iGEM 2019 — Team PuiChing_Macau

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# Team:PuiChing Macau/Notebook

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2019/thumb/e/e0/T--PuiChing_Macau--Logo--Mono--White.png/320px-T--PuiChing_Macau--Logo--Mono--White.png)](https://2019.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2019.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2019.igem.org/Team%3APuiChing_Macau/Design)
  + [Modelling](https://2019.igem.org/Team%3APuiChing_Macau/Model)
  + [Result](https://2019.igem.org/Team%3APuiChing_Macau/Results)
  + [Parts](https://2019.igem.org/Team%3APuiChing_Macau/Parts)
  + [Hardware](https://2019.igem.org/Team%3APuiChing_Macau/Hardware)
  + [Demonstration](https://2019.igem.org/Team%3APuiChing_Macau/Demonstrate)
* Outreach

  + [Integrated Human Practices](https://2019.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2019.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Public Engagment](https://2019.igem.org/Team%3APuiChing_Macau/Public_Engagement)
* Lab

  + [Notebook](https://2019.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2019.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2019.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us

  + [Team](https://2019.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2019.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3021)

![](https://static.igem.org/mediawiki/2019/8/84/T--PuiChing_Macau--OWEN--3.jpg)

# Notebook

This page documents all of the lab work that we have done this year.

## [Phase I: Characterization (Click here to open the document in a separate tab)](https://static.igem.org/mediawiki/2019/6/63/T--PuiChing_Macau--notebook--3.pdf)

## [Phase II: Engineered E.coli strain with Laccase secretion](https://static.igem.org/mediawiki/2019/3/33/T--PuiChing_Macau--notebook--2.pdf)

## [Phase III: Engineered Cyanobacteria strain with Laccase secretion](https://static.igem.org/mediawiki/2019/3/37/T--PuiChing_Macau--notebook--1.pdf)

* [![](https://static.igem.org/mediawiki/2019/f/fe/T--PuiChing_Macau--Sponsor--PuiChing.png)](http://www.puiching.edu.mo/index.php/en/main/)
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* [![](https://static.igem.org/mediawiki/2019/b/b3/T--PuiChing_Macau--Sponsor--FDCT.png)](http://www.fdct.gov.mo/en/index.html)
* [![](https://static.igem.org/mediawiki/2019/thumb/6/66/T--PuiChing_Macau--Sponsor--IDT.png/799px-T--PuiChing_Macau--Sponsor--IDT.png)](https://www.idtdna.com/pages)
* [![](https://static.igem.org/mediawiki/2019/thumb/e/ee/T--PuiChing_Macau--Sponsor--Twist.png/320px-T--PuiChing_Macau--Sponsor--Twist.png)](https://twistbioscience.com/)
* [![](https://static.igem.org/mediawiki/2019/0/00/T--PuiChing_Macau--Sponsor--NEB.png)](https://www.neb.com/)

* Project
  + [Description](https://2019.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2019.igem.org/Team%3APuiChing_Macau/Design)
  + [Modelling](https://2019.igem.org/Team%3APuiChing_Macau/Model)
  + [Result](https://2019.igem.org/Team%3APuiChing_Macau/Results)
  + [Parts](https://2019.igem.org/Team%3APuiChing_Macau/Parts)
  + [Hardware](https://2019.igem.org/Team%3APuiChing_Macau/Hardware)
  + [Demonstration](https://2019.igem.org/Team%3APuiChing_Macau/Demonstrate)
* Outreach
  + [Integrated Human Practices](https://2019.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2019.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Public Engagment](https://2019.igem.org/Team%3APuiChing_Macau/Public_Engagement)
* Lab
  + [Notebook](https://2019.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2019.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2019.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us
  + [Team](https://2019.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2019.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3021)

* @iGEMPuiChingMacau

* igem.wmc@g.puiching.edu.mo

© 2019 iGEM PuiChing Macau Media Team


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

# Team:PuiChing Macau/Experiments

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2019/thumb/e/e0/T--PuiChing_Macau--Logo--Mono--White.png/320px-T--PuiChing_Macau--Logo--Mono--White.png)](https://2019.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2019.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2019.igem.org/Team%3APuiChing_Macau/Design)
  + [Modelling](https://2019.igem.org/Team%3APuiChing_Macau/Model)
  + [Result](https://2019.igem.org/Team%3APuiChing_Macau/Results)
  + [Parts](https://2019.igem.org/Team%3APuiChing_Macau/Parts)
  + [Hardware](https://2019.igem.org/Team%3APuiChing_Macau/Hardware)
  + [Demonstration](https://2019.igem.org/Team%3APuiChing_Macau/Demonstrate)
* Outreach

  + [Integrated Human Practices](https://2019.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2019.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Public Engagment](https://2019.igem.org/Team%3APuiChing_Macau/Public_Engagement)
* Lab

  + [Notebook](https://2019.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2019.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2019.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us

  + [Team](https://2019.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2019.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3021)

# Protocols

## Material List

### 1. PCR

* New England BioLabs Q5® Hot Start High-Fidelity 2X Master Mix. Catalog number: M0494S

### 2. Gel electrophoresis

* Bio Rad 50x TAE Buffer Catalog number: 166-0742
* Bio Rad Certified™ Molecular Biology Agarose
* Invitrogen SYBR® Safe DNA gel stain. Catalog number: S33102
* New England BioLabs Gel Loading Dye Purple (6x). Catalog number: B7024S
* Invitrogen 1 Kb Plus DNA Ladder. Catalog number: 10787018

### 3. DNA and Gel Purification

* illustra™ GFX™ PCR DNA and Gel Band Purification Kit

### 4. DNA Assembly

* New England BioLabs NEBuilder® HiFi DNA Assembly Master Mix. Catalog number: M5520AA

### 5. Transformation of Escherichia coli

### 6. Miniprep plasmid purification

* QIAprep® Spin Miniprep Kit

### 7. Exfection™ Plasmid EF midi

* GeneAll® Exfection™ Plasmid EF midi

### 8. Blue-Green Medium (BG11) preparation

### 9. Nitrocellulose Membrane preparation

* Bio Rad Nitrocellulose Membrane, 0.2um

### 10. Transformation of Cyanobacteria (Synechococcus sp.)

* SIGMA Spectinomycin dihydrochloride penta-hydrate

### 11. Western bolt

## Experiment Protocol

### 1. PCR

|  |  |
| --- | --- |
|  | 1x |
| forward primer | 2.5µL |
| reverse primer | 2.5µL |
| Template | 1µL |
| H2O | 19µL |
| NEB Q5® Hot Start High-Fidelity 2X Master Mix | 25µL |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| step 1 | step 2 (35cycles) | | | step 3 | step 4 |
| initial denaturation | denaturation | anneal | extension | final extension | stock |
| 98°C | 98°C | 55°C | 72°C | 72°C | 4°C |
| 3mins | 10s | 30s | \* | 2mins | ∾ |

\*30 seconds/kb

### 2. Gel electrophoresis

1.Gel preparation

|  |  |
| --- | --- |
|  | 1x |
| Bio Rad TAE Buffer | 30mL |
| Bio Rad Certified™ Molecular Biology Agarose | \* |
| Invitrogen SYBR® Safe DNA gel stain | \*\* |

\*depend on the desired gel concentration

\*\*1µL SYBR® Safe per 1mL TAE Buffer

2.Sample loading

DNA sample : Gel Loading Dye Purple (6x) =5:1

### 3. DNA and Gel Purification

1. Cut gel and measure the weight.
2. Add Capture Buffer 3 (10µL/10mg), shake and heat at 60°C, 1000rpm for 10mins with ThermoMixer.
3. Transfer 750µL mixture to a microspin column, centrifuge at 16000×g for 30s.
4. Add 600µL Wash Buffer 1 and centrifuge at 16000×g for 30s twice.
5. Add 50µL Elution Buffer and centrifuge at 16000×g for 30s.

### 4. DNA Assembly

1. Calculations

Calculation rules: Vector:Insert=1:2 (in pmol)

Total DNA amount: 0.03pmol~0.2pmol

Total DNA volume<=10µL

pmol=ng\*1000/(bp\*650)

2. According to the calculations, mix the DNA samples and 10µL of NEBuilder® HiFi DNA Assembly Master Mix M5520AA.
3. Put the mixture at 50°C for 15 mins.

### 5. Transformation of Escherichia coli

1. Add the assembled product to 100µL of competent cells (*Escherichia coli*), place on ice for 20mins.
2. Heat shock at 42°C for 1min.
3. Transfer to ice for 2mins.
4. Add 900µL of LB to the tube, incubate at 37°C for 1 hour. Shake at 250rpm.
5. Warm the LB agar plate with antibiotics\* to 37°C.
6. Centrifuge at 12000rpm for 30s. Concentrate the cells to 100µL.
7. Spread 100µL cells onto the plate.
8. Incubate overnight at 37°C.

\*antibiotics (spectinomycin/ampicillin/cam): 50µg/mL

### 6. Miniprep plasmid purification

1. Pellet 1.5mL bacteria each tube and centrifuge at 6800×g for 3mins at room temperature for 3 times.
2. Resuspend pelleted bacteria in 250µL Buffer P1.
3. Add 250µL Buffer P2 and mix 6 times.
4. Add 350µL Buffer N3 and mix 6 times.
5. Centrifuge for 10mins at 13000rpm.
6. Apply 800µL supernatant to the column, centrifuge for 30s.
7. Add 0.5mL Buffer PB and centrifuge for 30s.
8. Add 0.75mL Buffer PE and centrifuge for 60s.
9. Centrifuge for 1min.
10. Add 30µL Buffer EB and stand for 1min. Centrifuge for 1min. Stock at -20°C.

### 7. Exfection™ Plasmid EF midi

1. Pellet 200mL (50mL each tube) of bacterial culture by centrifugation for 10mins at 5000×g in a tabletop centrifuge. Discard the supernatant as much as possible.
2. Resuspend pelleted bacterial cells thoroughly in 4mL of Buffer P1. Combine into 2 tubes.
3. Add 4mL of Buffer P2 and mix by inverting the tube 5~6 times (do not vortex). Incubate until the cell suspension becomes clear. Do not incubate for more than 5mins.
4. Add 4mL of Buffer P3 and thoroughly but gently mix by inverting the tube 7~8 times (do not vortex).
5. Centrifuge at 4500×g for 10mins, then transfer the supernatant into Ezclear™ filter (blue ring) sitting on a 50mL conical collection tube (provided). Let it stand for 2mins and centrifuge for 2mins at 1500×g (2800rpm).
6. Apply 500µL of Buffer ER to the filtrate and close the cap of 50mL conical tube.
7. Vortex to mix and incubate for 15mins on ice.
8. Incubate for 15mins at 37ºC and centrifuge for 2mins at 1500×g (2800rpm).
9. Transfer carefully the upper phase (clear) into a fresh 15mL conical tube (provided) by pipetting.
10. Add ½ volume of Buffer EG to the transfer and invert several times to mix completely.
11. Transfer all the mixture to SV midi column (clear ring) by decanting or pipetting. Centrifuge for 2mins at 1500×g. Remove the column, discard the pass-through, and re-insert the column to the collection tube.
12. Apply 10mL of Buffer EW1, centrifuge for 2mins at 1500×g. Remove the column, discard the pass-through and re-insert the column to the collection tube.
13. Apply 10mL of Buffer EW2, centrifuge for 2mins at 1500×g. Remove the column, discard the pass-through, and re-insert the column to the collection tube.
14. Apply 3mL of Buffer EW2, centrifuge for 15mins at 4500×g. Transfer the column to a new 50mL conical tube (provided).
15. Add 0.6mL of Buffer EF directly onto the centre of the column membrane and close the cap. Incubate for 5mins at room temperature and centrifuge for 5mins at 4500×g.

### 8. Blue-Green Medium (BG11) preparation

1. Stocks

|  |  |  |
| --- | --- | --- |
|  |  | per 500mL |
| 1 | NaNO3 | 75.0g |

|  |  |  |
| --- | --- | --- |
|  |  | per 500mL |
| 2 | H2HPO4 | 2.0g |
| 3 | MgSO4•7H2O | 3.75g |
| 4 | CaCl2•2H2O | 1.80g |
| 5 | Citric Acid | 0.30g |
| 6 | Ammonium ferric citrate green | 0.30g |
| 7 | EDTA Na2 | 0.05g |
| 8 | Na2CO3 | 1.00g |

|  |  |  |
| --- | --- | --- |
| 9 |  |  |
|  | Trace metal solution | per liter |
|  | H3BO3 | 2.86g |
|  | MnCl2•4H2O | 1.81g |
|  | ZnSO4•7H2O | 0.22g |
|  | Na2MoO4•2H2O | 0.39g |
|  | CuSO4•5H2O | 0.08g |
|  | Co(NO3)2•6H2O | 0.05g |

2. Medium

|  |  |
| --- | --- |
|  | per liter |
| Stock solution 1-8 | 10.0mL |
| Stock solution 9 | 1.0mL |

3. Agar

|  |  |
| --- | --- |
|  | per liter |
| BG11 medium | 1000mL |
| Agarose | 25g |

### 9. Nitrocellulose Membrane preparation

1. Cut the membrane into circles.
2. Soak the membranes into distilled water for 5mins and pour it out. Repeat this step for three times.
3. Soak the membranes into distilled water overnight.
4. Heat the membranes using a microwave oven for 3mins and autoclave.

### 10. Transformation of Cyanobacteria (Synechococcus sp.)

1. Collect 15 mL cell (OD730=0.8-1.0) from the conical flask.
2. Centrifuge at 4000rpm for 10mins, discard the supernatant.
3. Using the same volume of BG11 medium to resuspend the cells and centrifuge at 4000rpm for 10mins again to wash the cells.
4. Separate the cells into 200µL portions, add plasmids (conc. 10µg/mL) to each portion. Light incubate for 4 hours.
5. Spread 100µL of the mix on the nitrocellulose membranes which are soaked in BG11 agar plates without antibiotics. Light incubate overnight (for 18-20 hours).
6. Transfer the nitrocellulose membranes with the cells to a new BG11 agar plate containing antibiotics\*. Light incubate for 7-10 days until the single colonies appear. Check periodically for the growth condition and water condensation to prevent other bacteria growing on the plate.
7. Take a single colony, spread it on a new BG11 agar plate containing antibiotics and incubate under light.
8. Extract the cells growing on the new BG11 agar plate, culture in fresh BG11 medium containing antibiotics for 4 days (until logarithmic phase).
9. PCR validation: Pellet 1-2mL of cells to a microcentrifuge tube, lysis the cells by shaking with quartz sands to extract the DNA. Take 0.5µL of the DNA as a template to conduct PCR and take 3µL of the PCR product to conduct gel electrophoresis. Use the DNA of wild type Cyanobacteria as a control.

\*spectinomycin (10µg/mL)

### 11. Protein extraction

1. Pellet 100mL of bacterial culture by centrifugation for 10mins at 6000×g in a tabletop centrifuge. Discard the supernatant as much as possible.
2. Resuspend pelleted bacterial cells in 250µL of 50M HEPES.
3. Microtip sonication: 40%AMPL, 45s processing, 1s on-off. (3 times)
4. Centrifuge at 1200×g for 10mins.
5. Transfer the supernatant to a microspin tube, stock at -20ºC.

### 12. Western bolt

1. Protein processing: the extracted proteins were added into 4xloading, then boiled in water for 10min
2. Sds page preparing and runing: 12% sds-gel refer to **SDS gel preparation**.
3. Transfer the gel into PDFV membrane: refer to **Transfer membrane**.
4. Antibody incubation : primary antibody : anti-His rabbit developed(1:2000 diluted); second antibody: anti-rabbit conjugated HRP(1:5000 diluted) refer to **Antibiotic staining**.

### 13. SDS gel preparation

1. 10% resolving gel solution

|  |  |
| --- | --- |
|  | 1x |
| SureCast™ Acrylamide (40%) | 2.0mL |
| SureCast™ Resolving Buffer | 2.0mL |
| Distilled water | 3.9mL |
| 10% SureCast™ APS | 80µL |
| Total | 8mL |

2. Add 8 μL of SureCast™ TEMED for every 8 mL of resolving gel solution. Mix well (but gently) and proceed immediately to Pour resolving gel.
3. Tilt the handcast station to recline on the heel.
4. Add resolving gel solution to the glass plates until the solution reaches the level of the fill line.
5. Carefully overlay the resolving gel solution with butanol, or isopropanol.
6. Set the handcast station back to the upright position.
7. Allow the resolving gel to polymerize (5–10 min). The interface becomes more distinct as the gel polymerizes.
8. Verify polymerization by examining left over acrylamide in the tube.
9. If overlay was used, pour off the overlay solution and rinse with water (dispose of waste in the appropriate manner). Wick out any remaining liquid with a piece of blotting paper, and proceed to Prepare stacking gel solution.
10. 4% stacking gel solution

|  |  |
| --- | --- |
|  | 1x |
| SureCast™ Acrylamide (40%) | 0.30mL |
| SureCast™ Staking Buffer | 0.75mL |
| Distilled water | 1.92mL |
| 10% SureCast™ APS | 30µL |
| Total | 3mL |

11. Add 3 μL of SureCast TEMED for every 3 mL of resolving gel solution. Mix well and proceed immediately to Pour stacking gel.
12. Tilt the handcast station to recline on the heel.
13. Add the stacking gel solution until it reaches the upper edge of the front glass plate.
14. Set the handcast station back to the upright position.
15. Insert the comb slowly by starting at one end and sliding it between the glass plates until both ends are in place.
16. Allow the stacking gel to polymerize (5–10 min).
17. Verify polymerization by examining left over acrylamide in the tube.
18. Release the clamp and remove the plate. The gel can be used immediately, or wrapped in a damp paper towel and stored at 4°C for future use.

### 14. Transfer membrane

1. Place the cathode core (–) on a flat surface.
2. Add 5–10 mL of 1X Transfer Buffer to the cathode core (–).
3. Place a soaked sponge pad into the cathode core (–) of the blot module. Remove any bubbles with the Blotting Roller.
4. Take the gel with filter paper (from “Prepare gel for transfer” step 11, page 12) and place it on top of the sponge pad, with the gel side on top.
5. Wet the surface of the gel with 1X Transfer Buffer and remove any bubbles with the Blotting Roller.
6. Place a pre-soaked transfer membrane on the gel. Remove any bubbles with the Blotting Roller.
7. Wet a piece of filter paper in 1X Transfer Buffer and place it on top of the transfer membrane. Remove any bubbles with the Blotting Roller.
8. Place a pre-soaked sponge pad onto the filter paper. Remove any bubbles with the Blotting Roller.
9. Complete the module assembly by placing the anode core (+) on top of the assembled blot sandwich.
10. Press the two module halves together.
11. Snap the electrophoresis tank into the base. Make sure any Cassette Clamps are removed from the chambers.
12. Insert the blot module with the cathode core (–) facing the front. The blot module should be seated so that the electrodes clips of the module make contact with the electrode bar of the electrophoresis tank.
13. If necessary, add 1X Transfer Buffer to the module core to completely submerge the blot sandwich. Note: do not fill above the level of the gasket in the blot module.
14. Make sure the power supply is off and place the cover on the tank. Push all the way down so that the cover is firmly in place.
15. Plug the power leads into your power supply.
16. Turn on the power supply, program settings (see “Transfer Conditions”) as needed, and start transfer.
17. After protein transfer is complete, turn the power supply off, unplug the power leads, and remove the lid.
18. Remove the blot module from the chamber and empty the transfer buffer into an appropriate hazardous waste disposal container.
19. Open the module assembly.
20. Disassemble the blot sandwich and carefully remove the membrane with Blotting Tweezers.
21. Wash the membrane using 20 mL of ultra-pure water for 5 minutes, two times.

### 15. Antibiotic staining

1. Block the membrane for 1 h at room temperature or overnight at 4°C using blocking buffer.
2. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer. We recommend overnight incubation at 4°C; other conditions can be optimized.
3. Wash the membrane in three washes of TBST, 5 min each.
4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.
5. Wash the membrane in three washes of TBST, 5 min each.
6. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
7. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.

### 16. Dye decolorization

1. Without ABTS:

|  |  |
| --- | --- |
|  | 1x |
| BR Buffer\* | 20µL |
| 500mg/L Dye\*\* | 4µL |
| Sample\*\*\* | 33.4µL |
| 50mM HEPES | 142.6µL |

With ABTS:

|  |  |
| --- | --- |
|  | 1x |
| BR Buffer | 20µL |
| 500mg/L Dye | 4µL |
| Sample | 33.4µL |
| 100mM ABTS | 2µL |
| 50mM HEPES | 140.6µL |

\*pH=5

\*\*Coomassie Blue, Congo Red, Bromophenol Blue, Indigo Carmine

\*\*\*3µg/µL total protein/50mM HEPES (blank of protein)/LB (blank of supernatant)

2. Apply the configured mixture to a 96-well plate with three identical replicates for each experimental group.
3. Measure the optical density\* of each well.
4. Seal the 96-well plate and incubate at 37ºC. Remeasure the optical density every 24 hours.

\*Coomassie Blue:465nm, 595nm; Congo Red: 488nm; Bromophenol Blue: 593nm; Indigo Carmine: 610nm.

### 17. Nickel pull down

1. Make up 50mL of the following binding buffer: 20mM Tris 7.5, 150mM NaCl, 1%
   Triton-X100.
2. Thaw 20mL medium on ice.
3. Prepare nickel resin
4. Pipette 500μL medium to resuspend the resin, and pipette all into the 20mL medium.
5. Incubate and rotate it at 4°C for 45 min.
6. Spin at 3900 rpm for 10min, remove supernatant carefully (store this, just in case!), leave ~ 1mL fraction to resuspend the resin, transfer resin to a 1.5mL tube. 4000g, 2min spin down the resin, decant the supernatant.
7. Wash the resin with 750uL binding buffer (X3) (Note: don’t try to remove all the flow through or buffer in every wash, you will lose resin that way, just in the very last.
8. wash try and removes as much as buffer as possible.
9. Add 30μL 2X SDS dye, 98°C, 10min.

### 18. EDC degradation

|  |  |
| --- | --- |
|  | 1x |
| sodium citrate buffer (pH=5) | 500µL |
| 3µg/µL protein sample / Ni-pulldown product | 500µL |
| 200µM ABTS | 1.6µL |
| 100µM Beta-estradiol | 0.8µL |

2. Shake the mixture above at 25°C for 36hr.
3. Mix 100µL of the mixture with 400µL Acetonitrile. Centrifuge the sample at >10000rpm for 5mins to remove precipitated protein.
4. Transfer the supernatant to another tube and dry it by nitrogen blow.
5. Re-dissolve the dried sample in an appropriate volume (depends on concentration of targeted molecule in your sample) of acetonitrile by vivid vortex.
6. Centrifuge the sample at >10000rpm for 5mins to remove insoluble salt. The supernatant is then ready for LCMS analysis.

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# Team:PuiChing Macau/Results

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# Results

## Laccase secretion from recombinant E.coli

     Endocrine disrupting chemicals (EDC) are mostly man-made compound, which would be found in various daily materials such as pesticides, metals, additives or contaminants in food, and personal care products. It has been proposed by World Health Organization that EDCs would affect humans later in life and associated with an increased incidence of breast cancer, abnormal growth patterns and neurodevelopmental delays in children, as well as changes in immune function.

     Laccases are copper-containing enzyme, which would be found in many plants, fungi, and microorganisms. Due to its function in oxidizing phenolic and nonphenolic aromatic compounds, Laccases are increasingly being used in different industries, including food, pulping, textile and wastewater treatment. Regarding EDCs, phenolic compounds have constituted a part of this family and previous studies have already demonstrated Laccase's ability in bioremediation such chemicals.

     Current wastewater treatment technologies are yet to satisfy the need of EDCs removal. The secretion of Laccases would potentially improve the current treatment and increase. The present section focuses on the secretion of recombinant Laccases from engineered E.coli.

### Overview

     This study describes the construction and characterization of several new BioBricks, which will allow the future investigation of Laccases secretion by the SRP and Sec pathway, as the proposed mechanism of NSP4-Novel Signal Peptide 4. The findings of present studies suggest that our new Bio-Bricks are a success and would be expressed, and fully functional.

### Background

     The type II secretion pathway is a two-step process, which proteins are secreted out of the cell by periplasmic translocation. This is included the SecB-dependent (Sec), signal recognition particle (SRP), and twin-arginine translocation (TAT) pathways, and are commonly found in gram-negative bacteria. pelB and dsbA are sequences important to the Sec and SRP pathway. Through swapping individual domains of modifying dsbA and pelB signal peptides, a series of novel signal peptides were designed and demonstrated to have improved secretion efficiency in E. coli.

### Aim

     In this sub-project, we aim to improve Laccase production by introducing a inducible secretion system. We investigated the effect of the new Biobricks in dye decolorization and EDC degradation.

### Result

### 1. Successful assembly of gene constructs

![](https://static.igem.org/mediawiki/parts/b/b7/T--PuiChing_Macau--results_23.png)

Figure 1: Agarose gel electrophoresis(1% agarose) of PCR amplified products.

Lane 1: Invitrogen 1Kb plus DNA Ladder.

Lane 2: PCR product with T7 short and T7 term primers using K3021004\_pET11a (NSP4\_Lac1326\_pET11a) as a template.

Lanes 3-8: PCR products with T7 short and T7 term primers using K3021002\_pET11a (NSP4\_K863030\_pET11a) from different plasmids as templates.

Lanes 9: Negative control.

Figure 1 shows the agarose Gel electrophoresis of PCR. We first extracted DNA from the antibiotic-resistant clones, and then validation through PCR with T7 short and T7 term primers. The product of PCR in lane 2 shows a signal band at 1522 bp while products from lane 3 to 8 show a band at 1850 bp. The size match expected size of K3021004\_pET11a (NSP4\_Lac1326\_pET11a\_6xHis) and K3021002\_pET11a (NSP4\_K863030\_pET11a\_6xHis) respectively. These results indicated the successful assembly of the insert part (NSP4\_Lac1326\_6xHis and NSP4\_K863030\_6xHis) and the expression vector (pET-11a).

T7 short: TAATACGACTCACTATAGG

T7 term: CTAGTTATTGCTCAGCGGT

### 2. Constructs have correct sequences (check with sequencing and sequence alignment)

K3021004\_pET11a (NSP4\_Lac1326\_pET11a):
![](https://static.igem.org/mediawiki/2019/5/59/T--PuiChing_Macau--results_3.png)

Figure 2. Representative sequencing results of K3021004\_pET11a (NSP4\_Lac1326\_pET11a)

K3021002\_pET11a (NSP4\_K863030\_pET11a):
![](https://static.igem.org/mediawiki/2019/6/62/T--PuiChing_Macau--results_4.png)

Figure 3. Representative sequencing results of K3021002\_pET11a (NSP4\_K863030\_pET11a)

To futher confirm that our constructs have correct sequence, we send our clones (with positive PCR results) out to sequencing.

The sequencing results show that we have successfully assembled target genes K3021004 (NSP4\_Lac1326\_6xHis; Figure 2) and K3021002 (NSP4\_K863030\_6xHis; Figure 3) to the vector (pET-11a).

### 3. Successful expression of laccase protein

To validate the protein expression of K3021004 (with Lac1326 laccase), K863000 and K3021002 (with K863030 laccase), we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make Lac1326, K863000 and K3021002(with K863030 laccase) easily detectable via Western Blot, we designed our constructs to include a “His tag” which is targeted by a commercially available antibody. With the SDS-PAGE and western blot (anti-his for his-tagged protein), we found successful expression of K3021004 (with Lac1326 laccase), K863000 and K3021002 (with K863030 laccase).

Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K3021004 (with Lac1326 laccase), K863000 and K3021002 (K863030) under control of the T7 promoter. Cells were induced with 0.4mM, 1.0mM or 1.2mM IPTG until either OD0.4 or OD1.2 (wavelength: 600nm) at 25°C or 30°C. After expression, cells were then lysed according to our protein extraction protocol, and supernatant and pellet were collected for the SDS-PAGE.

For more experimental details of the Western Blot and SDS-PAGE, please see [protocol](https://2019.igem.org/Team%3APuiChing_Macau/Protocol).
![](https://static.igem.org/mediawiki/2019/6/64/T--PuiChing_Macau--results_5.png)

Figure 4. Western Blot results of inducible expression of K3021004 (Lac1326), K3021002 (K863030) and K863000.

(a)

Lane1: pET-11a empty vector;* Lane2: pET-11a empty vector+IPTG induction
* Lane3: K3021004 (Lac1326) colony1;
* Lane4:K3021004 (Lac1326) colony1+IPTG induction
* Lane5: K3021004 (Lac1326) colony2;
* Lane6:K3021004 (Lac1326) colony2+IPTG induction
* Lane7: K3021004 (Lac1326) colony3;
* Lane8:K3021004 (Lac1326) colony3+IPTG induction
* Lane9: K3021002 (K863030) colony1;
* Lane10: K3021002 (K863030) colony1+IPTG induction

(b)

* Lane1: K863000 colony2;
* Lane2: K863000 colony2+IPTG induction
* Lane3: K863000 colony3;
* Lane4: K863000 colony3+IPTG induction
* Lane5: K3021002 (K863030) colony1;
* Lane6: K3021002 (K863030) colony1+IPTG induction
* Lane7: K3021002 (K863030) colony2;
* Lane8: K3021002 (K863030) colony2+IPTG induction
* Lane9: K3021002 (K863030) colony3;
* Lane10: K3021002 (K863030) colony3+IPTG induction

As shown in Figure 4, Western Blot was performed to validate the presence of the his-tagged K3021004 (with Lac1326 laccase), K863000 and K3021002 (with K863030 laccase) protein. Anti-RNA polymersase beta was used as a loading control.
At 58 kD, we found a band in lane 3 to lane 8 which corresponds to the approximate expected size of K3021004 (with Lac1326 laccase). We also found a band at 64kD in lanes 9 and 10, which is the expected size of K3021002 (with K863030 laccase).

![](https://static.igem.org/mediawiki/2019/8/8f/T--PuiChing_Macau--results_6.png)

Figure 5. The figure represents the expression of different laccases under a variable of IPTG. The protein expression is calculated by the ratio of anti-His and anti-RNA polymerase beta (Loading control).

As we used IPTG inducible promoter (T7) in our study, we further analyze the results of Western Blot to compare the laccase expression with/without IPTG. As shown in Figure 5, the expressions of different laccases under a variable of IPTG were compared. K3021004 (Lac1326), K3021002 (K863030) and K863000 with 1mM IPTG have exhibited higher expression levels (33.97, 41.66 and 28.75 folds, respectively) than K3021004 (Lac1326), K3021002 (K863030) and K863000 without IPTG (5.53, 21.99 and 15.53 folds). There was an enhanced expression of the laccases with IPTG as expected.

![](https://static.igem.org/mediawiki/2019/1/12/T--PuiChing_Macau--results_7.png)
![](https://static.igem.org/mediawiki/2019/2/20/T--PuiChing_Macau--results_8.png)

Figure 6. The figure represents expression of laccases under differnt conditions. Anti-RNA polymerase beta was used as loading control.

* Lane1: K3021004 (Lac1326): OD=0.4, IPTG=0.4mM, 30°C
* Lane2: K3021004 (Lac1326): OD=0.4, IPTG=0.4mM, 25 °C
* Lane3:K3021004 (Lac1326): OD=0.4, IPTG=1mM, 30 °C
* Lane4:K3021004 (Lac1326): OD=1.2, IPTG=1mM, 30 °C
* Lane5:K863000: OD=0.4, IPTG=0.4mM, 30 °C
* Lane6:K863000: OD=0.4, IPTG=0.4mM, 25 °C
* Lane7:K863000: OD=0.4, IPTG=1mM, 30 °C
* Lane8:K863000: OD=1.2, IPTG=1mM, 30 °C
* Lane9:K3021002 (K863030): OD=0.4, IPTG=0.4mM, 30 °C
* Lane10:K3021002 (K863030): OD=0.4, IPTG=0.4mM, 25 °C
* Lane11:K3021002 (K863030): OD=0.4, IPTG=1mM, 30 °C
* Lane12:K3021002 (K863030): OD=1.2, IPTG=1mM, 30 °C
* Lane13: pET-11a empty vector without IPTG

After confirming the expression of laccases and confirming the laccases under T7 promoter can be regulated by IPTG, we further tested how laccases vary across different conditions (cell growth phase, temperatures, IPTG concentration), as shown in Figure 6.

![](https://static.igem.org/mediawiki/2019/a/a8/T--PuiChing_Macau--results_9.png)

Figure 7. The figure represents the expression of (a) K3021004 (Lac1326) (b) K863000 (c) K3021002 (K863030) with different temperatures, E. coli concentration and iPTG concentration. The protein expression is calculated by the ratio of anti-his and anti-RNA polymerase beta (Loading control).

The figure compares the expression of K3021004 (Lac1326), K863000 and K3021002 (K863030) under different conditions including temperature, E coli and IPTG concentration. K3021004 (Lac1326) expressed under OD=0.4, IPTG=0.4mM at 25°C, K863000 expressed under OD=0.4, IPTG=0.4mM at 30°C and K3021002 (K863030) expressed under OD=0.4, IPTG=0.4mM at 30°C had displayed the highest fold changes of 24.58, 51.33 and 44.16 respectively, compared with the pET-11a empty vector, signifying that OD=0.4, IPTG=0.4mM are comparably the best expression condition for all of the laccases while 25°C is the best expression temperature for K3021004 (Lac1326) and 30°C for K863000 and K3021002 (K863030).

### 4. K863000 and K863010 induction results

![](https://static.igem.org/mediawiki/2019/4/42/T--PuiChing_Macau--results_21.png)

Figure 8. The figure represents the Western Blot and SDS-PAGE (coomassie blue) of K863000 and K863010 laccase. Both laccase should be with His-tag.

Consistent with characterization by previous iGEM teams, we found no expression of laccase with K863010 plasmid (contains a fungal laccase, expected to be IPTG regulated), as shown in Figure 8.

To the best of our knowledge, there is no functional IPTG-regulated fungal laccase in the iGEM part registry. We here successfully add one (K3021002), as an improvement of the part K863010, which cannot be expressed.

### 5. Secretion signal peptides are functional in laccases

![](https://static.igem.org/mediawiki/2019/a/a2/T--PuiChing_Macau--results_11.png)

Figure 9. Expression of laccase with his-tag and secretion signal peptide in cell-free medium

* Lane1: K3021004 (Lac1326): OD=0.4, IPTG=0.4mM, 30°C
* Lane2: K3021004 (Lac1326): OD=0.4, IPTG=0.4mM, 25°C
* Lane3:K3021004 (Lac1326): OD=0.4, IPTG=1mM, 30°C
* Lane4:K3021004 (Lac1326): OD=1.2, IPTG=1mM, 30°C
* Lane5:K3021002 (K863030): OD=0.4, IPTG=0.4mM, 30°C
* Lane6:K3021002 (K863030): OD=0.4, IPTG=0.4mM, 25°C
* Lane7:K3021002 (K863030): OD=0.4, IPTG=1mM, 30°C
* Lane8:K3021002 (K863030): OD=1.2, IPTG=1mM, 30°C
* Lane9: pET-11a empty vector without IPTG

![](https://static.igem.org/mediawiki/2019/4/47/T--PuiChing_Macau--results_12.png)

Figure 10. The figure shows the laccases (K3021004 (Lac1326) and K3021002 (K863030)) secretion from the engineered E. coli by Nickel-pulldown assay. Tested with a one-sample student t-test. \*\*=p value=0.002739

Since we designed a secret form for the two laccases, we hypothesized that there would be his-tagged protein in the medium in which two genetically engineered E. coli were cultured.

First of all, we obtained the cell-free medium by spinning down the cells. We then used the Nickel-pulldown Assay and Western Blot to confirm the expression of his-tagged laccase. The results (Figure 9 and Figure 10) showed that the fold changes of K3021004 (with Lac1326 laccase) and K3021002 (with K863030 laccase) was 389.5 and 62.12 (compared to pET-11a control), indicating expression of K3021004 (with Lac1326 laccase) (significant) and K3021002 (with K863030 laccase) in the medium .

For detailed Nickel-pulldown protocol, please refer to our [protocol](https://2019.igem.org/Team%3APuiChing_Macau/Protocol) page.

### 6. Laccases expressed can decolorize dye

![](https://static.igem.org/mediawiki/2019/8/8c/T--PuiChing_Macau--results_13.jpeg)
![](https://static.igem.org/mediawiki/2019/9/92/T--PuiChing_Macau--results_14.jpeg)

Figure 11. Representative experiment of dye decolorization

![](https://static.igem.org/mediawiki/2019/f/f4/T--PuiChing_Macau--results_15.png)

Figure 12. Decolorization of Indigo Carmine (initial concentration of 10 mg/L) by different laccases (OD 0.4, IPTG 0.4mM, 25 °C) at different times with ABTS.

In order to investigate the biodegradable reactivity of the laccases, dye (Indigo Carmine) decolorization experiments were conducted (Figure 11).

As shown in Figure 12, the laccase K863000 from Bielefied-Germany 2012 was the most efficient enzyme with 81.09% removal of Indigo Carmine (initial concentration: 10 mg/L) after 96 hours incubation with ABTS at 37°C comparing to K3021004 (Lac1326) and K3021002 (K863030) which decolorized 47.62% and 22.01% of Indigo Carmine after 96 hours.

### 7. Successful EDC degradation by laccases

![](https://static.igem.org/mediawiki/2019/2/27/T--PuiChing_Macau--results_16.png)

Figure 13. Chromatogram of Beta-estradiol (Quantitative ion pair: 297.049/106.906) in various protein (a) Blank in ACN (b) K3021002 (K863030) (c) K863000 (d) K3021004 (Lac1326) (e) pET-11a

![](https://static.igem.org/mediawiki/2019/a/ab/T--PuiChing_Macau--results_17.png)

Figure 14. Liquid Chromatography–Mass Spectrometry analysis for Beta-estradiol degradation by different laccases expressed under the condition of OD=0.4, IPTG=0.4mM and 25°C.

In order to validate our concept of using laccases to degrade Endocrine Disrupting Chemicals, biodegradation test were conducted. We used beta-estradiol (one of the EDCs) as our the target substrate. After incubation of laccases with beta-estradiol, we analyzed the amount beta-estradiol with Liquid Chromatography-Mass Spectrometry.

As shown in Figure 13 and Figure 14, the concentration of beta-estradiol decreased after treatment with K3021004 (with Lac1326 laccase), K863000 and K3021002 (with K863030 laccase) proteins, compared with pET-11a (empty vector), which successfully demonstrated our concept of utilizing K3021004 (with Lac1326 laccase) for EDC degradation. Among all of the laccases, K863000 and K3021004 (with Lac1326 laccase) had respectively displayed the lowest and second lowest intensity of 2137.657 and 2421.598, signifying K863000 possesses the highest biodegradation activity of all.

Moreover, we found that the results of dye decolorization and EDC degradation are consistent. Protein K863000 has the highest laccase activity, followed by K3021004 (with Lac1326 laccase) (OD0.4, IPTG=0.4mM, 25&degC;) and then K3021002 (with K863030 laccase).

For detailed EDC degradation protocol, please refer to our [protocol](https://2019.igem.org/Team%3APuiChing_Macau/Protocol) page.

### Conclusion

Overall, we confirmed that laccases with signal peptides (K3021002 and K3021004) can be successfully expressed in E. coli. We also confirmed that laccases with signal peptides are functional in decolorization and EDC degradation. Moreover, we proved that our signal peptides can export his-tagged laccase to the medium, which may help to degrade EDCs outside the cell. We believe that our project significantly contributes to utilizing laccase in EDC degradation. We hope that with these kinds of engineered laccases, we could help to eliminate EDC pollution in the future.

## Laccase secretion from recombinant Cyanobacteria

     EDCs pollution is not solely an issue in wastewater. EDCs are bioaccumulation compounds, which would affect the ecology system and our drinking water source. Since the effect of EDCs to nature may not become evident until later, such pollution has remained as unsolved. To address this issue, we propose an engineered cyanobacteria strain to secret Laccase, which would harvest sunlight as an extra energy source and provide a possible solution to achieve nature bioremediation.

### Overview

     This study describes the construction and characterization of several new BioBricks, which will allow the future investigation of Laccases secretion by the pila pathway. However, due to the limited time, this sub-project was yet to finish.

### Background

     Cyanobacteria are prokaryotic autotrophic organisms, which would be found in a different water source, ranging from pristine hot springs to eutrophic aquatic water bodies. They are frequently found in polluted sites and wastewater since cyanobacteria have high N, P requirements. Through the massive intake of such high loads of these nutrients from sewage, the water quality would be improved. In additional, cyanobacteria would also remove and tolerant pollutants like heavy metals and inorganic and organic contaminants, which allow them to adapt to the harsh environment.

### Aims

     In this sub-project, we aim to develop an engineered cyanobacteria strain to produce and secretion laccases, which sunlight would be harvest as an extra energy to 1) further boost the productivity, or 2) to adapt the harsh environment in nature.

### Results

#### PCR and Gel Electrophoresis

![](https://static.igem.org/mediawiki/2019/c/c7/T--PuiChing_Macau--Results_22.png)

Figure 15. The figure shows a variety of gel electrophoresis results of different DNA fragments.

(a)BBa\_K3021006 (pilA\_Lac1326\_pSCBe):

* Lane 1: 1Kb Plus Ladder;
* Lane 2: pilA\_Lac1326\_0;
* Lane 3: pilA\_Lac1326;
* Lane 4: L\_pSCBe\_0;
* Lane 5: L\_pSCBe

(b)BBa\_K3021007 (pilA\_Lac1326\_mCherry\_pSCBe):

* Lane 1: 1Kb Plus Ladder;
* Lane 2: pilA\_Lac1326\_pSCBe\_mC\_0;
* Lane 3: L\_mCherry\_0;
* Lane 4: pilA\_lac1326\_pSCBe\_mC;
* Lane 5: L\_mCherry.

The figure shows the PCR results. The band sizes in the figure are consistent with our expectations, which preliminarily indicates our success in PCR.

![](https://static.igem.org/mediawiki/2019/8/86/T--PuiChing_Macau--Results_23.png)

Figure 16. Different E. coli single colonies formed on the antibiotic plates.

(a) BBa\_K3021006 (pilA\_Lac1326\_pSCBe), (b) BBa\_K3021005 (pilA\_K863030\_pSCBe), (c) BBa\_K3021007 (pilA\_Lac1326\_mCherry\_pSCBe).

This figure shows that there are single colonies on the antibiotic plates, which indicates that both of them had an antibiotic resistance.

#### Check clone

![](https://static.igem.org/mediawiki/parts/8/8f/T--PuiChing_Macau--results_22.png)

Figure 17: Agarose gel electrophoresis(1% agarose) of PCR amplified products.

* Lane 1: Invitrogen 1Kb plus DNA Ladder.
* Lane 2: negative control.
* Lanes 3-10: PCR products with pSCBe\_seqF and pSCBe\_seqR2 primers using K3021005\_pSCBe(pilA\_k863030\_pSCBe\_1) from different plasmids as templates.
* Lanes 11-12: Empty.

Figure 15 shows the agarose Gel electrophoresis of PCR with pSCBe\_seqF and pSCBe\_seqR2 primers. We first extracted DNA from the antibiotic-resistant clones and then validated them with PCR. The product of PCR from lane 3 to 10 shows a band at 1850 bp which matches the length of K3021002\_pSCBe (pilA\_K863030\_pSCBe) PCR product, indicating the successful assembly of the insert part (pilA\_K863030\_6xHis) and the expression vector (pSCBe).

pSCBe-seqF: GGCTCGTATAATGTGTGGAA

pSCBe\_seqR2: TAGTAGAGAGCGTTCACCGAC

#### Alignment

![](https://static.igem.org/mediawiki/2019/a/ac/T--PuiChing_Macau--Results_24.png)

Figure 18. Representative sequencing results of K3021006\_pSCBe (pilA\_Lac1326 \_pSCBe).

![](https://static.igem.org/mediawiki/2019/5/5a/T--PuiChing_Macau--Results_25.jpg)

Figure 19. Representative sequencing results of K3021005\_pSCBe (pilA\_K863030 \_pSCBe).

To future confirm that our constructs have correct sequence, we send our clones out to sequencing.

The sequencing results show that we have successfully assembled target genes BBa\_K3021006 (pilA\_Lac1326\_6xHis; Figure 18) , BBa\_K3021005 (pilA\_K863030\_6xHis; Figure 19) and BBa\_K3021007 (pilA\_Lac1326\_mCherry\_6xHis; Figure 20) to the vector (pSCBe).

![](https://static.igem.org/mediawiki/2019/8/87/T--PuiChing_Macau--Results_26.jpg)

Figure20. Representative sequencing results of K3021007\_pSCBe (pilA\_Lac1326\_mCherry\_pSCBe).

#### Transformation of Cyanobacteria (Synechococcus sp.)

![](https://static.igem.org/mediawiki/2019/6/6f/T--PuiChing_Macau--Results_27.jpg)

Figure 21. Different concentrations of cyanobacteria culturing in the light incubator.

![](https://static.igem.org/mediawiki/2019/1/18/T--PuiChing_Macau--Results_28.png)

Figure 22. Transformed cyanobacteria on the spectinomycin plates.

After confirming the sequences of our constructs, we intended to transform the plasmids into cyanobacteria. We saught help from Dr. Song for the method of transformation and try our best, but because of the time limit, we didn’t achieve our goal.

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* Project
  + [Description](https://2019.igem.org/Team%3APuiChing_Macau/Description)
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