered that the SYBR Green dye packed in small tubes may have problems, and the dye should be replaced on the second day.

#### 2019.7.29

1. Replace the nucleic acid dye and repeat yesterday's colony PCR experiment

2. Pet28a-rpij plasmid was tested

#### 2019.7.30

1. Check whether there is any problem with the plasmid. Pet28a-rpij is correct after re-sequencing

#### 2019.7.31

1. Reprepare 5xTBE, use imidazole and Na2HPO4, prepare loading buffer, 0.22um filter membrane, and filter PBS buffer.

#### 2019.8.5

1. Heat shock transformation of pet28a-apx3-1 (zooxanthellae), the receptive state used was DE3, and the next day, it was small shake (no bacteria) according to the situation.

2. Colony PCR (no positive bands) was performed for T4 junction products.

#### 2019.8.6

1. Preparation of plasmid maxiprep

2. Reheat control plasmid and APX3-1

3. The vector pet28a-apx (arabidopsis thaliana) -his was constructed

#### 2019.8.7

1. Bacteria culture of pet28a-apx3-1 (zooxanthellae)

2. Pet28a-apx (arabidopsis thaliana) plasmid was extracted

#### 2019.8.18

0. Sterilization of all required materials (tri-steam water, blue nozzle, large nozzle, blue cover centrifuge tube, glass tube, 2-YT)

1. Parts： shake five bacteria (1O,8P,9L,10C+pSB1C3), two tubes each, 5mL LB (Chl) each

2. There are already zooxanthellae APX, arabidopsis APX, liquid which contain cultivated bacterias, and sterilizing sealed 500mL conical flask (using four), each of which can shake 150mL 2-yt (Kana) and inoculate 1.5ml bacterial liquid

→ apx3-1 cultivate again, other samples have been induced completely → apx3-1 is inducing overnight (8.20.)

→ measure precipitation and supernatant, using a large colorimetric cup

5. Decolorization and review of PAGE glue samples, it seems that there is a certain amount of exogenous protein expression in precipitation and supernatant

#### 2019.8.19

1. Parts plasmid extraction, the concentration is almost the same as that extracted yesterday, the reason is unclear (kit?) (pollution?)

2.2-YT preparation (ZMX)

3.LB (Chl) prepare (DZH) on plate

Heat shock transformation 9L, 10C to BL21, heat shock transformation apx3-1, apx3-2 to BL21 and DE3)

5. Heat shock transformation 10L to BL21, 8P, heat shock transformation apx3-3, arabidopsis APX to BL21 and DE3

#### 2019.8.20

1. Take precipitation and supernatant to measure, using a large colorimetric cup to do APX enzyme activity

2. Four parts plasmids were sent to test in the morning. Only testing one reaction to exclude the possibility of miscellaneous bacteria

3. The united gold award parts primers are checked and gived to the company for synthesis

4. Cultivate the bacteria according to the growth of bacteria, or re-transform the coating plate

From August to September, we continue doing Prokaryotic protein expression and measurement of enzyme activity.

© 2019 Team:GZHS-United
[IGEM](https://2019.igem.org/Team%3AGZHS-United)

*arrow\_upward*


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:GZHS-United/Experiments

[![](https://static.igem.org/mediawiki/2019/e/e8/T--GZHS-United--logo-1.png)](https://2019.igem.org/Team%3AGZHS-United)

* Team
* Project
* Parts
* [Safety](https://2019.igem.org/Team%3AGZHS-United/Safety)
* Human Practices
* Awards

* [Team Members](https://2019.igem.org/Team%3AGZHS-United/Team)
* [Collaborations](https://2019.igem.org/Team%3AGZHS-United/Collaborations)

* [Description](https://2019.igem.org/Team%3AGZHS-United/Description)
* [Design](https://2019.igem.org/Team%3AGZHS-United/Design)
* [Experiments](https://2019.igem.org/Team%3AGZHS-United/Experiments)
* [Notebook](https://2019.igem.org/Team%3AGZHS-United/Notebook)
* [Contribution](https://2019.igem.org/Team%3AGZHS-United/Contribution)
* [Results](https://2019.igem.org/Team%3AGZHS-United/Results)
* [Demonstrate](https://2019.igem.org/Team%3AGZHS-United/Demonstrate)
* [Improve](https://2019.igem.org/Team%3AGZHS-United/Improve)
* [Attributions](https://2019.igem.org/Team%3AGZHS-United/Attributions)

* [Parts Overview](https://2019.igem.org/Team%3AGZHS-United/Parts)
* [Basic Part](https://2019.igem.org/Team%3AGZHS-United/Basic_Part)
* [Composite Part](https://2019.igem.org/Team%3AGZHS-United/Composite_Part)
* [Part Collection](https://2019.igem.org/Team%3AGZHS-United/Part_Collection)

* [Human Practices](https://2019.igem.org/Team%3AGZHS-United/Human_Practices)
* [Education & Engagement](https://2019.igem.org/Team%3AGZHS-United/Public_Engagement)

* [Model](https://2019.igem.org/Team%3AGZHS-United/Model)

## A. Experiment

#### 1. Selection of research objects

According to mechanism part, peroxide is one of the direct signal molecule clusters which cause coral bleaching, and ascorbic acid peroxidase (APX) is the most effective enzyme in peroxide degradation. By researching and analyzing various zooxanthella APX clades, we chose SymAPX3 as the research object, and download sequence information in the KEGG website.

By reading the scientific essays, we found that arabidopsis, as a higher plant, can withstand much more severe stress conditions such as high temperature, strong illumination and extreme pH stress than zooxanthellae. Therefore, we hypothesized that the APX enzyme of higher plant(arabidopsis) can also withstand high temperature, strong illumination and extreme pH stress than zooxanthellae and overexpression of higher plant APX gene in algae could solve the problem of inactivation and loss of algal APX under high temperature conditions. We also designed the in vitro expression and enzyme activity measurement of arabidopsis APX.

We compared the sequence, predicted function and mark the part with high similarity and conservatism in the sequence in functional sites such as heme binding site and H2O2 binding site which are mentioned in the scientific report. We took H2O2 binding site as the entry point, designing and doing experiment in vitro expression, measure the enzyme activity, and verified the prediction of this site on the level of biological experiment.

#### 2.1 APX gene acquisition of arabidopsis thaliana

We extracted the whole mRNA genome of arabidopsis using Trizol measure by kit. Then, the arabidopsis cDNA was obtained by rt-pcr. The gene fragment was amplified by PCR and connected to pet28b-rplJ to obtain the ATAPX1 connected to pET28b-rplJ.

#### 2.2 APX gene acquisition of zooxanthella

To verify the H2O2 binding site, we designed to measure and compare the enzyme activity of wild type and mutant enzyme proteins by mutating this site. If the enzyme activity data conform to the prediction (the activity difference is large), it indicates that the H2O2 binding site predicted in the text is accurate and can be used for further research, such as structural optimization.

The article pointed out that amino acid RL was conserved amino acid, so we chose the first case where mutating RL to AA. At the same time, it was pointed out that the H2O2 binding site had 7 amino acids (RLSWHDA), so the second case we chose was to mutate all the charged amino acids (R, H, D) to alanine (A), and the nucleotide sequence was mutated from "cggttgtcctggcatgatgca" to "gcgttgtcctgggccgcggca". Then, we connected these sequences into plasmid pET28b

After modifying the sequence, we sent the sequence information to the biological company for synthesis, and obtained SymAPX3, SymAPX-mut1 and SymAPX-mut2 genes which respectively connected to plasmid pET28b.

#### 3. Expression of prokaryotic protein and his-tag protein purification

The recombinant plasmid was transferred into DH5a for amplification. After doing plasmid extraction, it transferred into BL21 strain to do prokaryotic protein expression. The cell efflux was obtained after ultrasonically broken bacteria solution and the protein purification was performed. (see Protocols on the Experiments page for details)

#### 4. Determination of ascorbate peroxidase activity

After obtaining the purified protein solution, AsA oxidation rate was measured by using APX activity detection kit. By putting it into the enzyme activity calculation formula, we gain the results.

## B. Protocol

## C. Development of Project

1) Decide the topic of research project and read scientific essays

2)Amplified the cDNA from arabidopsis thaliana to get the target sequence (ATAPX1) . Select the target sequence (SymAPX3) and give it to the biological company for synthesis to transfer into the plasmids(SymAPX3-mut1&SymAPX3-mut2) after mutation.

3) Prokaryotic protein expression and His-tag protein purification

4) Measurement of ascorbate peroxidase activity

* [Experiment](#!)
* [Protocol](#!)
* [Development of Project](#!)

© 2019 Team:GZHS-United
[IGEM](https://2019.igem.org/Team%3AGZHS-United)

*arrow\_upward*

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:GZHS-United/Results

[![](https://static.igem.org/mediawiki/2019/e/e8/T--GZHS-United--logo-1.png)](https://2019.igem.org/Team%3AGZHS-United)

* Team
* Project
* Parts
* [Safety](https://2019.igem.org/Team%3AGZHS-United/Safety)
* Human Practices
* Awards

* [Team Members](https://2019.igem.org/Team%3AGZHS-United/Team)
* [Collaborations](https://2019.igem.org/Team%3AGZHS-United/Collaborations)

* [Description](https://2019.igem.org/Team%3AGZHS-United/Description)
* [Design](https://2019.igem.org/Team%3AGZHS-United/Design)
* [Experiments](https://2019.igem.org/Team%3AGZHS-United/Experiments)
* [Notebook](https://2019.igem.org/Team%3AGZHS-United/Notebook)
* [Contribution](https://2019.igem.org/Team%3AGZHS-United/Contribution)
* [Results](https://2019.igem.org/Team%3AGZHS-United/Results)
* [Demonstrate](https://2019.igem.org/Team%3AGZHS-United/Demonstrate)
* [Improve](https://2019.igem.org/Team%3AGZHS-United/Improve)
* [Attributions](https://2019.igem.org/Team%3AGZHS-United/Attributions)

* [Parts Overview](https://2019.igem.org/Team%3AGZHS-United/Parts)
* [Basic Part](https://2019.igem.org/Team%3AGZHS-United/Basic_Part)
* [Composite Part](https://2019.igem.org/Team%3AGZHS-United/Composite_Part)
* [Part Collection](https://2019.igem.org/Team%3AGZHS-United/Part_Collection)

* [Human Practices](https://2019.igem.org/Team%3AGZHS-United/Human_Practices)
* [Education & Engagement](https://2019.igem.org/Team%3AGZHS-United/Public_Engagement)

* [Model](https://2019.igem.org/Team%3AGZHS-United/Model)

## The Result

#### 1.Experiment a

To find the difference of tolerance between zooxanthella APX and arabidopsis APX under three stress conditions: high temperature, strong illumination and different pH values. Firstly, we treat the protein samples respectively in different temperature gradients and measuring the activity of sample protein enzyme. Enzyme activity data of SymAPX3, SymAPX3-mut1, SymAPX3-mut2 and ATAPX1 were measured at room temperature, and the results were summarized as follows:

![](https://static.igem.org/mediawiki/2019/2/2c/T--GZHS-United--re-1.jpg)

To find the difference of tolerance between zooxanthella APX and arabidopsis APX under illumination stress, we treat the protein samples respectively in different illumination intensity and measuring the activity of sample protein enzyme. The data of enzyme activities is showing below:

![](https://static.igem.org/mediawiki/2019/3/39/T--GZHS-United--re-2.jpg)

To find the difference of tolerance between zooxanthella APX and arabidopsis APX in different pH value, we treat the protein samples respectively in environment that have different pH values and measuring the activity of sample protein enzyme. The data of enzyme activities is showing below:

![](https://static.igem.org/mediawiki/2019/b/be/T--GZHS-United--re-3.jpg)

According to the line diagram, the enzyme activity of ATAPX1 is significantly higher than SymAPX3, SymAPX3-mut1 and SymAPX3-mut2 at all gradient temperatures, illumination intensity and pH values. It has a strong resistance to under three stress conditions: high temperature, strong illumination and pH value.

Therefore, we concluded that APX activity of arabidopsis has significant advantages in eliminating peroxides under various stress conditions especially high temperature, strong illumination and changing pH values.

#### 2.Experiment b

To discover and compare the ways of increasing zooxanthellae APX enzyme activity, we further design experiment b. We mutated the predicted H2O2 binding on zooxanthellae APX enzyme directly and measure the enzyme activity of mutant proteins under three stress conditions: high temperature, strong illumination and different pH values. The data is shown below:

![](https://static.igem.org/mediawiki/2019/2/2c/T--GZHS-United--re-1.jpg)

![](https://static.igem.org/mediawiki/2019/3/39/T--GZHS-United--re-2.jpg)

![](https://static.igem.org/mediawiki/2019/b/be/T--GZHS-United--re-3.jpg)

We found that the enzyme activity of the APX mutant of zooxanthellae was not significantly different from that of the wild type.

In addition, the activity of wild-type arabidopsis APX was significantly higher than that of zooxanthellae

From this, we come to two conclusions:

A. The predicted functional correlation between H2O2 binding site and SymAPX3 in the data cannot be determined at present

B. arabidopsis APX has the potential to replace zooxanthella APX and help zooxanthella remove peroxides

#### 3.The Conclusion

To sum up, our project has completed the process of hypothesizing, designing experiments, processing and analyzing experimental data. There are three conclusions:

1.We compared the enzyme activity between zooxanthellae APX and arabidopsis APX at different temperatures. We believe that arabidopsis APX have high potential of replacing zooxanthellae APX and helping algae remove peroxides.

2. While the enzyme activity of zooxanthelae apx after mutating the H2O2 binding site is nearly the same as before, we concluded that it is more difficult to rise the apx enzyme activity by changing the genes of zooxanthellae itself than by directly transplanting the genes related to higher plants

3. We expected to verify the functional site hypothesis from the biological experiment level. The experimental results partially indicated that there were doubts about the H2O2 binding site predicted by bioinformatics methods, at least in terms of functional correlation.

We hope that our project can contributed to further study of coral bleaching. For example, for teams that developed zooxanthella transformation system, they can choose to transform our "pET28b rplJ - ATAPX1" parts into zooxanthella and observe the reaction of zooxanthella - coral symbionts under high temperature stress. We hope that our research will help to improve the heat resistance of the zooxanthella-coral symbionts corals and help the coral to regain their color.

* [Experiment a](#!)
* [Experiment b](#!)
* [The Conclusion](#!)

© 2019 Team:GZHS-United
[IGEM](https://2019.igem.org/Team%3AGZHS-United)

*arrow\_upward*