iGEM 2019 — Team Hong_Kong_LFC_PC

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

# Team:Hong Kong LFC PC/Notebook

[Home](https://2019.igem.org/Team%3AHong_Kong_LFC_PC)

Team

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

Project

[Description](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Description)
[Design](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Design)
[Experiments](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Experiments)
[Model](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Model)
[Notebook](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Notebook)
[Characterization](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Contribution)
[Results](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Results)
[Attributions](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Parts)

Safety

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

Human Practice

[Integrated Human Practice](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Public_Engagement)

Award

[Medal](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Measurement)

 [Judging Form](https://igem.org/2019_Judging_Form?id=3058)

PLKLFC\_PC

# Notebook

| Time period | Administrative Work | Laboratory Work | Human Practice |
| April 2019 | Started to have regular meet-up with teammates and teachers every week Discussed which topic was our project’s focus, we had to consider two options: therapeutic approach for brain cancer or for gout. | NA | NA |
| May 2019 | Settled laboratory duties and positions Checked for any laboratory devices and chemicals which we needed to buy | NA | Met Professor Chan, who opposed us to investigate brain cancer cells, as it is too difficult to find suitable receptor cell in secondary school level. However, he appreciated the topic of uricase. |
| June 2019 | Read relevant essays to learn more about the mechanism of uricase, uric acid, HIU and variety of plasmids etc. | Cultivate two kinds of competent cells, DH5α and BL21. Prepare large amount of agar plates with antibiotics. | Met Professor Chui and discuss to learn more details about uricase and decide to adopt intracellular digestion. |
| July 2019 | Ordered materials such as gene sequence and pET-22. However, unlike a journal suggested, pET-22 didn’t ligate with gene of uricase well. Therefore, we order pSB-13 for our experiment after a few weeks. | We started our lab work according to our lab workflow, such as PCR, Restriction enzyme digestion, gel electrophoresis, ligation and bacterial transformation | Met Shenzhen IGEM Great Bay Team and they encouraged us to use Nissle 1917 instead of yeast as our host cell, since most yeast cannot replicate in the digestive tract. Asked for doctors’ opinions about our project. Worked on a questionnaire about gout to general public. |
| August 2019 | Replenished materials which were used up completely. Ordered new materials for new experiments such as SDS Page, Uric Acid Assay Started to do mathematical modelling. | Successfully transformed plasmid with gene of uricase into bacteria. | NA |
| September 2019 | Do protein analysis (SDS Page). After discovering some of our genes are not expressed well, we do problem shooting to check whether our SDS page percentage is accurate. We have succeeded this part after modifying the experiment in late September. | Analyzed the feedback of questionnaires done in August. Co-organized a workshop with CUHK to share ideas with local secondary and university teams. |
| October 2019 | Finalized and finished mathematical modelling. Completed our wiki page, poster and powerpoint. | Ran uric acid assay to prove if ygfu (transporter gene) is present in E.coli. | After results of the experiments were finalized, we met Entrepreneur Mr Ricky Chiu for potential business cooperation. Met lawyer Mr Gilbert Chan to check for patent about our project such as uricase. Organized activities such as paying a visit to local elderly centre in order to persuade the general public to accept the probiotic E.coli as a new type of treatment method. |


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

# Team:Hong Kong LFC PC/Experiments

{

[Home](https://2019.igem.org/Team%3AHong_Kong_LFC_PC)

Team

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

Project

[Description](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Description)
[Design](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Design)
[Experiments](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Experiments)
[Model](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Model)
[Notebook](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Notebook)
[Characterization](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Contribution)
[Results](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Results)
[Attributions](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Parts)

Safety

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

Human Practice

[Integrated Human Practice](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Public_Engagement)

Award

[Medal](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Measurement)

 [Judging Form](https://igem.org/2019_Judging_Form?id=3058)

PLKLFC\_PC

![](https://static.igem.org/mediawiki/2019/8/8d/T--Hong_Kong_LFC_PC--123456.jpg)

## Flow of experiments

![](https://static.igem.org/mediawiki/2019/e/e6/T--Hong_Kong_LFC_PC--flow.jpg)

dsDNA were ordered from IDT. The dsDNA was amplified by PCR and cloned into standard iGEM backbone pSB1C3. Colony PCR was used to check the insertion of dsDNA. SDS-PAGE was used to check the overexpression of protein. The uricase activity of the biobricks was then validated by commercial uric acid kit.

## Colony PCR confirms insertion of URI and HIU

![](https://static.igem.org/mediawiki/2019/8/81/T--Hong_Kong_LFC_PC--Cloninggg.jpg)

The cloning of both uricase and HIU hydrolase are successful. Cloning PCR revealed correct number of base pairs of both uricase and HIU hydrolase. A 1090bp band and a 538bp band could be observed, which are uricase and HIU hydrolase repectivily.

## SDS PAGE confirms the translation of uricase

![](https://static.igem.org/mediawiki/2019/5/5a/T--Hong_Kong_LFC_PC--SDSPAGE.jpg)

Protein expression of both uricase and HIU hydrolase were also successful.
A single colony of BBa\_K3058000 and BBa\_K3058001 were picked to grow a 5mL overnight starter solution. Untranformed cells were used as control. After 1% inoculation, the cells were allowed to grow for 16 hours. The cells were then harvested and SDS-PAGE was conducted. When comparing to the control, a overexpressed band could be observed in uricase-expressing and HIU hydrolase-expressing E.coli. The location matches the theoretical size, which are 34.2kDa and 13.56kDa respectively. This indicated that our biobrick could express our expected protein.

## Construction of uric-acid-inducible uricase generator (BBa\_K3058002) using Gibson Assembly

![](https://static.igem.org/mediawiki/2019/0/05/T--Hong_Kong_LFC_PC--GIBSON.jpg)

Initially, we could not synthesize a biobrick(BBa\_K3058002) because it was longer than 3000bp. Luckily, through collaboration,  [Hong\_Kong\_JSS 2019 team](https://2019.igem.org/Team%3AHong_Kong_JSS/Collaborations)  helped us to synthesize the DNA using Gibson assembly. The expected size was correctly synthesized. The validation of the biobrick is still in progress.

## Endogenous uric acid transporter may allow cellular uptake of uric acid.

![](https://static.igem.org/mediawiki/2019/4/45/T--Hong_Kong_LFC_PC--ygfuuu.jpg)

To check if E.coli could intake uric acid, we mix E.coli with uric acid-containing limited-medium and obtained the change in extracellular and intracellular uric acid concentration. At the beginning, 100% of the uric acid was located in the supernatant but not in the cell pellet after centrifugation. After 2 days, arround 50% of the uric acid is translocated into the cell pellet. This indicated that our intracellular approach is possible.

## Experimental Protocols

PCR

1. Set up PCR mixture as follow :

| Component | Volume |
| --- | --- |
| Green Master Mix | 25 μl |
| Primer Mix (10 μM) | 2 μl |
| Insert | 1 μl |
| Milli Q | 22 μl |

2. Mix reagents in tubes by pipetting the solution up and down slowly.

3. Quick spin the PCR tube to ensure the mixture is in the bottom of the tube.

4. Put it in the thermocycler (PCR machine) and set the cycle information.

5. Start the cycle and wait till it is finished.

Gel Electrophoresis

1. Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.

2. Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber.

3. Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times.

4. Load your samples and appropriate marker into your wells

5. Incubate at 37°C and 250 rpm for 60-120 minutes.

6. Apply 130 volts to the chamber for 20 mins.

7. Check your gel using a transilluminator or other UV emitting device.

8. Re-suspend cells by light vortexing

9. Plate resuspended cells as above

10. Incubate overnight at 37°C with plates upside down.

DNA Gel Purification

1. Trimp the DNA fragment from the agarose gel and transfer the gel to 1.5 mL
   microfuge tube followed by weighing the gel slice.

2. 4 volumes of gel dissolving buffer is transferred to the gel slice.

3. The sample is incubated between 37 o C – 55 o C and vortex the sample
   periodically until the gel slice is completely dissolved.

4. Insert the column into collection tube and the sample is loaded onto the
   column. The column is the spin for 1 minute, the discard the flow-through.

5. The column is re-inserted into collection tube. 200 μL DNA Wash Buffer and
   spin for 1 minute and repeat this step.

6. The column is transferred to a clean 1.5 mL microfuge tube.

7. 6 μL DNA elution buffer is added to the center of the matrix. After 1 minute,
   spin the sample to elute DNA.

Restriction Digestion

1. Prepare the PSB1C3 plasmid:

| Component | Volume |
| --- | --- |
| Buffer | 5 μl |
| EcoRI | 0.5 μl |
| PST1 | 0.5 μl |
| PSB1C3 | 4 μl |
| Milli Q | 40 μl |

2. Prepare the Insert:

| Component | Volume |
| --- | --- |
| Buffer | 5 μl |
| EcoRI | 0.5 μl |
| PST1 | 0.5 μl |
| Insert | 20 μl |
| Milli Q | 24 μl |

3. Pipette the solution up and down to ensure all reagents are mixed well.

4. Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours.

5. Purify the DNA by PCR purification kit/gel extraction kit for downstream process.

Ligation

1. Mix the solution mixture according to the contents below:

2. Prepare the Insert:

| Component | Volume |
| --- | --- |
| Buffer | 4 μl |
| Plasmid | 3 μl |
| Insert | 10 μl |
| Ligase | 1 μl |
| Milli Q | 2 μl |

3. Prepare the Control:

| Component | Volume |
| --- | --- |
| Buffer | 4 μl |
| Plasmid | 3 μl |
| Insert | 0 μl |
| Ligase | 1 μl |
| Milli Q | 12 μl |

4. The solution mixtures is then incubated in 4 o C for 16 hours

Competent Cell Preparation

1. Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.

| Composition | Volume |
| --- | --- |
| 0.6 M CaCl2 | 10 ml |
| 0.5 M PIPES pH7.0 | 2 ml |
| Glycerol | 15 ml |
| H2O | 73 ml |
| Total | 100 ml |

2. Spread the bacterial cell (DH5α, BL21 and Nissle 1917) for plasmid replication on the LB.

3. Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm.

4. Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h)

5. Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes.

6. Collect the cell by centrifuging at 1000 g for 10 min at 4°C.

7. Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. \* Cells must remain clod for the rest of the procedures!

8. Collect the cell by centrifuging at 1000 g for 10 min at 4 °C.

9. Centrifuge at 13,000 rpm for 1 min.

10. Gently resuspend the cell in each tube with 1.25 ml ice-old Ca/glycerol buffer.

11. Centrifuge at 13,000 rpm for 1 min.

12. Column dring with centrifuge 13.000rpm for 1 min.

13. Transfer all cells to one tube.

14. Dispense 100 μl aliquots of competent cells into each Eppendorf.

15. Store at -80 °C.

16. Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.

Transformation

1. Pre-heat the water to 42 o C in water bath and warm the LB solution in incubator
   at 37 o C.

2. Obtain -80 o C DH5α and remove from ice bath, to allow it to liquefy.

3. Add DNA in a ratio of 100 µL DH5α to 20 µL of DNA in the Eppendorf.

4. Wait 10-20 minutes in Ice Bath in order to allow DNA to enclose the cell
   membrane of DH5α.

5. Immerse the Eppendorf containing the mixture into water bath of 42 o C for 1
   min exactly as a heat shock procedure. The Eppendorf is then taken to an ice
   bath for 1-2 minutes to allow the pores on the cell membrane to close up.

6. Add 1 mL of LB solution at 37 o C to the Eppendorf, the mixture will then be
   incubated at 37 DC for 1.5 hours.

7. Centrifuge the Eppendorf for 1 minute 25 o C at 13,000 rpm.

8. Remove excess liquids and remain 100 µL of liquid in the centrifuge tube.

9. Pipette up and down to wash the pellet.

10. Use a pipette to extract 100 µL of the liquid then transfer and spread the liquid
    on the agar plate.

Picking Clone

1. 5 mL with 5 μL chloramphenicol of LB solution is placed inside 15 mL falcon

2. A colony is picked from the cultured plate after transformation and
   transferred to the falcon containing the solution under fire.

3. Loosen the cover of the flacon.

4. Incubate the solution in the flacon at 37 o C for a day..

SDS Page Protein Identification

1. Dilute the well mixed solution containing the bacteria and LB solution to 0.5
   A of OD600 from UV spectrophotometer.

2. 500 μL of sample is collected and centrifuged at 13000 r.p.m. in a micro-
   centrifuge tube.

3. Discard the LB solution.

4. Re-suspend in 50 μL of Milli Q water and transfer further 50 μL of laminin
   sample buffer.

5. Incubate the solution mixture in PCR at 95 o C for 15 minutes.

6. Run the SDS page using precision plus protein kaleidoscope standards ladder

Gibson Assembly

1. Prepare the following reaction mixture as the table shown below

| Component | Volume |
| --- | --- |
| Amount of DNA fragment | 3.45 μL of fragment 1 + 3.57 μL of fragment 2 |
| NEBuilder HiFi DNA Assembly Master Mix | 10 μL |
| Deionized Water | 2 μL |
| Total volume | 10 μl |
| Milli Q | 2 μl |

2. The reaction mixture is then incubated in a thermocycler at 50 o C for 15
   minutes.

3. After incubation, PCR is carried out.

Uric Acid Assay

1. Pipette 50 μL of the samples into separate wells of a microplate.

2. Begin the reactions by adding 50 μL of the Amplex® Red reagent.

3. Incubate the reactions for 30 minutes or longer at 37°C, protected from light. The absorbance may be measured at multiple time points to follow the kinetics of the reactions.

4. Measure the absorbance in a microplate reader using excitation with absorbance at ~560 nm.

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

# Team:Hong Kong LFC PC/Results

{

[Home](https://2019.igem.org/Team%3AHong_Kong_LFC_PC)

Team

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

Project

[Description](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Description)
[Design](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Design)
[Experiments](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Experiments)
[Model](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Model)
[Notebook](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Notebook)
[Characterization](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Contribution)
[Results](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Results)
[Attributions](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Parts)

Safety

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

Human Practice

[Integrated Human Practice](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Public_Engagement)

Award

[Medal](https://2019.igem.org/Team%3AHong_Kong_LFC_PC/Measurement)

 [Judging Form](https://igem.org/2019_Judging_Form?id=3058)

PLKLFC\_PC

![](https://static.igem.org/mediawiki/2019/7/78/T--Hong_Kong_LFC_PC--result.jpg)

## Method: Colorimetric Uricase and Uric Acid Assay Kit

![](https://static.igem.org/mediawiki/2019/c/ce/T--Hong_Kong_LFC_PC--H2O2.jpg)

Since we are measuring the uricase activity and the concentration of uric acid, a commercial uricase assay kit from Abcam (ab65344)is used. In the assay, uricase activity can convert uric acid to allantonin and H2O2. Under the catalyzation of horseradish peroxidase (HRP), the H2O2 produced will react with a fluorescence probe to give colorimetric signal. A red color would be observed if there is H2O2. The absorbance can then be read by plate reader at 570 nm.
![](https://static.igem.org/mediawiki/2019/c/cc/T--Hong_Kong_LFC_PC--URIUASTDC.jpg)

Two standard curves were plotted to covert the absorbance reading to the concentration of uricase and uric acid later. The equations of the curves were obtained, with R square nearly equals to 1.

## Result:[BBa\_K3058000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3058000) expressed Functional Uricase

![](https://static.igem.org/mediawiki/2019/3/3f/T--Hong_Kong_LFC_PC--UricaseCOM.jpg)
![](https://static.igem.org/mediawiki/2019/6/67/T--Hong_Kong_LFC_PC--UricaseTP.jpg)

Uricase assay was used to determine the uricase activity of our biobrick BBa\_K3058000. Briefly, BBa\_K3058000 DH5a colonies were picked to grow in 5ml LB solution overnight. The starter cultures were inoculated into 200ml LB and allowed to grow for 6 hours. When the OD600 reached to 0.9, cells were harvested by centrifugation. The total protein was extracted by freeze-thaw method and finally diluted into 1XPBS . Uricase assay was then performed according to the manufacturer's protocol. The absorbance of uricase and controls were being monitored at regular time intervals. Compare with the control, our BBa\_K3058000-expressing E.coli could give a bright red color in the assay, indicating a strong uricase activity. The absorbance was qualified. According to student t’s test, the difference between the control and our sample is significant, with p-value smaller than 0.05. According to the standard curve plotted, the concentration of uricase is about 25uM/mL on average. This assay proved that this biobrick worked as expected.

## Result: [BBa\_K3058001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3058001) expressed HIU

![](https://static.igem.org/mediawiki/2019/1/18/T--Hong_Kong_LFC_PC--hiuTP.jpg)

Uricase assay was also used to determine the effect of HIU hydrolase expressed by BBa\_K3058001. Using the same steps above, BBa\_K3058001 colonies were cultured and harvested by centrifugation. The total protein was extracted and Uricase assay was then performed. The absorbance of uricase and controls were being monitored at regular time intervals. The result revealed that HIU hydrolase does not have significant result on the uricase activity.