iGEM 2019 — Team GDSYZX

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# Team:GDSYZX/Notebook

GDSYZX\_Description

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Experimental record

7.20

Make LB culture medium, TBE, EDTA

7.21

Commence the PCR of ugt33 target fragment.

7.22

Perform agarose gel electrophoresis test, gel recycling and purification. (ugt33)

7.23

Morning: Purpose piece recycling. (ugt33)

Afternoon: Enzyme digestion. (ugt33)

7.24 (ugt33)

Electrophoresis test, re-purification of DNA solution

No positive clones were detected by the enzyme, and the enzyme may not be cleaned because of the poor quality of the enzyme.

7.25 (ugt33)

DNA conversion.

Plate culture.

7.26 (ugt33)

Colony PCR did not get positive results, and the sequence was wrong

Plasmid transformation

7.27 (ugt33)

Re-heat-induced transformation, plating, re-enzyme.

2019.7.28 (ugt33)

1. Today's electrophoresis (colon PCR, PCR purification product to marker) did not show a band, after re-changing the dye, we re-run electrophoresis and found a band. After analysis, it may be considered that there is a problem with the SYBR Green dye in the small tube, and the dye should be replaced on the second day.

2. Really re-do the transformation of UGT33 gene ligation product

2019.7.29. (ugt33)

1. Replaced the nucleic acid dye, the two teams repeated yesterday's colony PCR experiment

2. Extracted the plasmid containing the positive colonies of RrUGT33. The plasmid concentration is generally about 200 ng/ul. Has been sent for testing. (result is negative)

3. Really carry out the colony PCR on the transformed strain plate yesterday, there is a positive band, and 4 shakes have been selected.

2019.7.30 plan

waiting for the sequencing results, and another colony PCR after the transformation of the recombinant product (If Miao teacher can do the conversion tonight)

2019.7.30. Summary

1. after extracting plasmid of RrUGT33 of PUC-HA, conduct enzyme digestion verification, seven are background, not connected.

2. Amplification of RrUGT33, Rr4HPAAS, no banding in electrophoresis, after analysis, it is considered that there may be problems with Q5 enzyme activity or other reagents.

2019.7.31. Tomorrow's plan

1. Reconstitute 5xTBE

2. Imidazole and Na2HPO4 are required to prepare a loading buffer.

3. A 0.22 um filter is required and the PBS buffer is filtered.

4. Purchase SDS-PAGE gel preparation kit.

5. If the new Q5 enzyme is delivered, arrange for two teams to re-amplify the fragment that failed the amplification.

7.31 summary

1. UGT33 colony PCR, the result is positive

2. 4HPAAS gel recovery

3. Escherichia coli carrying UGT33

4. Take the plasmid and prepare for the subsequent pruning/transfer assay to detect whether it is a PUC-HA of ugt33 plasmid.

On August 3, the experiment concluded that UGT-33 PUC-HA carried out colony PCR and no positive bands.

2019.8.5. Summary

Realize the use of recombinant primers to amplify the RrUGT33 gene fragment and construct a PUC-4HPAAS-FLAG vector.

2019.8.6. Summary

1. Provincial implementation of plasmid preparation, collection

2. preparation of plasmids

2019.8.7. Plan.

Extract the 4HPAAS plasmid.

8.14

Make LB culture medium

8.15

1.Heat shock transformation of gold and bronze parts. (DH5 alpha of E. coli)

8.16

1. shake E. coli culture (3ml ×4 tubes) (gold and bronze parts)

8.17

1. plasmid extraction of gold and bronze parts. （failed）

2. Heat shock transformation of gold and bronze parts. (Transetta DE3 of E.coli)（canceled because we found out that Transetta DE3 bring Chl resistance.）.

8.18

1.make 2×YT sterile water and ddH2O.

2.shake E. coli culture (5ml LB culture with Chl ×2 tubes each) (gold and bronze parts)

8.19

1. plasmid re-extraction of gold and bronze parts.

2. Heat shock transformation of gold and bronze parts. (transBL 21 of E.coli) and make overnight culture in 37 degree Celsius.

3. plate cultivation (Chl and Kana resistance)

4. Confirm the presentation of Bronze Award Parts data, understand normalization, and communicate data processing with the modeling team

8.20

1. shake E. coli culture (3ml ×2 tubes) (bronze parts). (transBL 21 of E.coli)

2. make 5 different LB culture with PH value of 5,6,7,8, and 9.

3. 9L, 10C, due to the resistance error of No. 19 coating plate, use residual resuscitation bacterial solution coating plate in the morning

4. In the evening, 9L, 10C Bacterial Plate Bacteria, Select Monoclonal Shaker Bacteria

5. Send 1O, 8P, 9L, 10C, pSB1C3 (no sequencing results, inquire customer service, reply reason is low plasmid concentration)

6. Confirm the exact protocol of bronze medal.

8.21

1.In the morning, after 24 hours of frugal 1O culture, the concentration of bacterial solution reached a transferable shake.

2. Shake E. coli culture (100ml × 2tubes) (bronze parts) (transBL 21 of E.coli)

3. Save 1O sample continuously, measure OD600 to 0.6 to 0.8, then sample according to protocol time point to measure fluorescence (no effective fluorescence photo due to poor equipment condition)

8.23

Construct the vector of PUC-RrUGT33-HA successfully（use Gibson Assembly instead）.

8.24~ 9.22

1. Preparation and transfection of protoplasts from *Arabidopsis thaliana*.

2. Observe and measure protein expression rate by Western blot experiment.

3. Extraction and detection of target products (1.get cells expressing the target segments. 2. Conduct ethanol extraction. 3. detect target products by liquid chromatography)

9.22~

Compile and analyze result data.

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# Team:GDSYZX/Experiments

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TEAM

[Team Members](https://2019.igem.org/Team%3AGDSYZX/Team)
[Collaborations](https://2019.igem.org/Team%3AGDSYZX/Collaborations)

PROJECT

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

PARTS

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

[SAFETY](https://2019.igem.org/Team%3AGDSYZX/Safety)

HUMAN PRACTICES

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

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AGDSYZX/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AGDSYZX/Hardware)
[Measurement](https://2019.igem.org/Team%3AGDSYZX/Measurement)
[Model](https://2019.igem.org/Team%3AGDSYZX/Model)
[Plant](https://2019.igem.org/Team%3AGDSYZX/Plant)
[Software](https://2019.igem.org/Team%3AGDSYZX/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=GDSYZX)

# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### 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:GDSYZX/Results

Collection

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Result

1. Gene cloning

We first carried out codon optimization for our two target genes 4HPAAS and UGT33 according to the preference of *Arabidopsis* codon. After obtaining the target gene, we obtained a large number of target gene fragments by PCR gene cloning in vitro.

![](https://static.igem.org/mediawiki/2019/3/32/T--GDSYZX--RESULT1.jpg)

2. Carrier construction

As shown in the figures below, the carrier puc-ha and the carrier puc-flag were digested and glued. From the results of running gel, the enzyme digestion products of both vectors were 4000bp. Therefore, we judge we got purpose fragment.

![](https://static.igem.org/mediawiki/2019/4/4f/T--GDSYZX--RESULT2.jpg)

3. The colony PCR

After the gene was transferred to the vector, we transferred the plasmid to e. coli overnight. The next day, PCR was performed to test whether the gene transfer was successful. The figure shows that the size of both UGT33 gene and 4HPAAS gene is around 500dp, and the result is positive. After the PCR result of bacterial liquid was positive, we made a small plasmid and sent it to test, and obtained the transferred escherichia coli successfully.

![](https://static.igem.org/mediawiki/2019/b/ba/T--GDSYZX--RESULT3.jpg)

4. Test the expression products

We transfected two genes into the protoplast simultaneously. For the transformed protoplast, we first performed western blot using protoplast. Western blotting was performed at 4 hours, 8 hours, 10 hours, 12 hours and 14 hours, respectively, and the gray values were compared. We found that the highest expression efficiency of UGT33 and 4HPAAS was 12 hours after they were transferred into the protoplast. This proves that the protein can be expressed successfully and provides reference time for liquid chromatography.

![](https://static.igem.org/mediawiki/2019/0/0e/T--GDSYZX--RESULT5.jpg)

Below is the result of a liquid chromatograph. The first row is the wild type, the second row is the protoplast samples of *Arabidopsis Thaliana*after transfection, and the third row is the standard sample of salidroside. The peak value of product measured in the second sample was basically consistent with the peak value of salidroside standard sample, while there was no corresponding peak in the detection results of wild type. Therefore, we can determine that the protoplast of the untransfected *Arabidopsis Thaliana* does not contain salidroside, while the protoplast of the transfected *Arabidopsis Thaliana* can produce salidroside.

![](https://static.igem.org/mediawiki/2019/5/55/T--GDSYZX--RESULT6.jpg)

5. To summarize

Based on the above results, 4HPAAS and UGT33 are the key genes for producing salidroside. In the case that the salidroside anabolism pathway in *Arabidopsis* has not been explored, we successfully predicted two key genes and obtained the expression of salidroside after transferring into *Arabidopsis*.

![](https://static.igem.org/mediawiki/2019/0/07/T--GDSYZX--RESULT7.jpg)

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