iGEM 2019 — Team Nanjing

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# Team:Nanjing/Notebook

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TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

![](https://static.igem.org/mediawiki/2014/c/c6/OUC-China_Content_IGEM.png)
![](https://static.igem.org/mediawiki/2019/4/47/T--Nanjing--Title.png)

# Notebook

##### Hey guys! Please enjoy our process in the lab. It was really a meaningful experience, full of laugh and joy. Hope you enjoy our daily Diary!

### Day 1:

![](https://static.igem.org/mediawiki/2019/5/5c/T--Nanjing--Day1.png)

### Day 2:

![](https://static.igem.org/mediawiki/2019/3/39/T--Nanjing--Day2.png)

### Day 3:

![](https://static.igem.org/mediawiki/2019/9/99/T--Nanjing--Day3.png)

### Day 4:

![](https://static.igem.org/mediawiki/2019/0/0b/T--Nanjing--Day4.png)

### Day 5:

![](https://static.igem.org/mediawiki/2019/3/3f/T--Nanjing--Day5.png)

### Day 6:

![](https://static.igem.org/mediawiki/2019/0/0a/T--Nanjing--Day6.png)

### Day 7:

![](https://static.igem.org/mediawiki/2019/e/e9/T--Nanjing--Day7.png)

We performed restriction today, still a not-too-difficult process with a long time of waiting. It was surprising that restriction was performed in PCR machine as well. The PCR machine was actually a thermal chamber and we can design the temperature changes. Like before, we couldn’t see what happened inside the machine. What we can do was simply praying that it can work.

### Day 8:

![](https://static.igem.org/mediawiki/2019/e/e0/T--Nanjing--Day8.png)

Taking out the tube from the machine, it’s time for us to test our result. Another electrophoresis was performed, but we used 50μL system this time. The lab didn’t have such large model to make the grooves, so we stick two “broaches” together to make a larger one. We did the electrophoresis correctly this time. The image seemed to be good, and then we recycle the gel using gel recycling kit. The Brother helped us cut the gel with genes and put the bar into a centrifuge tube. I measured the weight. He but them so well that all the bars were exactly the same mass to the digit of 2. He didn’t even use any measuring tool during the process! The recycle was like PCR recycle, but a lot more complicated. Also, the plasmid is about 11kb. Although our kit showed it can recycle up to 40kb effectively, the senior told me it would still waste a lot of genes. After testing the concentration, the plasmid was only about 5ng/μL. The GOI was better. It was not a very satisfying concentration, but it still can be used to do ligation.

### Day 9:

![](https://static.igem.org/mediawiki/2019/c/c2/T--Nanjing--Day9.png)

We did ligation today. Again, in the PCR machine. After calculating the amount of vectors and GOIs used in ligation, we put the tiny tube under 4 degree Celsius for the whole day. Tomorrow we will transform the new plasmids into bacteria again to see whether it worked.

### Day 11:

It’s so sad we failed. No colonies were growing on the medium, which means the ligation didn’t work. It’s time for us to find out the problems during the process. We first checked the protocol to see whether there were errors. Interestingly, there wasn’t. We were confused. How can the result be like that? We thought maybe the concentration of vectors and GOI in ligation affected the process and we managed to increase the concentration and redo the whole process.

### Day 13:

So sad we failed again, and it showed the concentration may not be the key factor. The Sister asked me to give her our primer design. The primers were designed by company Beyotime as sponsorship. Surprisingly, the Sister found that the error was in the primer design. The company didn’t add protective bases into the sequence, making PCR fail. We contacted the company immediately and asked them to synthesize the primers again. It will take about five days for the new primers to arrive.

### Day 19:

Our new primer arrived and it was time for us to repeat almost all the process. I hope we can catch up with the time.


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# Team:Nanjing/Experiments

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TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

![](https://static.igem.org/mediawiki/2014/c/c6/OUC-China_Content_IGEM.png)
![](https://static.igem.org/mediawiki/2019/4/47/T--Nanjing--Title.png)

![](https://static.igem.org/mediawiki/2019/9/90/T--Nanjing--Plasmid_Transformation.png)

1. Prepare for plasmid pCAMBIA 2301 (with antibiotics-resistance mark Kan):

a. Centrifuge the collection tube with plasmid powder for 1 minutes under 500rpm to descend the powder to the bottom. Add 20μl sterile water to dissolve the plasmids, and rest it on the lab table for 1min under room temperature;

b. Take the competent cell (DH5α E.coli) from the fridge (-80℃ storage condition) and place on the ice box for melting. During the time, mark the tube that will be used to put competent cells;

c. Take 2μl plasmid solution using pipette and add it into 2μL competent cells. Leave the ice-bath for 30 minutes. At the same time, heat the water-bath so we can save time;

d. Take out the tube from the ice-bath, and let the solution undergoes heat shock for 90 seconds under the temperature of 42℃. Leave it into ice-bath again for 2 minutes;

e. Add 500μL LB liquid culture medium without antibiotics into the solution. Put it on the shaker for 45 minutes at 180rpm to promote cell replication;

f. Centrifuge the solution for 5 minutes under 6000rpm and pour it to culture dish with LB solid medium (containing Kanamycin this time). Use the glass rod to distribute bacteria evenly across the medium surface and let it dry;

g. Leave the culture dish upside down and cultivate bacteria for 12 to 16 hours;
Pick the single colony and put it into liquid LB culture medium with Kanamycin. Cultivate bacteria on the shaker for 12 to 16 hours, and we can then extract the plasmid we need accordingly.

![](https://static.igem.org/mediawiki/2019/7/7d/T--Nanjing--PCR_Principle.png)

2. PCR：

Forward：ATGAGCTTTCTTCAGGCCTTG

Reverse：CTACATTGAGTCCACCGAGA

PCR system（50 µL system）

ddH2O                        14 µL

Phanta Max Buffer (2×)          25 µL

ddNTP Mix (10mM each)         1μL

UP                            2 µL

DN                            2 µL

c DNA (GOI)                     1 µL

PCR procedure

Step1 95 ℃                    3min

Step2 95 ℃                   15 s

Step3 56 ℃                   3 min

Step4 Go to 2 for additional      35 cycles

Step5 72 ℃                   10 min

Step6 4 ℃                    end

3. Recycle the PCR product using PCR recycle kit (provided by Beyotime)

![](https://static.igem.org/mediawiki/2019/b/b6/T--Nanjing--PCR_RecycleKit.png)

4. PCR (add the proper restriction sites)：

Forward：GAATTCATGAGCTTTCTTCAGGC （EcoR I）

Reverse：AAGCTTCTACATTGAGTCCACCG  (Hind III)

PCR system（50 µL system）

ddH2O                        14 µL

Phanta Max Buffer (2×)          25 µL

UP                            2 µL

DN                           2 µL

c DNA                         1 µL

PCR procedure

Step1 95 ℃                    3min

Step2 95 ℃                   15 s

Step3 56 ℃                   3 min

Step4 Go to 2 for additional      35 cycles

Step5 72 ℃                   10 min

Step6 4 ℃                    end

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

5. Extract plasmid from Plasmid Extraction Kit (provided by TAKARA)

![](https://static.igem.org/mediawiki/2019/5/57/T--Nanjing--PlasmidExtract.png)

### EcoRI

![](https://static.igem.org/mediawiki/2019/9/9a/T--Nanjing--EcoRI.png)

### Hindlll

![](https://static.igem.org/mediawiki/2019/4/4d/T--Nanjing--Hindlll.png)

6. Use restriction enzyme to but open the plasmids and the GOI

a. The restriction enzymes we use were EcoR I and Hind III (provided by TAKARA). The reaction system is shown below:

ddH2O                        16 µL

plasmid DNA                   1 µL

10×K buffer                    1 µL

EcoR I                         1 µL

Hind III                        1 µL

Total volume                   20 µL

Add all these materials according to the above sequence into sterile PCR tube. Mix them and centrifuge the solution for several seconds until no bubbles exist. Keep the temperature at 37℃ for 3.5 hours to react.

7. Perform an electrophoresis to separate gene fragments.

a. Prepare agarose gel with dilute TAE (0.1%);

b. Add fluorescence marker into the agarose gel and leave the liquid gel under room temperature for 30 minutes to let it solidify;

c. Add gene fragments and plasmids into the groove, immerse the gel with dilute TAE (0.1%);

d. Put the gel in the correct direction (grooves at the positive pole). Connect to electricity and leave the gel for 1 hour;

e. Turn off the power and view the gel under UV light.

![](https://static.igem.org/mediawiki/2019/2/26/T--Nanjing--Electrophoresis.png)

8. Recycle the product after electrophoresis

a. Cut open the gel under the UV light and put the gel bar containing gene fragments into centrifuge tubes;

b. Recycle the gene fragments using Gel Recycle kit for large DNA chains (provided by TAKARA);

![](https://static.igem.org/mediawiki/2019/d/d5/T--Nanjing--DNALigation.png)

9. DNA ligation

a. The ligation system is shown below (10 µL system):

GOI                                     4 µL

pCAMBIA 2301 cloning vector               0.5 µL

T4 DNA ligase (provided by TAKARA)         0.5 µL

10×T4 DNA ligase buffer               1 µL

ddH2O                              4 µL

keep the system under 16℃ for 3 hours to combine.

10. Transform ligation product into E.coli competent cells (DH5α) to amplify the plasmid

a. Take 10μl ligation product solution using pipette and add it into 2μL competent cells. Leave the ice-bath for 30 minutes. At the same time, heat the water-bath so we can save time;

b. Take out the tube from the ice-bath, and let the solution undergoes heat shock for 90 minutes under the temperature of 42℃. Leave it into ice-bath again for 2 minutes;

c. Add 1000μL LB liquid culture medium without antibiotics into the solution. Put it on the shaker for 45 minutes at 180rpm to promote cell replication;

d. Centrifuge the solution for 5 minutes under 6000rpm and pour it to culture dish with LB solid medium (containing Kanamycin this time). Use the glass rod to distribute bacteria evenly across the medium surface and let it dry;

e. Leave the culture dish upside down and cultivate bacteria for 12 to 16 hours;

f. Pick the single colony and put it into liquid LB culture medium with Kanamycin. Cultivate bacteria on the shaker for 12 to 16 hours

![](https://static.igem.org/mediawiki/2019/3/3b/T--Nanjing--Recombinant_Plasmid.png)

11. Extract plasmids using plasmid Extraction Kit (provided by TAKARA)

12. Transform the plasmids into Agrobacteria tumefacien (GV3101) electroporation competent cells (provided by Mitose)

a. Take out the competent cells from fridge (storage condition: -80℃) and place it on the ice box for melting;

b. Add 1μL plasmids into the competent cells, mix them and put the final solution into electroporation cuvette (20mm);

c. Impulses for 5ms;

d. Quickly add 1mL liquid LB cultivation medium and mix them;

e. Put the solution into EP tube under room temperature;

f. Add 70-80μL agrobacteria solution evenly onto LB solid medium containing antibiotics (Kan);

g. Use the glass rod to make the solution dry and turn the dish upside down to cultivate for 2 days (under 28 ℃);

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