iGEM 2019 — Team Shenzhen_SFLS

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

# Team:Shenzhen SFLS/Notebook

* [Home](https://2019.igem.org/Team%3AShenzhen_SFLS)
* Team
  + [Members](https://2019.igem.org/Team%3AShenzhen_SFLS/Team)
  + [Attributions](https://2019.igem.org/Team%3AShenzhen_SFLS/Attributions)
  + [Collaboration](https://2019.igem.org/Team%3AShenzhen_SFLS/Collaborations)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AShenzhen_SFLS/Description)
  + [Design](https://2019.igem.org/Team%3AShenzhen_SFLS/Design)
  + [Improvements](https://2019.igem.org/Team%3AShenzhen_SFLS/Improvements)
  + [Future Work](https://2019.igem.org/Team%3AShenzhen_SFLS/Future)
* Experiment
  + [Protocol](https://2019.igem.org/Team%3AShenzhen_SFLS/Protocol)
  + [Results](https://2019.igem.org/Team%3AShenzhen_SFLS/Results)
  + [Notebooks](https://2019.igem.org/Team%3AShenzhen_SFLS/Notebook)
* [Safety](https://2019.igem.org/Team%3AShenzhen_SFLS/Safety)
* Human Practice
  + [Human Practice&Integrated Human Practice](https://2019.igem.org/Team%3AShenzhen_SFLS/Human_Practices)
  + [Education & Puclic Engagement](https://2019.igem.org/Team%3AShenzhen_SFLS/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3148)

##### Notebooks

##### Notes about Mgfp-5

9.11 Transformation of Mgfp-5

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

9.12

* 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

9.13 Induction of Mgfp-5

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 4 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

9.14 Natural purification of Mgfp-5 and SDS-page

* 1.

  Purify the protein by natural protocol.
* 2.

  Produce protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

9.20 Transformation of Mgfp-5

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

9.21

* 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

9.22 Induction of Mgfp-5

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 6 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

9.23 Natural purification of Mgfp-5 and SDS-page

* 1.

  Purify the protein by Natural protocol.
* 2.

  Produce protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.3 Transformation of Mgfp-5

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.4

* 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.5 Induction of Mgfp-5

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.6 Denatured purification of Mgfp-5 and SDS-page

* 1.

  Purify the protein by denatured protocol.
* 2.

  Produce protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.9 Transformation of Mgfp-5

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cools down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.10

* 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.11 Induction of Mgfp-5

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.12 Denatured purification of Mgfp-5 and SDS-page

* 1.

  Purify the protein by denatured protocol.
* 2.

  Produce protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.14 Transformation of Mgfp-5

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.15

* 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.16 Induction of Mgfp-5

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.17 Denatured purification of Mgfp-5 and SDS-page

* 1.

  Purify the protein by denatured protocol.
* 2.

  Produce protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

##### Notes about Tyr

9.20

* Transform plasmid into BL21
* Culture for 16h

9.21

* Picking out single clone, inoculating into 5ml LB with K+ system x3
* Culturing for 16h

9.22

* Preserved the BL21 cells with tyrosinase plasmid
* \*Due to the mistakes of plasmid synthesis, we do not know that whether PSBGAS, the replacement of the PET28a, has the site of lac. So we decide not to induce.

9.24

* Correct plasmid of tyrosinase arrived, new experiment started on 9.26

9.26

* The transformation of plasmid into BL21 cells
* Produced three plate with spectinomycin of BL21 cells and one of them scraped
* \*Due to the mistakes of technician, the whole experiment is failed. 9.27 redo the experiment.

9.27

* The transformation of plasmid into BL21 cells
* Culturing 16h, 37°C, 220rpm

9.28

* Pick out the 3 monoclonal colonies into 5ml LB with K+
* Culture for 16h

9.29

* 12:25pm

  Inoculate 1ml solution into 100ml LB with K+ system x2
* 2ml solution into 200ml Lb with K+ system
* All cultured for 1.5h
* 14:00pm

  Add IPTG (100ul for 100ml; 200ul for 200ml)
* 14:08pm

  culture for 4h, 37°C, 220rpm
* 2x100ml were split into 2x50ml in 50ml centrifugation tube
* Give out 2 x dense 50ml E.coli solutions labeled 1and 2 x diluted 50ml E.coli solutions labeled 2
* centrifugate in 7400rpm, 4°C
* \*The centrifugal machine does not support low temperature centrifugation.
* Preserve the solution in -20°C
* \*Due to the changes of the laboratory, these solutions were scraped

10.2

* The transformation of plasmid into BL21 cells
* Culturing 16h, 37°C, 220rpm

10.3

* Pick out the 3 monoclonal colonies into 5ml LB with K+
* Culture for 16h

10.4

* 1. Inoculate 1ml solution into 100ml LB with K+ system x3 labeled 1-3 cultured for 1.5h
* Add IPTG 100ul for 100ml system
* culture for 4h, 37°C, 220rpm

The result solution from 1 were centrifuged

| Tyr | Precipitate/g | Lysate/ml |
| --- | --- | --- |
| 1 | 0.53 | 2 |
| 2 | 0.51 | 2 |
| 3 | 0.48 | 2 |

* 2. Bacteria SDS-PAGE

##### Notes about mgfp5-Masp1

10.3
(For simplicity, all terms Masp1 refers to the part mgfp5-masp1 here).
Transformation of Masp1

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.4

* 1.

  Select monoclonal of mgfp5-Masp1 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.5 Induction of Masp1

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 4 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.6 Natural purification of Masp1 and SDS-page

* 1.

  Purify the protein by Natural protocol.
* 2.

  Producing protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.9 Transformation of Masp1

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.10

* 1.

  Select monoclonal of Masp1 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.11 Induction of Masp1

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.12 Denatured purification of Masp1 and SDS-page

* 1.

  Purify the protein by denatured protocol.
* 2.

  Producing protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.13 Transformation of Masp1

* 1.

  Take 50ul competent cell into EP tube on the ice to melt.
* 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.
* 3.

  After that, heat shock them in 42℃ water for 90 seconds.
* 4.

  Put them into ice for 5 minutes.
* 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.
* 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.14

* 1.

  Select monoclonal colonies  of Masp1 according to the protocol and add into 5ml LB liquid medium.
* 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.15 Induction of Masp1

* 1.

  Add 1ml bacteria solution into 99ml K+ Lb.
* 2.

  Table concentrator in 37℃ for 1-1.5 hr.
* 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.
* 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.16 Denatured purification of Masp1 and SDS-page

* 1.

  Purify the protein by denatured protocol.
* 2.

  Produce protein sample and load into the gel.
* 3.

  Gel electrophoresis.
* 4.

  Dye and wash the gel 1hr and scan using gel imager.


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

# Team:Shenzhen SFLS/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.

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)

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

# Team:Shenzhen SFLS/Results

* [Home](https://2019.igem.org/Team%3AShenzhen_SFLS)
* Team
  + [Members](https://2019.igem.org/Team%3AShenzhen_SFLS/Team)
  + [Attributions](https://2019.igem.org/Team%3AShenzhen_SFLS/Attributions)
  + [Collaboration](https://2019.igem.org/Team%3AShenzhen_SFLS/Collaborations)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AShenzhen_SFLS/Description)
  + [Design](https://2019.igem.org/Team%3AShenzhen_SFLS/Design)
  + [Improvements](https://2019.igem.org/Team%3AShenzhen_SFLS/Improvements)
  + [Future Work](https://2019.igem.org/Team%3AShenzhen_SFLS/Future)
* Experiment
  + [Protocol](https://2019.igem.org/Team%3AShenzhen_SFLS/Protocol)
  + [Results](https://2019.igem.org/Team%3AShenzhen_SFLS/Results)
  + [Notebooks](https://2019.igem.org/Team%3AShenzhen_SFLS/Notebook)
* [Safety](https://2019.igem.org/Team%3AShenzhen_SFLS/Safety)
* Human Practice
  + [Human Practice&Integrated Human Practice](https://2019.igem.org/Team%3AShenzhen_SFLS/Human_Practices)
  + [Education & Puclic Engagement](https://2019.igem.org/Team%3AShenzhen_SFLS/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3148)

##### Results

The aim of our experiments is mainly to confirm the expression of our protein. As described in lab protocol, all sample from induction and his-tag purification is sds-paged and we can quickly distinguish between the his-tagged target protein and other irrelevant ones. Therefore, we observe the protein and marker strips on the sds-gel to assess our outcomes.

##### The results of plasmid PCR

We conducted Plasmid PCR to confirm the validity of our protein sequence. The sequence is as follows:

* Grid 1: DNA marker

  * Grid 2: mfp3

    * Grid 3: Tyr

      * Grid 4: mgfp5-masp1

        * Grid 5: mgfp5

![](https://static.igem.org/mediawiki/2019/8/89/T--Shenzhen_SFLS--result11.png)

Fig 0: The UV image of DNA gel

The correct result of this DNA electrophoresis should produce a travelling distance of mfp3< mgfp5< mgfp5-masp1< tyr. Unfortunately, the grid which contains mgfp5-masp1 is broken due to unknown reasons and the electrophoresis fails to record the strips of DNA. However, the strips of other three proteins corresponds to their own molecular weight and shows the travelling distance relationship of mfp3< mgfp 5

##### The results of sds-page analysis

Note:

For mgfp5, we uses Prestained Color Protein Molecular Weight Marker (270kD to 6.5kD) from Beyotime. For more information about the marker and the scaling, visit
<https://www.beyotime.com/product/P0071.htm>.

For tyr, we uses Prestained Color Protein Molecular Weight Marker (170kD to 10kD) from Beyotime. For more information about the marker and the scaling, visit
<https://www.beyotime.com/product/P0075FT.htm>

##### (A)The results of mgfp5 characterization

At first, the sds-page images fail to record any strips in protein elution.

![](https://static.igem.org/mediawiki/2019/d/de/T--Shenzhen_SFLS--result12.png)

Fig 2: A picture showing no strips at all.

However, after our integrated human practice, we found out the reason behind failure to be the formation of inclusion body, which blocks the his-tag. In order to destroy the inclusion body and release the his-tag, we need to denaturate the protein to change its structure. With the consideration in mind, we repeated the experiment for another two times with denatured induction product and successfully capture strips on the sds-gel from the elution of his-tag purification.

Note: Due to the denaturation process, the first ten grids show messy strips that cannot be analyzed.

![](https://static.igem.org/mediawiki/2019/c/c9/T--Shenzhen_SFLS--result13.png)

Fig 3: Denatured protein samples and wash buffer content

Grid one: Marker; Grid two: Cl from cell lysis; Grid three: Cl from supernatant; Grid four: Ft (sample run through tube); Grid five: Wash 1; Grid six: Wash 2; Grid seven: Wash 3; Grid eight: Wash 4; Grid nine: Wash 5

![](https://static.igem.org/mediawiki/2019/7/7a/T--Shenzhen_SFLS--result14.png)

Fig 4: Elution to Fig 1 samples

Grid 1: Elution 1; Grid 2: Elution 2; Grid 3: Elution 3; Grid 4: Elution 4; Grid 5: Elution 5; Grid 6: Elution 6. Grids 7-10 are intentionally left blank.

![](https://static.igem.org/mediawiki/2019/6/66/T--Shenzhen_SFLS--result15.png)

Fig5: Fig 3 scanning image

![](https://static.igem.org/mediawiki/2019/c/c2/T--Shenzhen_SFLS--result16.png)

Fig 6: Fig 4 scanning image

As shown in the purification figure (Fig 4), we observe strips of a bit above 6.5kD marker strip, which corresponds to the molecular weight of our mgfp5 protein (258 bp = 86 amino acids = 8.6 kD). Therefore, we can confirm that we have successfully produced the mgfp5 protein.

##### (B)The results of Tyr Characterization:

We conducted a few trails in effort to express the enzyme tyrosinase. The first two experiments, owing to the issue of inclusion bodies, failed to record any strips on the gel. After preparing for a third time. We successfully derive strips from the elution of his-tag purification.

(Note: We didn’t keep the failure record so only success data will be included. Also, as denatured protein Cl and washings will show messy strips during electrophoresis, we discarded Samples from Cl to W3 and keep only E1-E5)

![](https://static.igem.org/mediawiki/2019/f/fa/T--Shenzhen_SFLS--result17.png)

Fig 7: The elution of Tyr

Grid 1: Elution 1; Grid 2: Elution 2; Grid 3: Elution 3; Grid 4: Elution4; Grid 5: Elution 5.

##### The results for mfp3:

Owing to time limit, we could only conduct one experiment on mfp3 and we only managed to finish the induction process. However, we have conducted DNA gel analysis on mfp3 to confirm the senquence length of our DNA. (As shown in the first picture).

##### The results of mgfp5-masp1:

Two experiments were conducted on the mgfp5-masp1 part to characterize it. We haven’t derived visible strips to confirm the production of this part due to time limit. However, we confirmed the sequence length using the PCR automatic analysis. (As shown in the first picture).