Team:Calgary/Results
Results
Synthesis Results
Characterization of pET29b(+)-phaCJ
The objective of our project was to genetically engineer E. coli DH5α to synthesize PHB. We designed pET29b(+)-phaJC construct so that the E. coli uses the β-oxidation pathway to break down fatty acids such as VFAs and undigested long-chain fatty acids to synthesize PHB. We have successfully ligated and transformed this part into E.coli. The figure below shows the digestion confirmation of the vector pET29b(+)-phaCJ. We have also sequence verified this part.
To confirm the production of protein, we induced gene expression in cultures of the transformed E. coli that contained pET29b(+)-phaCJ with IPTG and performed SDS-PAGE on the proteins. Our results are shown in the picture below. The SDS-PAGE results indicated that our protein PhaJ was being expressed. It was difficult to distinguish PhaC amongst the other protein bands. However, because PhaJ is downstream of PhaC, it is likely that PhaC was being expressed as well. Furthermore, our PHB synthesis experiment with E. coli containing pET29b(+)-phaCJ produced PHB as shown in Figure 3. The successful synthesis of PHB indicated the functionality of the proteins.
To confirm the functionality of E. coli transformed with pET29b(+)-phaCJ to produce PHB, we grew O/N of our cultures for 24 hours and induced them with 0.1mM of IPTG while supplementing them with chemical media mimicking fermented synthetic poop supernatant(FSPS) To extract the PHB, we performed chemical extraction on the culture samples following the EXTRACTION PROTOCOL
To confirm the identity of the white PHB made by the transformed E. coli, we ran our results alongside a sample of pure industrial-grade PHB provided to use by PolyFerm Canada by dissolving PHB in sulfuric acid, which leads to the production of crotonic acid peaks. The analysis confirmed the identity of our PHB because the presence of crotonic acid was indicated by the peaks. Furthermore, our PHB was comparable to industrial-grade PHB because the peaks for the samples matched.
Detailed results of these experiments can be found on this part's Registry page.
Characterization of pET29b(+)-phaCBA
As part of our project, we also used a pathway that relied on acetyl CoA (produced from metabolism of glucose and some VFAs) to produce PHB. We designed pET29b(+)-phaCBA construct so that the E. coli breaks down acetyl Co-A and converts the products into PHB. We have successfully ligated and transformed this part into E.coli. The figure below shows the digestion confirmation of the vector pET29b(+)-phaCBA. We have also sequence verified this part.
To confirm the identity of the white PHB made by the transformed E. coli, we ran our results alongside a sample of pure industrial-grade PHB provided to use by PolyFerm Canada by dissolving PHB in sulfuric acid, which leads to the production of crotonic acid peaks. The analysis confirmed the identity of our PHB because the presence of crotonic acid was indicated by the peaks. Furthermore, our PHB was comparable to industrial-grade PHB because the peaks for the samples matched.
Detailed results can be found on this part's Registry page.
Secretion Results
Characterization of pSB1C3-Phasin-HlyA Tag
In order to secrete PHB, we engineered Phasin-HlyA Tag first into E. coliDH5α for plasmid propagation then into E. coliBL21(DE3) for protein expression. Our Phasin_hlyA Tag construct was designed for use in psB1C3. Figure 8 shows the successful digestion of Phasin-HlyA Tag with EcoRI-HF and SpeI then ligation into pSBiC3./p>
To confirm protein expression we carried out SDS-Page of E. coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA Tag. BL21(DE3) transformed with an empty pSB1C3 vector was used as a control. Both E. coli were incubated to an OD550 between 0.4-0.8 then induced with IPTG. IPTG is necessary to induce the expression of T7 RNA polymerase in BL21(DE3) so that our Phasin-HlyA-Tag could be transcribed by the T7 polymerase. The culture was then separated into soluble and insoluble fractions, as well as supernatant, which we hoped would contain the Phasin protein. Initial SDS-Page experiments showed too many protein bands to be able to see a band for Phasin-HlyA Tag (27 kDa), therefore we prepared anti-FLAG resin and incubated the sample fractions with it in order to separate our Phasin protein from the other proteins that were present. After incubation with the resin, the sample fraction were run on a SDS-Page gel, the results of which can be seen in Figure 9. All lanes of the gel appeared empty, even samples from our pSB1C3-Phasin-HlyA Tag, which has an expected band size around 27 kDa.
Our final attempt at SDS-Page gel used Immunoblotting. Samples from both E. coli variants were prepared as described above, however the sample fractions were incubated with an anti-FLAG rabbit antibody instead. Then, goat anti-rabbit with igG-HRP was used as a secondary antibody to detect the presence of FLAG-tagged proteins by a color change. HRP changes color to blue if the secondary antibody binds to the anti-FLAG rabbit antibody bound to a FLAG-tagged protein (our Phasin-HlyA Tag).
To confirm the functionality of Phasin-HlyA Tag, a "super" plasmid that contains both PHB-producing and PHB-secreting genes was made. This plasmid was created by ligating Phasin-HlyA Tag into pSB1C3-PhaCAB (PhaCAB is a PHB-producing part from Imperial College, 2013). This plasmid was transformed into E. coli BL21(DE3). The successful ligation of the "super" plasmid was confirmed with NotI-HF digestion, as shown in Figure 10.
For a secretion assay, triplicates of the "super" plasmid in BL21(DE3) were inoculated into LB media + chloramphenicol + 3 % glucose and triplicates of pSB1C3-PhaCAB in BL21(DE3) were used as a control. The bacteria were induced with IPTG then incubated at 37°C for either 24 hours or 48 hours. After that time, the samples were separated into intracellular and secreted PHB fractions and the efficiency and rate of secretion was calculated.
Detailed results can be found on this part's Registry page.
Process Development Results
Methods for VFA quantification and characterization
As mentioned in our journal, determination of the total VFA concentration in the solution was an important step in the process – knowing how to quantify total VFAs in the solution helped to prove that the fermentation of human feces with naturally occurring bacteria increases the VFA concentration, as well as it helped to prove VFA presence in both - fermented and unfermented synthetic feces.
Titration is commonly employed by the wastewater treatment plants to give a rapid estimate of the VFA concentration in the solutions. We were able to successfully perform “Simple titration” experiments. The results (table 1) indicate that the method tends to give a slight overestimate of the total concentration – yet it can be used for quick estimations, as well as for determination of VFA concentration increase/decrease.
| Trial 1 | Trial 2 | Trial 3 | |
|---|---|---|---|
| Actual VFA Conentration (mg/L) | 60 | 60 | 60 |
| Sample volume (mL) | 40 | 40 | 40 |
| Acid normality | 0.1 | 0.1 | 0.1 |
| Original pH | 6.61 | 6.6 | 6.61 |
| Volume of acid added to titrate to pH 5 (mL) | 0.53 | 0.53 | 0.536 |
| Volume of acid added to titrate to pH 4.3 (mL) | 0.745 | 0.75 | 0.785 |
| Volume of acid added to titrate to pH 4 (mL) | 0.825 | 0.830 | 0.858 |
| Calculated VFA concentration (mg/L) | 66.1 | 67.7 | 74.9 |
HPLC is another method commonly employed in laboratory setting for the VFA concentration determination. The advantage of the method is the fact that it provides the concentration of different volatile fatty acids in the solution.
Process results
VFA fermentation results
The first VFA fermentation experiment showed higher VFA production at 37°C than at 22°C (Figure 11). The control condition was E. coli BL21(DE3) transformed with pET29b(+) vector without any inserts (non-PHB producing), Imperial condition was PHB-producing E. coli transformed with the Imperial College 2013 part , and Tokyo condition was PHB-producing E. coli transformed with the Tokyo 2012 part . The reported results are an average of 3 titrations performed for each sample.
The second VFA fermentation experiment confirmed higher VFA production at 37°C than at 22°C (Figure 12). In addition to a 3-day fermentation (denoted as D3), a 5-day fermentation was also introduced during this experiment (denoted as D5). Higher VFA concentrations were observed after 5 days of fermentation. Similarly to the first experiment, the reported results are an average of 3 titrations performed for each sample.
In subsequent experiments, the supernatant from fermented synthetic feces was collected after the VFA fermentation experiments, sterilized, and cultured with PHB-producing bacteria for 2 – 3 days at 37°C. PHB-producing bacteria cultured in the supernatant from synthetic feces fermented at 37°C had little to no growth, resulting in little to no PHB produced. Although the supernatant from synthetic feces fermented at 37°C had higher VFA concentration (Figure 11, Figure 12), we believe the lack of growth at PHB production stage could be due to lower pH. Consequently, 22°C was selected as preferred VFA fermentation temperature due to optimal pH for bacterial growth, although lower VFA concentration.
Liquid-solid separation results
The very fist experiments for the solid-liquid separation were "Gravity driven sedimentation" and "Gravity driven filtration" experiments The results are summarized in the table 2.
|
Gravity driven filtration |
Weight of water present in sample (g) |
Weight of liquid recovered after 24 hours (g) |
Percent of liquid recovered (%) |
|---|---|---|---|
|
Sample 1 |
15 |
0 |
0.00 |
|
Sample 2 |
40 |
20.4 |
51.00 |
|
Sample 3 |
65 |
52.5 |
80.77 |
|
Gravity Driven sedimentation |
Weight of water present in sample (g) |
Weight of liquid pipetted out after 24 hours (g) |
Percent of liquid recovered (%) |
|
Sample 1 |
15 |
0 |
0.00 |
|
Sample 2 |
40 |
21.4 |
53.50 |
|
Sample 3 |
65 |
47.5 |
73.08 |
It as clear the gravity alone would not do the required job, hence the"Staged Filtration" experiment was conducted using 25g of synthetic feces (recipe 2). The original sample contained 15g of water, yet only 10% of it was recovered, meaning that a more advanced and power intensive technology has to be considered for this stage of the process.
|
Filtration type |
Weight of liquid recovered (g) |
Liquid lost due to transfer (g) |
comments |
|---|---|---|---|
|
Strainer |
18.6 |
1.1 |
Yellow thick liquid went through. Yeast bodies we visible in the filtrate. |
|
"Paper towel" filter |
13.9 |
1.8 |
A thick creamy-yellow sludgy layer remained on the filter and could be scraped down. Yeast bodies could still be visible |
|
Coffee filter |
8.6 |
1.5 |
Another similar looking creamy-yellow layer was scraped down. The yeast bodies were not visible in the liquid any more |
|
11 micron filter |
5.8 |
1.2 |
Had to press very hard on the top of the filter to push the liquid through. |
|
0.2micron filter |
1.5 |
|
The majority of the liquid was not recovered because the filter got clogged. The recovered liquid had a brown tint, but appeared clear and transparent. |
Finally, we decided to investigate the efficiency of centrifugal based extraction methods using the "Centrifugation for solid-liquid separation" experiment. When a 50g undiluted sample of synthetic feces (recipe 2) was tested, the mass of water recovered was 19.6g, while the mass of initial water present in the sample was 30g, meaning that there was 65% recovery of water. This result indicated that centrifugal based solid-liquid separation technology would be the best fit for our application.
PHB Extraction results
PHB characterization
HPLC
Nile red staining
