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

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.

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:

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.

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