Team:PuiChing Macau/Results
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), 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) 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
1a) Amylase (BBa_K3863006) qPCR
1b) Glucoamylase (BBa_K3863006) qPCR
1c)PhaC (BBa_K3863008) qPCR
1d) oPCT (BBa_K3863008) qPCR
1e) Phasin (BBa_K3863008) qPCR
1f) PhaC (BBa_K3863004) qPCR
1g) PCT(optimized) (BBa_K3863004) qPCR
1h) PHA (BBa_K3863007) qPCR
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).
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)
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)
Figure 3a. O.D.600 of BL21 culture with starch and without starch
Figure 3b. GFP signal intensity of BL21 culture with starch and without starch
Figure 3c. O.D.600 signal intensity of BL21 culture with starch and without starch (normalized)
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.
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), 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).
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.
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)
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)
Figure 4c. LB agar plate 50μg/mL (E. coli BL21(DE3) in pH 4
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.
Figure 5a IR spectrum of pETDuet vector
Figure 5b IR spectrum of BBa_K3863004
Figure 5c IR spectrum of BBa_K3863008
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.
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