iGEM 2021 — Team PuiChing_Macau

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:PuiChing Macau/Notebook

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2021/a/a9/T--Puiching_Macau--logo2021.png)](https://2021.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2021.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2021.igem.org/Team%3APuiChing_Macau/Design)
  + [Engineering Success](https://2021.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Results](https://2021.igem.org/Team%3APuiChing_Macau/Results)
  + [Hardware & Contributions](https://2021.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Model](https://2021.igem.org/Team%3APuiChing_Macau/Model)
  + [Implementation](https://2021.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Lab

  + [Parts](https://2021.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2021.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2021.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2021.igem.org/Team%3APuiChing_Macau/Protocol)
* Outreach

  + [Human Practices & Integrated HP](https://2021.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2021.igem.org/Team%3APuiChing_Macau/Education)
  + [SDG](https://2021.igem.org/Team%3APuiChing_Macau/SDG)
* About Us

  + [Team](https://2021.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2021.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3863)

---

# **⭐ Please click the buttons below to show detailed information ⭐**

Week 1
Week 2~3
Week 4
Week 5~6
Week 7
Week 8
Week 9

### Order Plasmid and Experiment Preparation

**[BBa\_K3863007 PHA-flag-PCT-HA](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)**

**[BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)**

**[BBa\_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863008)**

**[BBa\_K3863003 fabB-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863003)**

GFP (J364000)

### 1.Exfection ™ EF Midi:

### 2.RT-qPCR:

* **[a.BBa\_K3863007 PHA-flag-PCT-HA](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)**
* **[b.BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)**
* **[c.BBa\_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863008)**
* **[d.BBa\_K3863003 fabB-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863003)**
* **[e.BBa\_K3863006 a-amylase-HA-glucoamylase-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863006)**

### [BBa\_K3863006: a-amylase-HA-glucoamylase-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863006)

* Transformation of E.coli* Pick Colony & Bacteria Culture Preparation

### Testing amounts of monosaccharides:

Glucose test result to compare the change of the amount of glucose between day 1 and day 2 in the addition of amylase and glucoamylase

### E.coli growth analysis in food waste:

##### BBa\_J3640000: Green Fluorescence Protein (GFP) BL21 (control)

1.Establishing model of E.coli growth in food waste with GFP and O.D.600 (with starch and without starch)

### [BBa\_K3863003 fabB-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863003)

### BL21 (control)

### Investigating Acid-Tolerant System (through CFU colony-forming unit):

* 1.Transformation of E. coli
* 2.Pick colony
* 3.IPTG Induction and Bacterial Culture Preparation

+ **[a. BBa\_K3863003 fabB-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863003)**

- i. uninduced
- ii. 0.1mM IPTG

+ b. BL21

- i. uninduced
- ii. 0.1mM IPTG

* 4.Spread plate and Count Colony

+ Investigating acid tolerant system, to see if the addition of fabB is capable for E.coli’s growth in acidic environment through colony forming unit (C.F.U).

### [BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)

### [BBa\_K3863007 PHA-flag-PCT-HA](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)

### pETDuet (vector control)

### Bioplastic production (without phasin):

* **[1. BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his (BL21)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)**

+ a. UN: uninduced
+ b. IN: 0.1mM IPTG

* **[2. BBa\_K3863007 PHA-flag-PCT-HA (BL21)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)**

+ a. UN: uninduced
+ b. IN: 0.1mM IPTG

* 3. pETDuet (vector control)

+ a. UN: uninduced
+ b. IN: 0.1mM IPTG

### Verifying Bioplastic Production through IR(Infrared Radiation) Spectroscopy:

* **[1.BBa\_K3863007 PHA-flag-PCT-HA](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)**
* **[2.BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)**

To test if PHA(K3863007), Phac(BBa\_K3863004) works for bioplastic production.

### [BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)

### [BBa\_K3863007 PHA-flag-PCT-HA](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)

### [BBa\_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863008)

### pETDuet (vector control)

### Bioplastic production (with phasin):

* Compare the result with and without phasin, to see if the yield of bioplastic production has increased with the addition of phasin.

+ 1. Produce Bioplastic Culture
+ 2. Producing Bioplastic
+ 3. Bioplastic Extraction

### Sample:

+ **[4. BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his (BL21)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)**

- a. UN: uninduced
- b. IN: 0.1mM IPTG

+ **[5. BBa\_K3863007 PHA-flag-PCT-HA (BL21)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)**

- a. UN: uninduced
- b. IN: 0.1mM IPTG

+ **[6. BBa\_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K38630048)**

- a. UN: uninduced
- b. IN: 0.1mM IPTG

+ 7. pETDuet (vector control)
  - a. UN: uninduced
  - b. IN: 0.1mM IPTG

### Nile Red - quantitative measurement of bioplastic production:

* **[1.BBa\_K3863004 Phac-flag-PCT(optimized)-HA-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)**
* **[2.BBa\_K3863007 PHA-flag-PCT-HA](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863007)**
* **[3.BBa\_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863008)**
* 4.pETDuet (vector control)

+ Compare the result with and without phasin, to see if the yield of bioplastic production has increased with the addition of phasin through quantitative measurement by measuring the intensity of Nile Red.

Go to Top

* [![](https://static.igem.org/mediawiki/2020/thumb/6/65/T--PuiChing_Macau--Wynn_Care.jpeg/598px-T--PuiChing_Macau--Wynn_Care.jpeg)](https://www.facebook.com/we.love.wynncare/)* * [![](https://static.igem.org/mediawiki/2019/f/fe/T--PuiChing_Macau--Sponsor--PuiChing.png)](http://www.puiching.edu.mo/index.php/en/main/)
    * [![](https://static.igem.org/mediawiki/2019/thumb/8/8e/T--PuiChing_Macau--Sponsor--MSAIEA.png/600px-T--PuiChing_Macau--Sponsor--MSAIEA.png)](https://www.facebook.com/MSAIEA/)
    * [![](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/2020/d/d0/T--PuiChing_Macau--Sponsor--UMFHS.jpg)](https://fhs.um.edu.mo/en/#/)
    * [![](https://static.igem.org/mediawiki/2019/thumb/e/ee/T--PuiChing_Macau--Sponsor--Twist.png/320px-T--PuiChing_Macau--Sponsor--Twist.png)](https://www.twistbioscience.com/)
    * [![](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/2019/0/00/T--PuiChing_Macau--Sponsor--NEB.png)](https://www.neb.com/)
    * [![](https://static.igem.org/mediawiki/2020/9/9d/T--PuiChing_Macau--Sponsor--MathWorks.png)](https://www.mathworks.com/)
    * [![](https://static.igem.org/mediawiki/2020/thumb/a/aa/T--PuiChing_Macau--Sponsor--Benchling.png/320px-T--PuiChing_Macau--Sponsor--Benchling.png)](https://www.benchling.com/)

* @iGEMPuiChing2021

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

© 2021 iGEM PuiChing Macau Media Team

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:PuiChing Macau/Engineering

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2021/a/a9/T--Puiching_Macau--logo2021.png)](https://2021.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2021.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2021.igem.org/Team%3APuiChing_Macau/Design)
  + [Engineering Success](https://2021.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Results](https://2021.igem.org/Team%3APuiChing_Macau/Results)
  + [Hardware & Contributions](https://2021.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Model](https://2021.igem.org/Team%3APuiChing_Macau/Model)
  + [Implementation](https://2021.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Lab

  + [Parts](https://2021.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2021.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2021.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2021.igem.org/Team%3APuiChing_Macau/Protocol)
* Outreach

  + [Human Practices & Integrated HP](https://2021.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2021.igem.org/Team%3APuiChing_Macau/Education)
  + [SDG](https://2021.igem.org/Team%3APuiChing_Macau/SDG)
* About Us

  + [Team](https://2021.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2021.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3863)

With the overall project goal to convert food waste into biodegradable and eco-friendly bioplastics, polylactic acid (PLA). Considering food waste contains different types, including Fruit & Vegetable, Meat, Carbohydrates etc. In order to achieve our goal of producing PLA in a one-step approach and promoting its efficiency, we tend to check different data of the food waste that we get. However, this requires innovative molecular design, sophisticated synthesis, and therefore long hour input and expensive resources. Therefore, we carried out the investigation to:

* To analyse the pH value of different type of food waste (Fruit & Vegetable, Meat, Carbohydrate)
* To analyse the growth of e.coli in different pH value
* To improve the production system and help increase the growth of e.coli in acidic environment

---

# **⭐ Please click the buttons below to show detailed information ⭐**

Build & Test (1st Cycle)
Learn & Improve (1st Cycle)
Build & Test (2nd Cycle)
Learn & Improve (2nd Cycle)

### Food Waste Analysis Protocol

### **Materials:**

---

* 60g of food waste
* 60mL of deionized water
* Blender
* Filter bag/gauze (for filtration of solid food waste)

### **Protocol:**

---

1. Collect 60 g of food waste (e.g. starch, vegetables, fruit, or trimmings)
2. Add 60mL of deionized water and 60g of food waste into a blender and blend it until most of the food waste has turned to liquid
3. Pour the mixture into a filter bag/gauze to obtain the liquid filtrate
4. Analyze the pH value of the liquid filtrate using a pH meter or pH test strips

![engineering success pic](https://static.igem.org/mediawiki/2021/2/2d/T--PuiChing_Macau--engineeringsuccesspic.jpg)

Figure 1. The average pH value of the three main categories of food ( Fruit & Vegetable, Meat, Carbohydrate)

Based on the data we got in the food waste analysis, the result shows that most of the food waste performs in the range of pH4-pH7 in acidic or neutral environments, which may inhibit the survival and growth of the E.coli. In this case, we have to confirm and analyse the effect on the growth of E.coli in the acidic environment, so we built up a test (CFU (WT4 & WT7) with IPTG to mimic the growth of E. coli)
To analyse if the pH value will inhibit the growth of the E.coli using colony-forming unit (CFU) to estimate the number of e.coli grown in the Agar plate. Counting with colony-forming units requires culturing the microbes and counts only viable cells.

### **Materials:**

---

1. Agar Plates in pH4 & pH7 (calculate the amount of agar plates needed depends on the dilution range)
2. Snap cap tubes
3. Sample ( BL21(DE3)induced)

### **Experimental steps:**

---

1. After induction overnight, add 200ul sample into 96 well plate and measure the OD of each sample, use LB as a blank.
2. calculate the amount of bacteria culture needed
   e.g. sample 1 OD: 0.5 sample 2 OD: 0.6 sample 3 OD: 0.55 Use 0.5 to calculate, sample 1—>2ml，sample 2—>1.667ml，sample 3—>1.818ml
   Equation: (lowest OD value among the samples)(Final volume) / (OD value)(sample volume)
3. Separate the samples into 2 groups: pH 7 and pH 4
   e.g. sample 1: 1ml for pH7 and 1ml for pH4
4. Centrifuge the samples at 5,000rpm to get the pellets
5. Wash cells twice with respective pH minimal medium (Add100ul of respective pH minimal medium to the sample and centrifuge with 5000rpm)
6. Resuspend the pellets in 2 ml minimal medium (pH 7 and pH 4 for each samples) in snap cap tubes
7. Take 25ul from each samples and do serial dilution (For 10-1: 25ul sample + 225 ul respective pH minimal medium)
8. Spread plate with the dilution 10-5, 10-6, 10-7, 10-8

### **Result:**

---

![engineering success pic1](https://static.igem.org/mediawiki/2021/0/0a/T--PuiChing_Macau--engineeringsuccesspic1.jpg)

Figure 2. Number of  E.coli colonies (WT: BL21 control) in pH4 and pH7

From the graph, we can find out in pH4 and pH7 the total number of e.coli have a big difference, which confirms that the acidic environment inhibits the growth of E. coli. To solve the problem our team has developed an acid-tolerant system using fabB genes to try to increase the production of unsaturated fatty acids by increasing the growth of E.coli in an acidic environment.

### Build & Test: CFU

---

CFU (WT4 & WT7) with IPTG to mimic the growth of E. coli

To analyse if the pH value will inhibit the growth of the E.coli useing colony-forming unit (CFU) to estimate the number of e.coli grown in the Agar plate, and check if the Acid-Tolerant System does help increase the growth of the E.coli in acidic environment. Counting with colony-forming units requires culturing the microbes and counts only viable cells.

### **Materials:**

---

Sample: **[BBa\_K3863005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863005)**

Control: BL21(DE3)

1. Agar Plates in pH4 & pH7 (calculate the amount of agar plates needed depends on the dilution range)
2. Snap cap tubes
3. Sample (uninduced & induced)

### **Experimental steps:**

---

1. After induction overnight, add 200ul sample into 96 well plate and measure the OD of each sample, use LB as a blank.
2. calculate the amount of bacteria culture needed
   e.g. sample 1 OD: 0.5 sample 2 OD: 0.6 sample 3 OD: 0.55 Use 0.5 to calculate, sample 1—>2ml，sample 2—>1.667ml，sample 3—>1.818ml
   Equation: (lowest OD value among the samples)(Final volume) / (OD value)(sample volume)
3. Separate the samples into 2 groups: pH 7 and pH 4
   e.g. sample 1: 1ml for pH7 and 1ml for pH4
4. Centrifuge the samples at 5,000rpm to get the pellets
5. Wash cells twice with respective pH minimal medium (Add100ul of respective pH minimal medium to the sample and centrifuge with 5000rpm)
6. Resuspend the pellets in 2 ml minimal medium (pH 7 and pH 4 for each sample) in snap cap tubes
7. Take 25ul from each sample and do serial dilution (For 10-1: 25ul sample + 225 ul respective pH minimal medium)
8. Spread plate with the dilution 10-5, 10-6, 10-7, 10-8

### **Learn**

---

After adding the Acid-Tolerant system that we have designed, e.coli growth have a significant increase compare to the e.coli without the system in pH4 ( the acidic environment), whereas in pH7, the Acid-Tolerant does not have negative impacts on the growth of the E.coli, the growth range is similar in both with and without the Acid-Tolerant system, which proves that the system does help increase E.coli growth in the acidic environment.

![](https://static.igem.org/mediawiki/2021/a/a1/T--PuiChing_Macau--learn.jpg)

Figure 3a. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 4 (N=4, error bar: SEM)

![](https://static.igem.org/mediawiki/2021/1/14/T--PuiChing_Macau--resultfig4b.jpg)

Figure 3b. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 7 (N=4, error bar: SEM)

### **Improve**

---

Based on the analysis of the first and second cycle mentioned, our data are slightly in line with the theory, we have proved that the Acid-Tolerant System we created does help to improve the growth of E.coli in pH4, for the further experiment, we aim to have more test on different pH value from pH4-pH7, more specific test to confirm the growth of the E.coli and the effect on our production system.
Furthermore, we found out that Acid-Tolerant System which comprised both FabB and FabA gene is proven to be effective for the growth of the E.coli in acidic environment, we hope that we can have further experiments on these and compare if it is more beneficial or not to add the FabA gene in our Acid-Tolerant System.

Go to Top

* [![](https://static.igem.org/mediawiki/2020/thumb/6/65/T--PuiChing_Macau--Wynn_Care.jpeg/598px-T--PuiChing_Macau--Wynn_Care.jpeg)](https://www.facebook.com/we.love.wynncare/)* * [![](https://static.igem.org/mediawiki/2019/f/fe/T--PuiChing_Macau--Sponsor--PuiChing.png)](http://www.puiching.edu.mo/index.php/en/main/)
    * [![](https://static.igem.org/mediawiki/2019/thumb/8/8e/T--PuiChing_Macau--Sponsor--MSAIEA.png/600px-T--PuiChing_Macau--Sponsor--MSAIEA.png)](https://www.facebook.com/MSAIEA/)
    * [![](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/2020/d/d0/T--PuiChing_Macau--Sponsor--UMFHS.jpg)](https://fhs.um.edu.mo/en/#/)
    * [![](https://static.igem.org/mediawiki/2019/thumb/e/ee/T--PuiChing_Macau--Sponsor--Twist.png/320px-T--PuiChing_Macau--Sponsor--Twist.png)](https://www.twistbioscience.com/)
    * [![](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/2019/0/00/T--PuiChing_Macau--Sponsor--NEB.png)](https://www.neb.com/)
    * [![](https://static.igem.org/mediawiki/2020/9/9d/T--PuiChing_Macau--Sponsor--MathWorks.png)](https://www.mathworks.com/)
    * [![](https://static.igem.org/mediawiki/2020/thumb/a/aa/T--PuiChing_Macau--Sponsor--Benchling.png/320px-T--PuiChing_Macau--Sponsor--Benchling.png)](https://www.benchling.com/)

* @iGEMPuiChing2021

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

© 2021 iGEM PuiChing Macau Media Team

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:PuiChing Macau/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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:PuiChing Macau/Results

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2021/a/a9/T--Puiching_Macau--logo2021.png)](https://2021.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2021.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2021.igem.org/Team%3APuiChing_Macau/Design)
  + [Engineering Success](https://2021.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Results](https://2021.igem.org/Team%3APuiChing_Macau/Results)
  + [Hardware & Contributions](https://2021.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Model](https://2021.igem.org/Team%3APuiChing_Macau/Model)
  + [Implementation](https://2021.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Lab

  + [Parts](https://2021.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2021.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2021.igem.org/Team%3APuiChing_Macau/Safety)
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# Overview

In this project, the conversion of food waste into biodegradable plastic is being focussed as it was shown to potentially provide a re-use opportunity for food waste. We have built our model with a bioplastic (polylactic acid (PLA) and polyhydroxyalkanoates (PHA)) production system and an acid-tolerant system, and validated our goal of producing PLA and PHA and promoting its efficiency. We transform the PhaC and PCT genes into *E.coli* so that we are able to convert food waste into polylactic acid PLA and polyhydroxyalkanoates PHA.
This study described the construction and characterization of a bioplastic production system using *E.coli*.the conversion of food waste into biodegradable plastic is being focussed as it was shown to potentially provide a secured, meaningful and marketable re-use opportunity for food waste. We verified our constructs (BBa\_K3863004, BBa\_K3863006, BBa\_K3863007, BBa\_K3863008), confirming their expressions using RT-qPCR. Furthermore, we have tested the amount of conversion of food waste to monosaccharide using the Glucose Test Kit (Suzhou Grace Biotechnology) for BBa\_K3863006. Also, we have designed an acid-tolerant system composed of the fabB gene to tackle the low pH value due to the presence of lactic acid and the low pH of the food waste, the raw material for our bioplastic production system, to ensure the growth of *E.coli* in different pH environments and the process’ estimated yield, we examined the acid resistance of the fabB gene through counting colonies of cultured *E.coli* with and without fabB in pH4 and pH7. We also test our system by qualitative and quantitative testing of bioplastic production, through IR-spectrum and Nile Red staining respectively. Our findings here suggest that our new Bio-Bricks were successfully expressed and were functional, which validates the idea of the bioplastic production system.

# Background

Based on the achievement of iGEM 2013 Yale((BBa\_K1211002), a PLA and PHA production system consisting of PhaC and PCT, was proposed, for which we here further improved. We also used the Biobricks (PhaC; BBa\_K2042001) from iGEM 2016 Evry to produce our target bioplastic. We have also optimized the PCT and PhaC gene for E. coli, and added a phasin enzyme to increase the rate of production. In addition, our project also tried to use food waste as the bacteria medium, in hope of turning food waste into bioplastics. Here we make use of amylase, glucoamylase ([BBa\_K3863006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863006)), an enzyme to break starch in food waste into monosaccharides. To achieve our goal of producing PLA and PHA in a one-step approach, we have also designed our Biobricks ([BBa\_K3863004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863004)) for PLA and PHA production. To further improve the efficiency, we added the bioplastic production gene phasin into our constructs to promote the efficiency and effectiveness of the bioplastic production system.

# Development of different bioplastic-producing E. coli strains

Our system consists of 3 three parts: (1) Conversion of food waste into monosaccharide using amylase and glucoamylase; (2) acid tolerant system in resisting acidic environment in food waste by fabB gene; (3) Bioplastic production system by PHA, PCT(optimized) and PhaC gene. DNA of all novel BioBricks were ordered from Integrated DNA Technologies (IDT, State of Lowa) using the iGEM sponsor credit. The DNA fragments of amylase and glucoamylase with kanamycin resistance were then cloned into the pCOLADuet vector (IDT, State of Lowa). Subsequently, fabB gene with Streptomycin resistance were cloned into pCDFDuet and PHA, PCT(optimized), PCT gene with ampicillin resistance were cloned into pETDuet vector, followed by transformation into *E.coli* strain BL21(DE3) and an antibiotic selection.

# Validation of construct expression using RT-qPCR

![](https://static.igem.org/mediawiki/2021/7/7e/T--PuiChing_Macau--1aa.jpg)

1a) Amylase (BBa\_K3863006) qPCR

![](https://static.igem.org/mediawiki/2021/9/92/T--PuiChing_Macau--1b.jpg)

1b) Glucoamylase (BBa\_K3863006) qPCR

![](https://static.igem.org/mediawiki/2021/c/cb/T--PuiChing_Macau--1c.jpg)

1c)PhaC (BBa\_K3863008) qPCR

![](https://static.igem.org/mediawiki/2021/2/2d/T--PuiChing_Macau--1d.jpg)

1d) oPCT (BBa\_K3863008) qPCR

![](https://static.igem.org/mediawiki/2021/3/3b/T--PuiChing_Macau--1e.jpg)

1e) Phasin (BBa\_K3863008) qPCR

![](https://static.igem.org/mediawiki/2021/7/72/T--PuiChing_Macau--1ff.jpg)

1f) PhaC (BBa\_K3863004) qPCR

![](https://static.igem.org/mediawiki/2021/c/cc/T--PuiChing_Macau--1g.jpg)

1g) PCT(optimized) (BBa\_K3863004) qPCR

![](https://static.igem.org/mediawiki/2021/3/35/T--PuiChing_Macau--1h.jpg)

1h) PHA (BBa\_K3863007) qPCR

![](https://static.igem.org/mediawiki/2021/8/81/T--PuiChing_Macau--1i.jpg)

1i) PCT (BBa\_K3863007) qPCR

Figure 1. Real time qPCR fold change of reference gene

Through qPCR, we were able to validate the RNA expression levels of our reference gene. As shown in Fig 1a and 1b, both amylase and glucoamylase have very strong expression after IPTG induction for 80 and 480 on average respectively, which validates the expression level of the BBa\_K3863006. In Fig 1c to 1i, PhaC, Phasin, PHA, PCT(optimized) and PCT gene had slight increase in expression after IPTG treatment with an increase of 0.2-0.6 fold change, which means our biobricks BBa\_K3863004, BBa\_K3863007 and BBa\_K3863008 were successfully expressed.

# *E.coli* growth in food waste analysis

To examine the growth of *E.coli* in food waste, Optical Density of *E.coli* is commonly used. Yet, in the presence of food waste (starch from rice as an example here), it is difficult to measure the Optical Density of *E.coli* growth at a wavelength of 600 nm (O.D.600), where determining the growth phase is almost impossible. Thus, we would like to use Green Fluorescent Protein (GFP) (488 nm and 510 nm) to establish a model of *E.coli*’s growth in food waste (starch).
We estimate the amount of bacteria in culture by assessing the emission wavelength for fluorescence signal at O.D.510 and O.D.600.
The tested samples are composed of 100ml of LB medium and 1.5ml of overnight bacteria culture. The time point measured are: 0hr, 4hr, 12hr, 24hr
As demonstrated by Fig.3 (3a, 3b, 3c, 3d), samples with GFP have shown higher absorbance compared with controls of BL21. And samples in the presence of food waste (starch) have also shown higher absorbance than those without food waste (starch).

![](https://static.igem.org/mediawiki/2021/5/5b/T--PuiChing_Macau--results4.jpg)

Figure 2a 2b Experiment culture (bacteria and GFP) centrifuged: 2a normal; 2b under UV light

(from left to right: 1. LB 2. LB+starch 3. GFP+starch 4. GFP 5. BL21+starch 6. BL21)

![](https://static.igem.org/mediawiki/2021/d/d2/T--PuiChing_Macau--result5.jpg)

Figure 2c 2d Experiment culture (bacteria and GFP) under UV light: 2c with starch; 2d without starch

(from left to right: 1. BL21+starch 2. GFP+starch 3. GFP 4. BL21)

![](https://static.igem.org/mediawiki/2021/0/01/T--PuiChing_Macau--result6.jpg)

Figure 3a. O.D.600 of BL21 culture with starch and without starch

![](https://static.igem.org/mediawiki/2021/7/76/T--PuiChing_Macau--resulttt.jpg)

Figure 3b. GFP signal intensity of BL21 culture with starch and without starch

![](https://static.igem.org/mediawiki/2021/2/27/T--PuiChing_Macau--result8.jpg)

Figure 3c. O.D.600 signal intensity of BL21 culture with starch and without starch (normalized)

![](https://static.igem.org/mediawiki/2021/5/5c/T--PuiChing_Macau--result9.jpg)

Figure 3d. GFP signal intensity of BL21 culture with starch and without starch (normalized)

# Validation of the efficiency of amylase

In acknowledgement, starch can be broken down into monosaccharides by amylase. In note that food waste contains large amount of starch, breaking down starch into monosaccharides should increase the contamination of monosaccharides in food waste. Hence, the raw materials of bioplastic production are generated from fermentation of monosaccharides. Therefore, in our project, through increasing the content of monosaccharides in food waste, the yield of bioplastic production should be increased.
IPTG induction is conducted to investigate our engineered protein, and the final concentration reaches 0.1mM.
To validate the efficiency of amylase, we have tried to conduct a test to find the amount of monosaccharides contained in food waste by Glucose Test Kit (Suzhou Grace Biotechnology), with the addition of overnight culture of amylase (single positive colony from our recombinant E.coli strains), together with the control.
Each sample consists of 2ml overnight culture (not needed for control), 250ml M9 medium, 250ul antibiotics (Kanamycin), 5g starch and 250ul IPTG (only needed for induced samples). Every sample is tested with the original sample and 2 dilutions.
In the test, glucose, together with the presence of enzyme complexes such as hexokinase, NADP+ is reduced to NADPH. By detecting the increase of NADPH at 340 nm, the contents of glucose is calculated.

![](https://static.igem.org/mediawiki/2021/8/86/T--PuiChing_Macau--resulttable1.jpg)

Table 1. Glucose test result to compare the change of the amount of glucose between day 1 and day 2 in the addition of amylase and glucoamylase

Theoretically, with the addition of amylase and glucoamylase([BBa\_K3863006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3863006)), the amount of glucose should have increased since starch should be broken down into monosaccharides. Yet, the results turn out to be unreasonable on both control and experimental groups. As shown by the table, the change of amount of glucose between day 1 and day 2 are less than 0, meaning that the amount of glucose has decreased instead of increasing.

Therefore, we presume that the glucose may have been taken up by *E.coli* and are not released. Thus, the content of glucose in the medium decrease beacuse of this. To find out more, further testing on this part (about amylase and glucoamylase), for instance, extracting proteins from cells to test the efficiency, is required.

# Investigation of the acid-tolerant system of our engineered protein

In food waste processing and fermentation, the pH of the environment will be changed due to the presence of lactic acid, which can easily inhibit the growth of *E.coli*. Indeed, results obtained from the measurement of the food waste show that many of the food waste from the daily basis state are in pH4-pH7, which can reduce the efficiency of our PLA production system. In order to solve this problem, we designed an acid tolerance system composed of the fabB gene.
To investigate the Acid-Tolerant system of our engineered protein, IPTG was added to the bacterial cultures. IPTG addition was achieved at a time of 4 hours to reach the concentration of 1mM and incubate overnight.
Briefly, to compare and analyse the growth of *E.coli* in different pH environments, we conducted overnight culture of a single positive colony from each of our recombinant *E.coli* strains and get re-transformation to agar plate with different pH environments (pH4 and pH7), and compare it with and without the Acid-Tolerant system (the fabB gene)
When comparing the data, colony forming units (CFU) were used to estimate the number of *E.coli* growing in agar plates. Colony forming units were used to count the microorganisms to be cultured and only live cells were counted. In order to ensure that our samples produce CFU within this range, the samples need to be diluted and plated with several diluents (10-5,10-6,10-7 and 10-8).

*Total colonies（CFU/mL) =

No. of colonies \*Total dilution factor/Volume of the culture plate in mL*

As shown in Figure 4, we can see that the pH environment does have effects on the growth of the *E.coli*, there are fewer colonies in pH4 compared to the one in pH7, therefore, it shows that it is necessary for us to add the Acid-Tolerant system. After adding the Acid-Tolerant system, containing with the fabB gene, the total number of *E.coli* have a significant increase compare to the *E.coli* without the system in pH4 ( the acidic environment), whereas in pH7, the fabB gene does not have negative impacts on the growth of the *E.coli*, the growth range is similar in both with and without the Acid-Tolerant system, which proves that the fabB gene does help increase *E.coli* growth in the acidic environment.

![](https://static.igem.org/mediawiki/2021/4/4b/T--PuiChing_Macau--resultfig4a.jpg)

Figure 4a. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 4
(N=4, error bar: SEM)

![](https://static.igem.org/mediawiki/2021/1/14/T--PuiChing_Macau--resultfig4b.jpg)

Figure 4b. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 7
(N=4, error bar: SEM)

![](https://static.igem.org/mediawiki/2021/b/b6/T--PuiChing_Macau--resultfig4c.jpg)

Figure 4c. LB agar plate 50μg/mL (E. coli BL21(DE3) in pH 4

![](https://static.igem.org/mediawiki/2021/5/53/T--PuiChing_Macau--result4d.jpg)

Figure 4d. LB agar plate 50μg/mL (E. coli BL21(DE3) in pH 7

# Confirmation of our produced bioplastic

In order to verify the production of PLA, infrared spectroscopic (IR) analysis was used. Since the bioplastic PLA has its particular functional group in chemical structure, and therefore can be detected with particular IR absorption wavelengths. The absorption peak of PLA is at 1081, 1188, 1364, 1452 , 1751 cm-1[1],  whereas the absorption peak is 979, 1057, 1100, 1282, 1723, 2934, 2977cm-1[2]. We have compared and analysed the peak of the wavelength to confirm the bioplastic product we have produced.
In Figure 5a, no absorption peak of PHA and PLA was identified, which indicated that the vector control (pETDuet Vector) cannot produce PLA as we would expect. On the other hand, in Figure 5b and 5c,  the absorption peaks of PHA and PLA were identified after incubation, which suggested that the BBa\_K3863004 (in Figure 5b) and BBa\_K3863008 (in Figure 5c), the bioplastic performs significantly in the transformation process. Furthermore, the absorption peaks of PHA in Figure 5d were found, which showed that BBa\_K3863007 performs well and was the product that we expected to have. These results have proven that the bioplastic can be produced with our constructs BBa\_K3863004, BBa\_K3863008 and BBa\_K3863007.

![](https://static.igem.org/mediawiki/2021/7/75/T--PuiChing_Macau--result13.jpg)

Figure 5a IR spectrum of pETDuet vector

![](https://static.igem.org/mediawiki/2021/e/ed/T--PuiChing_Macau--result14.jpg)

Figure 5b IR spectrum of BBa\_K3863004

![](https://static.igem.org/mediawiki/2021/1/18/T--PuiChing_Macau--result15.jpg)

Figure 5c IR spectrum of BBa\_K3863008

![](https://static.igem.org/mediawiki/2021/d/d0/T--PuiChing_Macau--result16.jpg)

Figure 5d IR spectrum of BBa\_K3863007

# Quantitative Measure of Bioplastic Production

In order to measure the amount of bioplastic produced, we used 20 microgram Nile Red (sigma aldrich N3013) per milliliter of agar to make a Nile agar plate and streak the cell culture (BBa\_K3863007, BBa\_K3863004, BBa\_K3863008) on the plate. After incubating for 2 days at 37 celsius in darkness. Stereomicroscope (Nikon SMZ18) is used to measure the intensity of Nile red.

![](https://static.igem.org/mediawiki/2021/c/c5/T--PuiChing_Macau--result0000.jpg)

Figure 6. Mean intensity of Nile red for quantitative measure of bioplastic production of (BBa\_K3863007(PHA), BBa\_K3863004(PhaC), BBa\_K3863008(Phasin))

As shown in the figure, it clearly indicated the mean intensity of Nile red and showed a significant difference compared with the vector with a mean intensity of about 70. It was important to note that (BBa\_K3863008(Phasin)) had the greatest mean intensity among 3 constructs of about 120, which indicated that the addition of Phasin allows us to promote the production of bioplastic. Furthermore, the mean intensity of (BBa\_K3863007(PHA)) and (BBa\_K3863004(PhaC)) lied at 90 and 80 respectively, which illustrated a successful plastic production.

# Conclusion

All in all, we are building up our project with the bioplastic (PLA and PHA) production system and the acid-tolerant system, in order to achieve our goal of producing PLA and PHA in a one-step approach and promoting its efficiency.

# Reference:

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3450024/pdf/12088\_2009\_Article\_31.pdf

[2]https://www.researchgate.net/figure/Fourier-transform-infrared-FTIR-spectra-of-PLA-PEG-PLA-PEG-blend-and-PLA-PEG-xGnP\_fig1\_277675367

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