Team:Calgary/Results

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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.

Figure 1: To confirm transformation of the pET29b(+) vector containing phaJ phaC into competent E. coli DH5α, a double digest confirmation was performed using restriction enzymes NotI and HindIII. DNA samples were run on 1% agarose gel run at 100V for 40 minutes.

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 liekly 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 indicates the functionality of the proteins

Figure 2: SDS-PAGE of soluble (S) and insoluble (I) proteins obtained from E. coli containing pET29b(+)-phaCJ induced with 0.1mM of IPTG at 37°C and lysed with lysozyme and sonication. The gel was run at 30mA for 50 minutes.

Figure 3: PHB extracted from PhaCJ-expressing cells cultured for 24 hours and inoculated with FSPS for 16 hours. PHB was extracted using TritonX-100, sodium hypochlorite, and ethanol in a series of washes and incubation. The negative control tube contained E. coli transformed with the pET29b(+) vector (without insert). This sample underwent the same extraction process as the phaCJ-expressing cells

Characterization of pET29b(+)-phaCBA

Figure 4: HPLC

Figure 5: To confirm transformation of the pET29b(+) vector containing phaCBA into competent E. coli DH5α, a double digest confirmation was performed using restriction enzymes NotI and KpnI. DNA samples were run on 1% agarose gel.

Figure 6: Extracted PHB from phaCBA-expressing cells cultured for 24 hours and inoculated with FSPS for 16 hours. PHB was extracted using TritonX-100, sodium hypochlorite, and ethanol in a series of washes and incubations.

Figure 7: HPLC analysis of pure PHB powders from POLYFERM and PHB powders produced from phaCBA-expressing bacteria digested with crotonic acid.

Secretion Results

Figure 3: Photograph of one of our SDS-PAGE gel electrophoresis apparatuses running with proteins from E.coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA tag, E.coli BL21(DE3) transformed with an empty pSB1C3 vector, and a protein ladder.

Figure 3: Photograph of one of our SDS-PAGE gel electrophoresis apparatuses running with proteins from E.coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA tag, E.coli BL21(DE3) transformed with an empty pSB1C3 vector, and a protein ladder.

Figure 3: Photograph of one of our SDS-PAGE gel electrophoresis apparatuses running with proteins from E.coli BL21(DE3) transformed with pSB1C3-Phasin-HlyA tag, E.coli BL21(DE3) transformed with an empty pSB1C3 vector, and a protein ladder.

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 (summarized below) 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 breakdown: the concentration of different volatile fatty acids in the solution.

Process results

VFA fermentation results

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 two tables below:

Gravity driven filtration	Weigt 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	Weigt 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
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 65% water recovery. Such 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