Week 2: May 8 - May 12

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Week 3: May 15 - May 19

An ordered list!

1. Secretion
2. Syncretion
3. Engineering
4. Janitorial duties
5. Fruit flies
6. Synthesis

Team Member Classifications:

• Coworkers
• Friends
• Bilal

Week 1 (May 1 - May 5, 2017): Uploaded Sequences onto Benchling

The synthesis group focused on completing lab safety courses and laboratory training, which involved following commonly used protocols, and took inventory of our lab supplies. We also looked into the genes needed for synthesizing PHA in E. coli. After finalizing the genes, the constructs were uploaded on benchling as follows:

• Promoter T7, spacer sequence, RBS (B0034), FadE (E. coli), FadD (E. coli), PhaJ4pp, PhaC (p.aeruginosa)
• Promoter T7, spacer sequence, RBS (B0034), FadEE. coli, FadDE. coli, PhaJ (a.caviae), PhaC (p. aeruginosa)
• LacO operator, spacer sequence, promoter T7, spacer, RBS (B0034), FadE (E. coli), FadD (E. coli), PhaJ4 (p. putida), PhaC (p. aeruginosa)
• LacO operator, spacer sequence, promoter T7, spacer, RBS (B0034), FadE (E. coli), FadD (E. coli), PhaJ (a.caviae), PhaC (p. aeruginosa)
• phaCAB with T7 (constitutive) on lac I
• phaCBA with T7 (constitutive) on lac I double vector
• Hybrid promoter with number 8
• T7 (constitutive) + SS + RBS + SS + PhaC1 ap + phaJ4 (p. putida) + fadE + fadD + 10nt SS + RBS + SS + phaCAB

Week 2 (May 8 - May 12, 2017): Designing constructs and protocol for media

This week, we worked on researching protocol for media to be used for bacterial growth. Our mentors and advisers raised questions concerning the containment of the bacteria. This led our group to look into an antibiotic-free selection system; a number of methods for antibiotic free selection were brought up. We also looked into the possibility of using Bacterial Artificial Chromosome (BAC) to deliver our inserts due to concern over the large size of the insert. Ultimately, we decided that, if time allows, we will consider pursuing the antibiotic free selection method and the BAC. The lab training we participated in this week allowed us to practice protocols for performing transformations, preparing competent cells, and performing cPCR using template DNA from colonies on a streak plate. We also practiced agarose gel electrophoresis.

This week, the details of the plasmid design were discussed. We decided to use pET29b(+) as our vector because it has an inducible lacI and a T7 promoter. We also planned characterization experiments for PhaJC + FadE, and compare PhaCBA with the PhaCAB Biobrick from Tokyo 2012. We decided to code our parts on benchling with the following template: junk-ENX (prefix)-RBS-cds-SNP (suffix)-kpn1 (restriction)-junk. We finalized the three plasmid inserts with T7 promoters (____ , ____ and _____) and double-checked the spacer sequences using BLAST. We also decided to add 6x-His tags after methionine to help with characterization in the later stages of the experiment. Restriction sites were decided on for each part so that the whole final biobrick can be ligated together (3 pieces for beta-oxidation PHA sequence and 2 pieces for glucose PHA synthesis).

Week 3 (May 15 - May 19,2017): Codon optimization and ordering sequences

The synthesis group optimized codons, removed a number of restriction sites, finalized our constructs, and ordered our sequences from IDT. We also researched the chemicals required for growth media and the characterization protocols we planned to perform. We continued formulating and editing protocols for post-synthesis experiments such as chemical cell lysis, PHA extraction and purification, and Nile Red Fluorescence quantification. A table was compiled to compare the pros and cons of different procedures for processes such as PHA extraction and quantification.

Week 4 & 5 (May 22 - June 2, 2017)

This week, the entire iGEM team took a field trip to a Calgary Wastewater Plant to learn about wastewater management. This trip informed us of the applications of our project. After evaluating the economic feasibility of implementing our waste-to-plastic system for use in the municipal wastewater treatment plant, the team started to discuss other possible applications of our project (wastewater treatment, developing countries, landfills, or space). Each member of the group researched a different application of our project to present and discuss at the weekly lab meeting. The group finalized the post-synthesis protocols that were researched last week and ordered our required chemicals for these experiments.

Week 6 (June 5 - June 9, 2017): Restriction digest + electrophoresis

While waiting for the rest of the ordered gblocks™ to arrive, the synthesis group assisted the efforts of the human practices and modelling groups. We practiced coding for the wiki and researched E. Coli infections in space to evaluate its virulence and containment.

Week 7 (June 12 - June 16, 2017): Plasmid mini prep

We performed single digests on our Pet29b(+) vectors using restriction enzymes HindIII, Sal1, EcoRI, and KpnI to check that each enzyme worked. After the DNA was digested we ran the samples on 1% agarose gel. The gel showed bands representing linearized plasmids on the gel, which informed us that our restriction enzymes were functional.

Week 8 (June 19 - June 23, 2017): Run Controls and Digest gBlocks

We received streak plates of E. coli BL21(DE3) and E. coli DH5α containing pET29b(+) vectors from Dr. Wong’s lab. These stock plates were used to streak fresh plates of the E. coli. Glycerol stocks of these cells were also made to preserve them. We made O/N cultures of the E. coli DH5α and performed plasmid miniprep to obtain PET29b(+) vectors. When we tested the plasmids on the NanoDrop, the samples were contaminated and did not show high concentrations. We decided to make O/N of the E. coli DH5α and then perform plasmid miniprep again. This time, the NanoDrop results showed a successful miniprep of the pET29b(+) vectors. These pET29b(+) vectors were stored in the -20°C freezer.

Week 9 (June 26 - June 30, 2017):

We performed diagnostic testing of NotI in CutSmart™ buffer and HindIII in Fast Digest Buffer with RFP plasmids from the iGEM registry. Diagnostic testing was done because the manuals outlined digestion of NotI in Fast Digest Buffer and HindIII in CutSmart™. We wanted to see if one of the buffers would work for both restriction enzymes. We then ran the digests on 1% agarose gel. The results showed that the digest of NotI in CutSmart™ worked and that FastDigest Buffer did not work. This meant that we could use CutSmart™ buffer for both restriction enzymes. The resulting gel electrophoresis is shown below.

We then streaked a fresh plate of E. coli DH5α containing pET29b(+), made O/N, and performed miniprep to get more pET vectors to use for future experiments.

Week 10 (July 3 - July 7, 2017): Ligated gBlocks

This week we ligated the digested PhaC and PhaBA each into pET29b(+) vectors. After ligation we transformed our cells and incubated the plates O/N. The next day we chose four colonies from PhaC and PhaBA-transformed plates to make O/N cultures and a master plate from. We isolated the plasmid from our O/N cultures to perform a confirmation digest of the transformed cells. We performed double or single digests of the plasmids from miniprep to check that the presence of the insert within the vector and to check the directionality of the insert. The restriction enzymes and the respective sites and expected bands on the plasmids are shown below.

We ran gels of undigested and digested plasmids. When we compared the predicted Benchling digests with our results, we found that colony 1 from the transformed PhaBA matched the expected bands, suggesting that the transformation was successful because the insert was in the plasmid and the direction was correct. However, the PhaC transformants did not match or show corresponding bands with the digests performed on a transformed vector. The results are shown in the figure below.

Week 11 (July 10 -July 14, 2017):

From the confirmation digest and gel electrophoresis from last week, PhaBA colony 1 seemed to have the correct insert. Therefore, we wanted to sequence the colony to confirm that the correct insert was in the plasmid. To do so, we designed and ordered primers for pET29b(+) PhaBA and prepped our samples for sequencing. The PhaC transformant colonies 1-4 did not pass the confirmation digest screening, but to double-check for the plasmids, we isolated, digested (NotI, HindIII and HincII), and ran gel electrophoresis on the plasmids from 6 more PhaC transformant colonies (5-11) using protocols ____ and ____. The results are shown below.

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We planned out the rest of our experiments for the summer and set tasks to complete. We transformed our DH5α competent cells with each of our plasmid inserts. We ran digests to confirm that the transformants actually carried our plasmids. We also developed a protocol for sequential digest of our pET29b(+) vectors because the NotI and HindIII restriction sites are very close together on plasmid. A Beta oxidation (PhaC-J) was digested with HindIII and SalI, ligated with pET29b(+) vectors, transformed into competent DH5α cells, plated on Kan resistant plates, and left to incubate O/N at 37°C. The culturing tubes of the transformants from the Transformation protocol were kept in the 4°C fridge. No colonies were observed in the plate the next day. To troubleshoot why the cells did not grow, plasmids were isolated from the inoculation tube with the transformed culture that was kept in the 4°C fridge. Confirmation digest was performed on these plasmids. The gel electrophoresis did not work. Therefore, we decided to redigest our gblock, ligate, and transform it again. B Beta Oxidation and C Beta Oxidation were digested with HindII/SalI and SalI/KpnI, respectively following the Restriction Digest protocol. A salt solution was made to adjust for the salt concentration. After heat inactivation, the digested DNA was stored at -20°C for the weekend.

Week 12 (July 17 -July 21, 2017):

The ligated PhaC pET29b(+) and A Beta oxidation pET29b(+) were transformed into competent DH5α cells and plated on Kan LB agar plates. Colonies appeared on these plates. A master plate and O/N cultures were made to screen these transformants. The O/N culture was used to isolate plasmids from. The plasmids were digested and screened for confirmation of insert and directionality. The enzymes used are shown below along with their respective restriction sites and expected band size. The screening results showed that there was no insert.

Last week, we digested B Beta Oxidation and C Beta Oxidation and stored them. This week, we ran the digested B beta oxidation part on an low melting point (LMP) gel and recovered the part by excising it from the gel. The gel containing the B Beta oxidation insert was melted at 65°C and ligation protocol was performed. We then transformed the plasmid into competent DH5α. C Beta Oxidation was also ligated with pET29b(+) vectors and transformed into competent DH5α. The B Beta Oxidation and C Beta Oxidation transformants colonies were observed after the O/N incubation. A master plate and O/N culture of selected colonies from each plate was made. The vectors were isolated, digested for confirmation of insert, and ran 1% agarose gel.

Week 13 (JULY 24 - JULY 28, 2017)

Because PhaC and A Beta oxidation did not transform with the inserts, the gBlocks were digested, ligated, and transformed this time in pET-RFP plasmids following protocol the pETRFP Digestion protocol. B Beta Oxidation colony 2 and C beta oxidation colony 3 seemed to have worked so the plasmids were isolated by miniprep and sent in for sequencing using the designed primers. The C beta oxidation sequencing results were incorrect. However, to double-check, we prepared a master plate and O/N of a few colonies from the plate of transformants. Miniprep was performed and the plasmids were digested and were run on 1% agarose gel. The results did not indicate the successful ligation of the insert. To keep the pET29b(+) E. coli DH5α fresh, a new streak plate was made. An O/N culture was made and plasmids were isolated by miniprep and stored in the -20 freezer for future use.

Week 14 (July 31 - Aug 4)

The sequencing result___. To check that the B beta oxidation transformed into DH5α on ___ was correct, an O/N was made. Miniprep was performed to isolate the plasmid. The plasmids were then digested and run on a 1.5% agarose gel at 70V for 90 minutes. The results showed that____

Week 1 (May 1-May 5, 2017)

We are all fully trained in laboratory safety! We each completed 6 online courses and 2 seminars on lab safety and biosafety. In the lab we practiced important protocols, which include media preparation, overnight culture inoculation, preparing chemically competent cells, and transforming chemically competent cells.

We also reserached ways that we will be able to use synthetic biology to extract synthesized Polyhydroxybutyrate (PHB) from cells, without the use of traditional chemical or mechanical lysis.

Week 2 (May 8-May 12, 2017)

We practiced more important laboratory techniques: plasmid minipreparation, preparing master plates of transformed E. coli, and agarose gel electrophoresis.

We also narrowed down our PHB extraction method to a hemolysin type I secretion system native to E. coli, like what was used by Team SDU-Denmark in 2016. We uploaded sequences of the hemolysin secretion tag, HlyA, fused to phasin (Part:BBa_K2018024), hemolysin membrane transport protein, HlyB (Part:BBa_K2018027), and hemolysin membrane transporter, HlyD (Part:BBa_K2018029) onto Benchling and began designing our parts to be ordered for synthesis from Integrated DNA Technologies (IDT). Each part will be upregulated by control under a T7 Promoter. Modifications to the parts we uploaded to Benchling were made: flag tags for easier protein expression validation were added to each coding sequence and restriction sites were removed from Part:BBa_K2018029 to make the parts RFC10,12,21,23,25, and 1000 compatible.

Week 3 (May 15-May 19, 2017)

This week we finished editing our secretion parts before ordering them from IDT. Our complete system with Phasin-HlyA tag, HlyB, and HlyD was split into two separate gBlocks: Secretion Part 1 (SP1) and Secretion Part 2 (SP2). A gBlock with just the phasin-HlyA tag was ordered as well so that we could compare secretion of the endogenous E. coli secretion system to our system with upregulated HlyB and HlyD. Restriction sites for the HindIII-HF enzyme were added to the ends of SP1 and SP2 so they could be easily ligated together. All stop codons were changed to TAA because it is the most effective stop codon in E. coli. Also, codons were optimized before ordering using this tool.

Week 4 (May 23- May 26, 2017)

Jacob and Sam began working on the Interlab Study. To begin, chemically competent DH5𝛼 E. coli were made and stored at -80°C in glycerol stocks, PSB buffer was made and diluted several times to develop a standard absorbance curve for the study, and the plate plate reader was calibrated.

Actual work on the Interlab Study was not very successful because there was little to no growth on plates of the transformed DH5𝛼, even though they were incubated at 37°C for two days.

The entire iGEM team was given very informative tour of the Pine Creek Wastewater Treatment Plant in Calgary. Many questions were answered and the information we have gained will be used in deciding which application route our project will take.

Week 5 (May 29- June 2, 2017)

Due to the lack of growth on the Interlab Study plates last week, chemically competent DH5𝛼 E. coli were made again because there may have been issues with the first set of competent cells created. However, there was still no growth on the plates inoculated with transformed cells.

On a positive note, our constructs from IDT that were ordered in week 3 arrived!

Outside of the lab, lots of work was dedicated to researching four possible applications of our project (space, wastewater treatment, landfill leachate, and developing countries. More about this research can be found on our Applied Design and Integrated Design pages).

Week 6 (June 5- June 9, 2017)

Again, Jacob and Sam’s work on the Interlab Study was unsuccessful. Different protocols for making chemically competent cells and transformation were used, but there was still no growth observed. This strongly suggests that there is an issue with our DH5𝛼 cells, so next week we will get new cells and try again. Due to our problematic cells, work has not yet begun with our actual secretion construct from IDT.

Lalit and Kaitlin prepared necessary supplies that will be needed on Lalit’s trip to Winston Churchill High School on Monday, June 12, 2017. During this outreach, he will discuss synthetic biology with Grade 11 students, practice gel loading, and perform strawberry DNA extraction experiments with them. .

June 11, 2017- Geekstarter iGEM Workshop, presented by MindFuel and Alberta Innovates

Today was an iGEM workshop hosted by Geekstarter that was attended by the University of Alberta, University of Calgary, Urban Tundra High School, and University of Lethbridge iGEM teams. There were four speakers who gave ½ hour presentations on an introduction to syn bio, mathematical modeling, wiki design, and integration of art into syn bio. Later, we had the chance to speak with each presenter individually and ask questions specifically related to our project. Also, there was a collaboration period where we got to mingle with the other teams and discuss our projects with them. Overall it was a successful day where we learned a lot from the presenters, and identified possible collaborations with the other teams.

Week 7 (June 12- June 16, 2017)

On Monday, June 12 Lalit and Sam visited Winston Churchill High School to present to Grade 11 students about Synthetic Biology (which we prepared for in Week 8).

We acquired new competent E. coli DH5α cells from Dr. Dong and transformed them for the Interlab study. Finally, transformation was successful and there was colony growth of GFP cells. This supports the theory that there was something wrong with the cells that we were previously using. Therefore, for our work throughout the summer we will continue using the DH5α cells from Dr. Wong. Jacob and Sam used the new cells to successfully complete the laboratory component of the Interlab study this week.

Also, we miniprepped pSB1c3 plasmids from RFP E. coli Top10 for use in cloning our secretion inserts in the following weeks.

Week 8 (June 19- June 23, 2017)

To prepare ourselves for work with our IDT constructs, we performed various diagnostic tests with restriction enzymes. pSB1C3-RFP and -GFP were digested SpeI and EcoR-HFI, and confirmed on a 1% agarose gel. After some troubleshooting, we obtained two distinct bands on the gel, representing the pSB1C3 backbone and RFP/GFP insert. This indicates that these enzymes are functional and can be used on our IDT parts.

Outside of the lab, Jacob and Sam completed and submitted the online portion of the Interlab Study this week. Kaitlin and Lalit attended various meetings with other members of the team.

Week 9 (June 26- June 30, 2017)

The digested pSB1c3-RFP and -GFP from last week were run on a 3% low melting point agarose gel and both the plasmid backbones and the RFP/GFP inserts were excised. Our T4 DNA Ligase was tested by ligating the GFP inserts to the RFP backbones, and the RFP inserts to the GFP backbones. As we expected, bacteria transformed with the GFP insert/RFP backbone grew green colonies and bacteria transformed with RFP insert/GFP backbone grew red colonies. This indicated that our T4 DNA Ligase functioned properly and can be used with our IDT parts.

Jacob and Sam tested out a new protocol for making chemically competent E. coli DH5α cells from Richard Moore, who is part of Dr. Dong’s lab here at the Foothills Hospital. The cells’ competency was tested with 100 ng/µL RFP. The transformed cells grew extremely well with numerous colonies and a high transformation efficiency.

Our IDT parts were digested and ligated into their corresponding backbones, then transformed into chemically competent E. coli DH5α cells.

Week 10 (July 3-July 7, 2017)

We isolated the plasmids of E. coli DH5α transformants from our ligated backbones/parts from last week. We screened 8 phasin-HlyA colonies, 8 SP1 colonies, and 2 SP2 colonies. Isolated plasmid of each colony was digested, then ran on a 1% confirmation gel. Some plasmid of each colony was left undigested as a control, double-digested with the same enzymes we used last week for ligation to check for the insert size, and digested with another enzyme to check for insert directionality:

We determined that only colony 1 of the phasin-HlyA tag had successfully received a plasmid with our part, as seen in Figure 1 below. Our other parts were not successfully transformed into our cells, which indicates that our ligation or tranformation into E. coli DH5α had failed.

Week 11 (July 10- July 14, 2017)

Colony 1 phasin-HlyA was sequenced and confirmed that these cells do in fact contain pSB1c3-phasin-HlyA. Therefore, psB1c3-phasin-HlyA was miniprepped from DH5α and transformed into E.coli BL21(DE3), which we will use for protein expression. We also transformed E. coli DH5ɑ with a PHB synthesis biobrick, PhaCAB(Part:BBa_K934001) present in the iGEM 2017 distribution kit. This was done to establish a PHB-producting cell line and we do not have to wait for the Synthesis subgroup to finish their molecular cloning before we can begin testing the secretion of PHB.

On Friday, July 14 Kaitlin and some of the other iGEM team members visited the Grades 7-9 Minds in Motion summer camp at the University of Calgary. With the children, they discussed the potential implications of genetic engineering for the future and performed a strawberry DNA extraction experiment.

Week 12 (July 17-July 21, 2017)

Our SP1 gBlock was re-digested with Xba1 and HindIII-HF and ligated into a linearized pET29B backbone with XbaI/HindIII sticky ends. Colonies were run on a confirmation gel and the transformation of colony 3 appeared successful, as shown in Figure 2. A sample of SP1-colony 3 was submitted for sequencing.

Rachelle developed a line of cells that contain pET29B-RFP This was done because HindIII and NotI restriction sites in pET29B are overlapping if there is no insert, causing our sequential digests for SP2 to fail. Thus, RFP was inserted to separate the two restriction sites and permit our digestion. Vector pET29B-RFP can be sequentially digested with HindIII-HF then NotI-HF to remove the RFP insert and leave the sticky ends intact for SP2 to be ligated to.

On the wiki, Sam and Kaitlin coded the protocols page. Furthermore, we took individual and team photos so that we could start completing the team page of the wiki.

Week 13 (July 24 - July 28, 2017)

The “protocols” page of the wiki was competed, and we uploaded all of the general protocols used by everyone.

Our sequencing results of SP1-colony 3 showed that we had successfully transformed SP1 with pET29B vector into E.coli DH5ɑ. With regards to SP2, we tried multiple times to perform our sequential restriction digest of the pET-RFP backbone with HindIII-Hf and NotI-Hf, and ran into multiple roadblocks. After various troubleshooting techniques, we were finally successful upon using a DNA isolation protocol in between the two digestion steps. Following this, we excised the pET29B backbone from an LMP gel, and ligated with our HindIII/NotI-digested SP2 part. We also managed to create our first batch of PHB using a the PHB synthesis biobrick from the registry, and confirmed it using nile red staining. The PHB was extracted with chloroform and poured into a sheet (Figure 3).

Week 14 (July 31 - August 4, 2017)

Work on cloning in our SP2 part was continued this week, using the same DNA isolation protocol as before in between the HindIII and NotI phases of our sequential digest of the pET29B vector. Using this technique, we successfully digested, ligated and transformed pET29B-SP2 into DH5α. 11 Colonies grew and these will be screened next week.

Lalit performed an SDS-PAGE in an attempt to qualify phasin production in our phasin-HlyA tag-transformed cells. Results were inconclusive because there were too many other bands around the same size as phasin, and the SDS-PAGE will be repeated at a later date.

Jacob transformed DH5α with a PhaCAB biobrick from Imperial College (Part:BBa_K1149052) that he will compare to the part from Tokyo Tech (Part:BBa_K934001) that he used in Week 13.

Week 15 (August 8 - August 11, 2017)

All of the pET29B-SP2 colonies from last week were digested and screened, however none contained our insert, indicating that the ligation had failed. We re-digested our SP2 gBlock with NotI-HF and HindIII-HF and ligated it into one of pET29B backbones prepared last week. We also began a new approach of ligating SP2 directly into pET29B with SP1 insert (originally obtained in Week 12). To do this, both pET29B-SP1 and SP2 were digested with XhoI and HindIII-HF then ligated together. Both ligation products were transformed into chemically competent E.coli DH5ɑ cells.

Jacob Made LB plates with Nile Red and with them, he confirmed the presence of PHB granules in the cells transformed in Weeks 13 and 14 with PhaCAB biobricks from both the Imperial College and the Tokyo Tech teams. After overnight incubation in LB media with 3% glucose, he also successfully extracted the PHB granules from these cells with sodium hypochlorite (bleach) (Figure 4).

Week 16 (August 14 - August 18, 2017)

Kaitlin and Sam re-digested pET29B-RFP sequentially with NotI-HF and HindIII-HF, as was done in weeks 13-15. The digests were run on a 1% low melting point agarose gel, then the backbones were excised from the gel. 10 replicates of the backbone were excised so that it could be used by our group and the synthesis group. This backbone was ligated to our SP2 gBlock digested with HindIII-HF and NotI-HF. Also, the new approach for cloning in SP2 discussed in week 15 was tested. We digested pET29B-SP1 and our SP2 gBlock with HindIII-HF and XhoI, then ligated these together to create pET29B-SP1-SP2.

Week 17 (August 21 - August 25, 2017)

There were plenty of colonies on both the pET29B-SP2 and pET29B-SP1-SP2 plates that Kaitlin and Sam had prepared last week. However, when overnight cultures and masterplates of these colonies were prepared for screening, none of the preparations showed any sign of growth. It was suspected that there was a viral contamination in the LB + kanamycin broth used for overnight preparation. New LB + kanamycin was made, but nonetheless, overnight cultures and masterplates of these colonies still showed no growth. From this, it was deduced that the LB + kanamycin broth was not the issue, therefore, the same HindIII-HF/XhoI digests as in Week 16 were carried out in order to prepare a new set of pET29B-SP1-SP2 colonies to be screened.

Lalit worked on another SDS-Page experiment to qualify phasin production in our BL21 cells transformed with pSB1c3-phasin-HlyA tag. BL21 transformed with empty pSB1c3 were used as controls. He sub-cultured overnights of the cells, induced them with 1mM IPTG for 4 hours, then lysed them in order to separate them into soluble and insoluble protein fractions, as well as a supernatant fraction. First, he will run the purified fractions through a Sigma-Aldrich anti-FLAG M2 affinity gels (because our phasin-HlyA has FLAG tags) to isolate the phasin before running the SDS-page gels.

With the Process subgroup, Jacob carried out experiments to determine if PHB could be produced with synthetic feces supernatant that had been fermented at either 23°C or 37°C for 3 days. After the fermentation, the supernatant was inoculated with E.coli DH5𝛼 that had been transformed with pSB1c3-PhaCAB from Imperial College. E.coli DH5ɑ transformed with an empty pSB1c3 backbone was used as a control. Then, after overnight incubation at 37°C, PHB was extracted with bleach. Supernatant fermented at 23°C yielded significantly more PHB than supernatant fermented at 37°C.

Week 18 (August 28 - September 1, 2017)

Overnights and masterplates of the second set of pET29B-SP1-SP2 colonies from last week did not show any signs of growth. Since two sets of pET29B-SP1-SP2 colonies and two sets of pET29B-SP2 colonies were unable to grow in overnights or on masterplates it was deduced that our SP2 was somehow harmful to the E.coli and it would be very difficult to transform this part into our bacteria. We decided to move past cloning our SP2 part in and instead focus on carrying out assays with the pSB1c3-phasin-HlyA (cloned in week 10) and pET29B-SP1 (cloned in week 12).

Week 19 (September 5 - September 8, 2017)

Jacob repeated the experiment from Week 17 with 3-day fermented synthetic feces supernatant. Again, results were similar, and supernatant fermented at 23°C yielded a much higher amount of PHB than supernatant fermented at 37°C.

Week 20 (September 11 - September 15, 2017)

In order to carry out assays of PHB secretion, E.coli BL21 must first be double transformed with a plasmid containing PHB-producing genes, as well a plasmid containing our secretion genes. So, this week Kaitlin worked on double transforming cells with iGEM registry pSB1c3-PhaCAB from Imperial College and pET29B-SP1 (isolated in week 12). Since synthesis has not yet completed ligating their PhaCBA genes together, we decided to use the part from imperial college, which we know can successfully produce PHB. Although we carried out two double transformation attempts, unfortunately neither showed any colony growth, indicating that the double transformations had failed.

Kaitlin also digested both pSB1a3-RFP and pSB1c3-phasin with EcoRI-HF and SpeI, then ligated the products together in order to produce pSB1a3-phasin. This is necessary because pSB1c3-phasin would not be able to be transformed into the same cells as pSB1c3-PhaCAB, since it is not possible to double transform a cell with two of the same plasmid backbones. Digest confirmation with NotI-Hf showed that the ligation was successful and pSB1a3-phasin that can be transformed into BL21 alongside pSb1c3-PhaCAB had been obtained.

Week 21 (September 18 - September 22, 2017)

Since the registry pSB1c3-PhaCAB from Imperial College was almost empty and double transformations with it last week had failed, we decided to isolate a fresh sample of pSB1c3-PhaCAB from the cells that Jacob had been working with earlier in the summer. This new pSB1c3-PhaCAB and pET29B-SP1 was transformed into chemically competent BL21. Also, the pSB1c3-PhaCAB and pSbBa3-phasin were transformed into chemically competent BL21. However, these transformations all failed and we decided to move forward with a new method of double transforming, in which chemically competent BL21 were transformed with pSB1c3-PhaCAB alone. Then, these cells were made competent and next week we will attempt to transform either pSb1a3-phasin or pET29B-SP1 into the chemically competent BL21(+pSB1c3-PhaCAB).

Week 22 (September 25 - September 29, 2017)

Two attempts of transforming chemically competent BL21BL21(+pSB1c3-PhaCAB) with either pSB1a3-phasin or pET29B-SP1 were made this week, however, both attempts failed and the cells were not successfully transformed.

Week 23 (October 2 - October 6, 2017)

After some research, we realized that pSB1a3 is incompatible with pSB1c3 and that transforming cells with both of these plasmids at the same time would not be possible. This lead us to attempt to create a large pSB1c3 containing both our phasin-HlyA genes and the PhaCAB from Imperial College. Sam used XbaI and SpeI to cut phasin-HlyA out of the pSB1a3 backbone and ligate it into pSB1c3-PhaCAB that had been linearized with Xba1. This ligation, however was unsuccessful and will be re-attempted next week.

Kaitlin and Lalit prepared the protein fractions collected by Lalit in Week 17 for running on an SDS-Page Gel. First, the Sigma-Aldrich anti-FLAG M2 affinity gel resin was prepared, as per manufacturer's instructions. Then each sample fraction was incubated with the anti-flag resin in order to help isolate our phasin-HlyA tag from the large volume of other proteins present in the samples.

Week 24 (October 10 - October 13, 2017)

Week 1: May 1 - May 5

During the first week, we checked the inventory and organized our lab supplies. We familiarized ourselves with the lab equipment and some of the protocols we might be using during the summer such as preparing LB agar plates, chemically competent E. coli cells, plasmid minipreps and running colony PCRs. We also completed the required lab safety and biosafety training. We started researching Alberta’s wastewater treatment process and conducted preliminary literature review of volatile fatty acid (VFA) production and polyhydroxybutyrate (PHB) extraction. We met with Dr. Peter Dunfield, a professor of microbiology at the University of Calgary, to discuss a novel approach of extracting PHB from bacteria to avoid using solvents such as chloroform for PHB extraction in our final process. Consequently, we brainstormed ideas about binding PHB granules to beads after autolysis of engineered E.coli.

Week 2: May 8 - May 12

We continued practicing common lab procedures and reviewed similar projects from the past to explore avenues for improvement and innovation. As a result, we decided on a secretion based system as opposed to a lysis system. Concerns were also raised this week about the use of pure cultures with sludge in the wastewater treatment process. Genetically engineered bacteria can be outcompeted if added directly to sludge, while sterilizing sludge before introducing engineered bacteria would result in additional costs. Since PHB production occurs in nutrient limited conditions, we also began to research ways to remove nutrients before inoculating sludge with the engineered bacteria. Our team started a draft outline of a proposed process from VFA production to PHB extraction and purification.

Week 3: May 15 - May 19

During this week, we discussed our sub-group’s role in the project and divided the work into subcategories: extraction and purification of PHB, quantification and characterization of PHB, process development/scale up. Objectives and milestones were then developed for each subcategory. Since extraction and purification of PHB step is one of the major contributors to high costs of PHB production (Jiang, Mikova, Kleerebezem, van der Wielen & Cuellar, 2015), we will research novel, feasible extraction and purification methods for the large scale process and will test proposed methods in the lab. The process development group will also support the synthesis group by quantifying and characterizing the PHB that they produce in the lab. This week, we looked into potential protocols and the availability of resources for lab-scale quantification and characterization of PHB. When designing a PHB production process, we will also consider scalability and feasibility at large by conducting a cost analysis for the proposed large scale PHB production process.

We also met with Dr. Christine Sharp, a postdoctoral fellow from the Energy Bioengineering Group supervised by Dr. Marc Strous. Their group is working on PHB production using alkaline soda lake biomass and designing their own bioreactors.

Week 4: May 22 - May 26

At the start of the week, the group met with Dr. Nashaat Nassar, an Assistant Professor in Chemical and Petroleum Engineering, to discuss PHB extraction and purification techniques. He recommended a combination of coagulation and settling to separate out PHB. We also met with Dr. Saurabh Sarma, a postdoctoral fellow in Civil Engineering, to discuss VFA production in wastewater treatment plants (WWTP). He advised us on integrating our process with the desired applications. We also discussed the composition of VFA commonly found in WWTP and parameters that impact VFA composition. Dr. Sarma suggested reaching out to Daniel Larson, a laboratory technician in Civil Engineering, to discuss VFA and PHB quantification using gas chromatography.

The entire team visited one of Calgary’s WWTP this week, where we learned about the current wastewater treatment process. After the tour, we met with representatives from ACWA (Advancing Canadian Water Assets) Research Facility at the Pine Creek Wastewater Treatment plant, who are working on wastewater treatment research. However, they work with the output from the wastewater treatment plant, while most of our questions were on the wastewater treatment process itself. They directed us to the City of Calgary chemistry laboratory that performs analysis on intermediate samples from various stages of the process. We have contacted representatives from the City of Calgary.

Week 5: May 29 - June 2

This week, the entire team focused on evaluating the feasibility of the 4 proposed project applications: production of PHB on Mars from human waste, integrating PHB production in a wastewater treatment plant, integrating PHB production with leachate treatment, and integrating PHB production in developing countries. Our subgroup calculated the approximate amounts of PHB that would be expected in each scenario.

We approximated that about 40 - 90 kg of PHB can be produced on Mars per year with a crew of 6 astronauts. A crew of 6 will generate about 6 tonnes of solid organic waste over 2.5 years (Zhang, Ylikorpi & Pepe, 2015). Reported COD content in feces was found to be 354 mg COD per gram of wet human waste (Rose, Parker, Jefferson & Cartmell, 2015). One study looking at PHA production from food waste estimated the yield of 0.05 g of PHA per g of COD applied (Rhu, Lee, Kim & Choi, 2003). Another study reported 0.11 kg of PHA produced per kg of effluent COD in a PHA production process from activated sludge (Bengtsson, Werker, Christensson & Welander, 2008). The predicted PHB range was based on COD to PHA conversion. Another member of the team assumed the average COD content in human excretions to be 61.75 g/cap/day (Rose, Parker, Jefferson & Cartmell, 2015), the COD to VFA ratio of 0.74 (Coats, VandeVoort, Darby & Loge, 2011)and the VFA to PHB conversion of 0.38 g PHA/g VFA ,(Coats, VandeVoort, Darby & Loge, 2011) which resulted in 41 kg of PHA per year per crew of 6. According to NASA, the cost of shipping supplies to space using SpaceX Dragon spacecraft is $27,000 per pound. The costs saved by producing 41 kg of PHA in space would then be about $2,440,000.

We also contacted a 3D printing company called 4G Vision Tech that uses selective laser sintering (SLS), which can be used to 3D print with PHB (Pereira et al., 2012). Howard from 4G Vision Tech approximated that the predicted amount of PHB can be used to create approximately 50 hydroponic systems and 20 general tools like wrenches, hammers, and scissors.

The integration of PHB production in leachate treatment would likely be unfeasible due to low volumes of leachate that are usually produced at landfills. In Calgary, a single landfill generates about 100,000 L of leachate per day. Although COD content in leachate is higher than in wastewater, the estimated amount of PHB produced in Calgary was about 8000 kg/year, based on COD content of 1977 mg/L in Calgary leachate (Kashef & Lungue, 2016). This would result in about $40,000 in revenue, assuming a price of $5 per kg of PHB (Manufacturing and properties of PHB, 2017). Another member of the team performed similar calculations assuming 0.38 grams of PHB produced per gram of VFA, which resulted in 22,250 kg of PHB per year and a potential revenue of $111,126 per year. The cost of implementing a PHB production process will likely be magnitudes larger. Leachate treatment in China is a more promising alternative. China generates a larger amount of leachate compared to many other countries("Leachate treatment in China: Technologies and Import Opportunities", 2015). Additionally, the COD content in Hong Kong, China ranges from 15,700 to 50,000 mg/L for young landfills ("Leachate treatment in China: Technologies and Import Opportunities", 2015), which is 8 to 25 times greater than in Calgary. We estimated that about 900,000 - 3,000,000 kg of PHB can be produced per year in Hong Kong depending on COD content and using PHA yield of 0.11 kg of PHA per kg of COD. PHB production from leachate was also considered for Vancouver, which generates 2,225,978 cubic meters of leachate per year (Vancouver landfill 2016 annual report, 2017). Based on our estimates, about 3,100,000 kg of PHB can be produced per year assuming the COD content of about 13,000 mg/L (Tao, Hall & Masbough, 2005)

For the wastewater treatment plant, we estimated 28,100,000 kg of PHB produced per year. The Pine Creek Wastewater Treatment Plant in Calgary processes about 1 million cubic meters of waste per day. The COD content and PHA yield were assumed to be 1000 mg/L and 0.11 kg of PHA per kg of COD, respectively, for the calculations.

In developing countries, we envisioned PHB production incorporated into scaled-down wastewater treatment systems in small communities that lacked established treatment methods. Selling PHB would provide monetary incentive to construct a wastewater treatment system, which, in turn, will reduce diseases due to poor sanitation. Additionally, we wanted to compare PHB production between genetically engineered bacteria and natural bacterial communities in sludge, which have been previously used to feasibly produce PHB. Assuming a community size of 2000 people, solid waste generation of 3.113 x 10-3 m3 /day/person (Palanivel & Sulaiman, 2014), COD content of 601 mg/L [14], COD to PHB conversion of 0.11 for mixed cultures and 0.88 for pure cultures (Rhu, Lee, Kim & Choi, 2003) and a price of $5 per kg of PHB (Choi & Lee, 1997), we found that using pure cultures results in additional $2,000 in revenues. However, the cost of sterilization of waste stream before inoculation with pure culture was estimated at $100,000 (Choi & Lee, 1997).

Week 6: June 5 - June 9

This week, our team decided to pursue PHB production on Mars from human waste as the main application for our project. Our subgroup revisited the scope of work and different stages of the required process and prioritised tasks. We first researched potential extraction and purification techniques and chose the top 3 methods for further testing in the lab. At the same time, we also looked at VFA production from feces. For safety reasons, we decided to use synthetic feces in preference to real samples. We found synthetic feces recipes (Wignarajah, Litwiller, Fisher & Hogan, 2006), (Colon, Forbis-Stokes & Deshusses, 2015) and purchased required materials. After preparing synthetic feces, we researched desired conditions for VFA production, separation of VFA from feces and VFA quantification methods.

Week 7: June 12 - June 16

This week, we met with professors in the Chemical and Petroleum Engineering department at the Schulich School of Engineering to discuss PHB extraction and purification techniques. One professor suggested growing bacteria in a biofilm while continuously collecting PHB. With this set up, dead bacteria can be filtered out by a 0.2 µm filter and harvest containing PHB can be separated from biomass without centrifugation. However, biofilms require a large surface area, which is not ideal for the space application where compact systems are desired. Another professor suggested continuous production of PHB in a membrane reactor that retains the bacteria, but lets the PHB through. This continuous PHB production system will also be compact compared to biofilms. We also discussed coagulation including electrocoagulation as a potential method for extraction and purification of PHB with Dr. Nashaat Nassar.

For lab scale experiments, we plan to test centrifugation and coagulation methods for PHB extraction. Centrifugation method would involve two centrifugation steps: the first step to separate supernatant containing PHB from biomass and other large particles and the second step to settle the PHB granules. Since bacteria and PHB have different settling velocities, the first centrifugation step can remove biomass while leaving PHB in suspension. The second step will likely require an ultracentrifuge but will allow to settle PHB granules and remove the supernatant. Adding a coagulant such as calcium ions can aid in settling PHB granules; we will experiment with coagulation to see whether is can eliminate the need for the second centrifugation step or reduce required centrifugation speeds.

Additionally, we discussed a fermentation experiment to produce VFA from synthetic feces. The experiment will aim to answer the following questions:

• Is it possible to make VFA by inoculating synthetic feces with E. coli?
• How much VFA can be made per amount of input feces and what types of VFA will be made?
• How long does it take to reach maximum VFA composition?
• Does temperature have an impact on VFA production?
• Does the addition of yeast to synthetic feces make a difference in VFA production?

The proposed conditions for the first VFA experiment are summarized in Table 1. We plan to run the fermentation process for 5 days. Samples for VFA analysis will be collected on day 1 and day 5.

We plan to detect VFA using gas chromatography. The first step is to test the gas chromatography method and determine the amount of VFA present in synthetic feces. To test this, we will take 4 samples of synthetic feces and spike 3 of the sample with known concentrations of VFA.

Week 8: June 19-23

This week, we researched microbial composition of human feces and the role of different microorganisms in the human gut and feces. It was found that gram-positive bacteria averaged 10 ^(10.5±0.4(sd)) organisms per gram of wet feces with significant variation from host to host (Dae Lee et al., 2010). The 5 major strains found in human feces are Bifidobacterium adolescentis, Eubacterium aerofaciens, Eubacterium rectale, Peptostreptococcus productus, and Ruminococcus bromii (Dae Lee et al., 2010). Short-chain fatty acids such as propionate, butyrate, and acetate are produced by Bifidobacteriu.

We also researched fermentation conditions that result in higher VFA production. Literature reported highest VFA production when the pH is controlled at 6.8 with NaOH additions (Lifschits, Wolin & Reeds, 1990). Additionally, we discussed the proposed VFA production experiment with Dan, one of our advisors. He suggested reducing the number of replicates and the number of samples we plan to take for the first experiment as our experiment might not work the first time. We also found an alternative method to measure VFA using High Performance Liquid Chromatography (HPLC) instead of gas chromatography. We contacted Christine Sharp, who is a postdoctoral fellow in Dr. Marc Strous’ research lab, as their lab uses HPLC to measure VFA, and they were willing to let us use their HPLC to measure VFA.

Together with our team members from other sub groups, we visited Bonnybrook wastewater treatment plan to discuss wastewater analysis conducted by City of Calgary. We were particularly interested in VFA and COD monitoring in the wastewater treatment plant. Marko Markicevic, the City of Calgary contact we met with, answered our questions about COD and VFA monitoring and suggested we submit a request form obtain COD and VFA data from the City of Calgary. Marko also mentioned a titration method that might be easier for us to use to measure VFA instead of gas chromatography or HPLC. A titration method, however, will only show the total concentration of VFA unlike the HPLC that can show concentrations of different types of VFA.

Lastly, we contacted nanoparticle manufacturers including US Research Nanomaterials Inc., Nanostructured & Amorphous Materials Inc., Sigma-Aldrich, and NN-Labs to inquire about PHB particles in 20 - 60 nm range. We wanted to obtain PHB nanoparticles to mimic the PHB particles we expect our engineered bacteria will make, in order to start testing our extraction and purification methods. Only NN-Labs might be able to produce PHB nanoparticles in the this size range.

Week 9 : June 26 - June 30

This week, we made synthetic feces in the lab following a recipe found in literature (Colon, A. Fobris-Stokes & A. Deshusses, 2015). However, we substituted oleic acid with peanut oil, which contains about 50% oleic acid and is much cheaper. Peanut oil was also used in a different synthetic feces recipe developed by NASA [23]. We prepared synthetic poop supernatant samples with 0.05 mM, 0.5 mM and 2 mM concentrations of 1:1:1 solution of acetic acid, butyric acid and propionic acid assuming no VFA were present in the samples initially. Although the HPLC working range for VFA quantification is 0.1 - 1 mM, we wanted to test concentrations above and below the limit as we are unsure of VFA concentrations we should expect from our fermentation experiments. Lastly, we also prepared a supernatant sample without added VFA. We then ran these samples on the HPLC.

While running HPLC samples in Dr. Strous’ lab, we also spoke with Karen, who is working on producing PHB from algae. Karen can provide us with samples of their PHB. They produce approximately 2 mg of PHB from 25 mg of dry weight, obtained from 15 mL of harvest and have approximately 700 mL of harvest per day.

NN-Labs, that we contacted last week, can make PHB granules in 20 - 60 nm range, but we may face suspendability issues. We contacted Dr. Nashaat Nassar, Dr. Maen Husein, & Dr. Giovanniantonio Natale about re-suspension and dispersion of nanoparticles. Dr. Natale recommended literature search on chemical functionalization (Sperling & Parak, 2010).

Week 10: July 3-7

The results of the first HPLC run to quantify VFA were inconclusive due to background noise, which made peak integration challenging, and issues with the standard curve, which most likely occurred due to a dilution mistake during preparation. We identified peanut oil as a potential source of the background noise; for the second run, we will not add oil when preparing synthetic feces and will dilute samples with milliQ water.

This week, we also broke down the process we would need to develop for Mars into multiple stages: collection and fermentation of solid human waste to obtain VFA, separation of VFA from fermented human waste, fermentation of genetically engineered bacteria with obtained VFA to produce PHB, and PHB extraction and purification. After discussing our project with Matthew Bamsey, who is a Chief Systems Engineer at the German Aerospace Center, we decided to use Equivalent System Mass (ESM) to evaluate all the stages of our process. In ESM analysis, pressurized volume, power generation, cooling power and crew time required for a proposed system are converted to an equivalent mass number and added to the system mass. Since the cost of transporting payload is proportional to its mass, ESM is used in Advanced Life Support (ALS) studies as a cost of transportation and as a way to compare different systems (Levri et al., 2003). To help with ESM analysis, Matt Bamsey suggested we also read the Life Support Baseline Values and Assumptions Document, which was created by NASA and provides common assumptions that can be made when developing life support systems.

We read NASA’s document with Equivalent System Mass Guidelines to familiarize ourselves with ESM analysis and factors we need to consider. We also read the Baseline Values and Assumptions Document to understanding what resources would be available on Mars. Among other useful assumptions, we learned about the power sources such as solar and nuclear power that would be available on Mars and how much power each source could provide.

This week, we also run the next set of HPLC samples to quantify VFA. Firstly, we made changes to synthetic poop recipe: live yeast was replaced with yeast extract and peanut oil was removed from the recipe. Secondly, we added a mixture of acetic acid, propionic acid, and butyric acid to synthetic feces supernatant to the final concentrations that were within the HPLC working range: 0.25 mM, 0.5 mM, and 0.75 mM assuming the initial sample had no VFA. We also run an undiluted sample of synthetic feces supernatant without additional VFA added. All samples were run in triplicates, as before.

Week 11: July 10-14

Although the second HPLC run still had background noise, we were able to manually integrate the peaks for acetic acid, propionic acid, and butyric acid.

Results from the second run of our VFA quantification experiments showed that there were approximately 109 mmol/L acetic acid , 197.51 mmol/L propionic acid, 12.65 mmol/L butyric acid in our supernatant, which was much larger than we had expected. This meant, we needed to dilute our samples before passing them through the HPLC.

We have also spent a considerable amount of time brainstorming the process flow: starting from the feces production and collection and finishing with the PHB extraction and purification. The goal of these discussions was to determine the main stages in the process and ensure all members of process development sub-group envision the process the same way. We have also discussed the milestones related to each stage of the process and created a timeline.

VFA extraction from synthetic feces protocols were created, a new synthetic feces recipe was chosen and all the required laboratory supplies were purchased to test those protocols next week. We have also found a sponsor for the PHB samples - PolyFerm Canada have kindly agreed to sent us a couple of hundred grams of PHB powder to test the PHB extraction procedures and to use as a standard.

Week 12: July 17-21

This week, the engineering team worked on three major project components: VFA separation from synthetic feces laboratory experiments, VFA quantification titration experiments and the HPLC experiments.

Titration is the industry employed method for continuous testing for the overall VFA concentration in the solution. It is based on the acid-base chemistry: the sample is titrated to the specified end points with sulfuric acid and the VFA concentration is found using a formula based on the amount of acid consumed to reach the end point. This method worked when the standards of 1mM acetic acid and 1mM of acetic, propionic and butyric (1:1:1) acids were tested. The titration method was then tested on synthetic poop supernatant and the total VFA concentration of about 80mM was found, which is in line with the range of VFA present in the human fees.

Four experiments were conducted to test different VFA separation methods: simple filtration, settlement, centrifugation and pressure-filtration. It was found that pressure filtration is the most efficient way of recovering liquid from synthetic feces. However, it required quite a lot of pressure and didn’t seem to be the most feasible solution for the solid human waste on Mars. So another experiment was conducted where a staged filtration approach was used - the feces passed through a series of filters which gradually decreased in size. The recovery past 0.2 micron filter was very small - 10% of the water present in synthetic feces. However, this was also a result of losses of liquids due to transfer between containers.

For this week's HPLC experiment, samples were diluted by three different dilution factors. The HPLC results were more reliable. The optimal dilution rates were determined from this experiment and will be used in the future.

Week 13: July 24-28

This week a couple of group members worked on developing a large scale method for VFA extraction based on conducted experiments. The proposed technologies included screw dewatering system, centrifuge, worm centrifuge and self-cleaning filters. We have also looked into the wastewater processing technologies employed on the International Space Station and the technologies proposed for Mars missions by NASA. This research generated new ideas such as multi-filtration, distillation and torrefaction. Torrefaction can be defined as a thermochemical treatment of biomass in the absence of oxygen. The two methodologies that the team chose to explore further were the screw press followed by multi-filtration and the torrefaction processing unit.

This week, we also focused on feces collection on Mars and fermentation of feces to increase the concentration of VFA, which would then be consumed by engineered bacteria to produce PHB. In our proposed system, astronaut’s feces would be collected using a vacuum toilet into a 10L storage tank. Although a vacuum toilet requires a small amount of water, it can be recovered at the end of the process. The volume of the storage tank was selected based on NASA requirements of accommodating production of up to 150 grams of feces per event for 2 events per crew member per day and also accommodating for potential diarrhea events that can result in up to 1.5L of liquid. In the next stage of the process, feces would be fermented with naturally occurring bacteria to increase the concentration of VFA. Although higher VFA production is expected to occur at 37C, experiments will be conducted to determine whether VFA production can also occur at room temperature.

Week 14: July 31 - August 4

To compare the two proposed methodologies for VFA and liquids extraction for large scale, the ESM parameters were found/estimated for different systems. Since the torrefaction process is a novel solution to solid waste management the ESM parameters for the equipment are not yet developed, so it was chosen to make estimations based on the vapour compression distillation (VCD) system, since torrefaction and distillation are governed by similar principles.

Table 2 summarises the ESM parameters. The data for multi-filtration and VCD system was found in the paper by (W. Jones, W. Fisher, D. Delzeit, T. Fynn & H. Kliss, 2016). The parameters for the screw dewatering systems were found while doing the market search.

Week 15: August 7- August 15

After researching the torrefaction of solid human waste process, we realised that the VCD estimation for the process is not reliable due to different resource requirements, feedstock size and temperature. A diagram (Figure 1) of the full-scale apparatus was created with all the materials, dimensions, and parameters. Equipment set-up, materials, and energy inputs were estimated based on experimental setup found in literature (A. Serio, E. Cosgrove & A. Wojtowicz, 2016). The suggested equipment set-up assumes batch processing once in 3 days, mild pyrolysis 45 minutes in duration, the maximum temperature of the reaction vessel of 280 degrees Celsius, and 2.5kW power supply. The above stated conditions should assure complete recovery of moisture (A. Serio, E. Cosgrove & A. Wojtowicz, 2016), some additional water production (pyrolytic water), modest reduction of the dry solid mass, and the evaporation of all the required VFA. The ESM estimates for the torrefaction processing unit are included in Table 3.

Final comparisons for the two proposed systems were made (Table 4) and the team chose to proceed with torrefaction processing unit.

This week we also planned our experiments for PHB extraction. We plan to make a 5 g/L PHB-co-HV suspension in water to be sonicated. The granule size of PHB-co-HV sample we received from PolyFerm was too large for our purposes, since we expect our secreted PHB to be in the 20-60 nm range (Rahman, Linton, Hatch, Sims & Miller, 2013). To make a representative mixture from which we would have to extract the PHB, we plan to dilute the sonicated PHB-co-HV suspension with synthetic feces supernatant to make a 3.5 g/L mixture, which is within the range of concentrations we might expect to be secreted (Rahman, Linton, Hatch, Sims & Miller, 2013)). We want to test 8 different conditions:

• PHB self-agglomeration (24 hrs, 48 hrs)
• Centrifugation (200 RPM, 500 RPM, 1000 RPM)
• PHB + Coagulant (CaCl2) + settling
• PHB + Coagulant (CaCl2) + centrifugation (500 RPM)

We plan to use 10 mM (final concentration) of CaCl2 as our coagulant because literature reported 95% of PHB granules were sedimentable by low speed centrifugation after addition of 10 mM (Resch et al., 1998).

This week, we also tested an HPLC method for PHB quantification. The same HPLC column we use for VFA quantification can also be used for PHB quantification. In the proposed method, PHB is converted to crotonic acid by dissolving in sulfuric acid for 30 min at 95C. For the first HPLC run, we prepared crotonic acid standards of various concentrations to see whether the HPLC method will be able to detect crotonic acid. The run was extended to 60 minutes as we were unsure of the retention time of crotonic acid. The method was successful and crotonic acid picked around 20 minutes.

Together with the secretion team, we started working on synthetic feces fermentation experiments to determine the optimal temperature for VFA fermentation step and to see whether engineered bacteria can produce PHB from synthetic feces. The two temperatures used in the experiment were 37°C and 22°C, which correspond to optimal bacterial growth temperature and room temperature in a Mars habitat, respectively. For the experiment, we used Imperial and Tokyo phaCAB constructs and E. coli without PHB-producing genes as a control.

Week 16: August 14-18

This week we worked on creating representative PHB samples for the extraction experiments. Our bacteria would be producing PHB in the 20-60nm range, and the PHB that we received from Polyferm is in the 0.1-1mm range. PHB was suspended in tap water to achieve 5 g/L concentration. PHB-co-HV was also suspended in tap water to achieve 5 g/L concentration. The samples were then sonicated using QSonica homogeniser. The sonication was performed on both samples at Amplitude -37 at 25 second intervals for 10 minutes. The particle size distribution was later measured on the NanoPlus HD device(Figure 2). It is clear that the finer PHB-co-HB powder was broken down better than the larger PHB particles, yet the required size range was still not achieved. More research would be done to improve the experimental procedure to achieve representative sizes.

Week 17: August 21-25

Week 18: August 28-September 1

Week 19: September 4-8

Week 20: September 11-15

Week 21: September 18-22

Week 22: September 25-29

Week 23: October 2-6

Week 24: October 9-13

Week 1 & 2: (May 15 - May 26)

Week 3 (May 29 - June 2)

To analyze the different mathematical models proposed in Week 1 & 2, we used the following criteria:

• Usefulness to the project
• Time required
• Skills
• Resources

Week 4: June 5 - June 16

The modelling group met with Dr. MacCullum to discuss the possible mathematical modelling methods. The group was advised that flux balance analysis and kinetic model would be the best to pursue for our project as it will inform our experiments and is feasible in the given time.OpenCobra toolbox for MATLAB was installed because it has functions for carrying out flux-balance analysis and visualizing the results.

Week 5: June 19 - June 23

This week we met with a group of postdocs working in Dr. Ian Lewis’s lab. We discussed how flux balance analysis could be helpful for our project. We also discussed flux variability analysis (FVA) in comparison to flux balance analysis. We decided that after finding optimal solutions using FBA, we can look into FVA. We were given some suggestions on some objectives we could look for in our model such as optimizing bacterial growth, optimizing PHB production using glycolysis only, and optimizing PHB production using beta-oxidation pathway only. This could be done by changing the parameters of the command optimizeCbModel(‘parameter’).

Week 6 & 7: June 26 - July 7

The modeling team had a meeting to discuss the milestones for flux balance analysis and kinetic model. We decided on the different models we plan to optimize using the flux balance analysis and kinetic modelling. We plan to study a number of models. One of the model will contain pathways for beta oxidation and PhaJ-C4 genes that can help produce PHB from medium-chain and long-chain fatty acids. The other model will contain CBA genes and PhaJ-C4, which can help produce PHB from glucose, short-chain, medium-chain, and long-chain fatty acids. The kinetic model will look into reactions that are rate-limiting in these pathways. Thus, FBA and kinetic model will work together to help optimize the production of PHB in E. coli (BL21) and modify substrate concentrations involved in the rate-limiting steps.

Week 8 & 9: July 10 - July 21

FBA: For the flux balance analysis we found an e coli model for our strain, which is BL21 (DE3). The model was found from the BiGG database. We looked into the genes and reactions it contained and the reactions that have to be added to the model. The genes of interest that the model already contained are:

• FadD
• FadE
• tolC
• LacY
• LacZ

• PhaA
• PhaB1
• PhaC1
• HlyA
• HlyB

We also used a plugin called Paint4Net to visualise the pathways/reactions in the model. A zoomed in section of the visual representation of FBA analysis resulting from calling the "draw_by_rxn" command for the coli_core_model is given in figure 1.

Kinetic: After deciding to do a kinetic model. We brainstormed what questions we would try to answer with our model. We wanted to compare PHB production via the beta oxidation pathway and the glycolysis pathway. Therefore we would create a model with phaJ and phaC and another with phaC, phaB and phaA. We also wanted to compare overexpressing fadD and fadE and see which one had a higher effect on PHB yield. The following are two other systems we hope to model. Some other questions we considered answering with our kinetic model include: What is the rate-limiting step in synthesis of PHB? What is the rate-limiting step in secretion of PHB? With those questions in mind we started searching for existing models for beta oxidation and PHB synthesis. We decided to base the PHB synthesis part of our model on the one created by the 2013 Imperial team. [1] For the beta oxidation part, since we could not find an established model we looked at the pathways for degradation of oleic acid (the main long-chain fatty acid in our media) via beta oxidation (Ren et al., 2004).

Week 10 & 11: July 24 - Aug 4

FBA: This week we looked into having a visual representation of the reactions/pathways in e coli BL21. However, the graph had too many nodes and the cobra toolbox could not visualise it. Thus, we looked into plotting a part or subsystems that included reactions of interest. This was done after optimizing the model and then calling the draw_by_rxn command. The command we used to select the specific reactions of interest such as the citric acid cycle was: fluxReactions = Model.rxns(ismember(Model.subSystems,'Citric Acid Cycle'));

Week 12: Aug 7 - Aug 11

Week 1: May 1 - May 5

In the planning phases of our project, our team envisioned a wastewater treatment application to our engineered bacteria; however, much of the planning work done before the start of May was focused on the synthetic biology and engineering aspects of our project. On May 2nd, our team began holding human practices meetings. These meetings, held throughout the summer, focused on both high-level policy aspects of human practices and the education and public engagement aspect of human practices. At our first human practices meeting, the human practices team brainstormed different ways to get involved with the community.

We made a list of bioplastic production companies to contact regarding the input they would have for our team. We sent emails to TELUS Spark, Minds in Motion, TedX, and the wastewater treatment plant in order to secure possible meetings with these organizations.

We also considered different aspects of policy to look into if we had chosen the wastewater treatment application: the safety of bioplastics, the impact of our engineered bacteria on the ecosystem, and the different applications to which our bioplastic is suited (medical implants, 3D printing, etc.) We reviewed the 2016 UofC Calgary team’s human practices efforts in order to understand possible avenues which we could take for our human practices efforts this summer.

Week 2: May 8 - May 12

We continued to follow up with contacts at the Pine Creek wastewater treatment plant. On May 9th, Michaela met with Magdalena Pop (Magda) from GeekStarter to discuss logistics for a workshop which GeekStarter was planning on holding at the University of Calgary for all of the Alberta iGEM teams. The list of requirements for the workshop were provided by Magda and were listed below:

• Room booking (lecture theatre for expert presentations and small rooms for breakout sessions)
• Catering (lunch, served buffet-style in the HRIC atrium)
• Printed signs and providing directions to the different rooms during the event (needed to know the number of signs and directions of arrows needed)
• Accommodating the final number of attendants (provided by Magda by May 23), as well as any dietary restrictions for the 50-70 planned attendees
• Help with snacks and coffee for the afternoon (which was not provided through catering company)
• No hotel rates for out-of-town guests could be arranged (as the number of attendees was not yet known)

Week 3: May 15 - May 19

We looked into sponsorship opportunities with Genome Alberta, based on past teams from Calgary receiving funding from the aforementioned organization. On May 18th, Michaela scheduled a meeting with Magda for May 30th at 2:00 PM (MST) in order to review and finalize the plan for the June 11th GeekStarter workshop. Michaela also contacted Lamiley Ludderodt , a caterer affiliated with the University of Calgary, regarding the catering for the workshop.

Our team began to read into microbead policies, as we were aware that microbeads were being phased out of cosmetic products in Canada, and the option of biodegradable microbeads was not widely known to the public. We wondered how increased public awareness of the possibility of biodegradable microbeads would impact government action on what is seen in Canada as a serious ecological issue. A summary of our findings can be found on our human practices page, as well as under Week 6 of this journal.

Week 4: May 22- May 26

We began emailing other Canadian iGEM teams in search of collaboration opportunities. UBC’s iGEM team replied to our email and asked for our CRISPR/Cas9 protocols. McMaster also replied to our email and expressed interest in a wet lab collaboration with our team.

We also inquired about their interest in possibly organizing a Canadian iGEM team Newsletter. Helen took on the initiative of managing the submissions, editing, and production of the newsletter, which she envisioned as an accessible platform for collaborations between Canadian teams.

On Thursday, May 25, the entire iGEM Calgary team attended a tour of the Pine Creek wastewater treatment plant. We learned about the different stages of wastewater treatment. A diagram, provided by the City of Calgary, was used to illustrate the wastewater treatment process which we saw firsthand. This diagram is shown below:

Week 5: May 29 - June 2

CRISPR and dCas9 protocols were obtained from the Childs Lab at the University of Calgary. These were then sent to the UBC iGEM team as per their request. The remaining Canadian iGEM teams were contacted to follow up on possible collaborations and the newsletter. All the Canadian teams got back to us and expressed their interest in establishing the newsletter. On May 30th, our team began researching the different applications for our project, as the wastewater treatment plant field trip made us rethink the direction of our project. The applications we chose to look further into were:

• Wastewater treatment
• Small-scale wastewater treatment in developing countries
• Landfill leachate treatment
• Outer space missions

A few team members were chosen to look into specific aspects of each application so as to gain a holistic understanding of the feasibility of each application and thus choose the application which was the best fit for our project. The aspects of each application which our team researched are listed as questions below:

1. Is there a significant demand for a solution to this problem?
2. What are the costs associated with implementing our process to solve this problem?
3. Do we have any resources or industry contacts in this field? Whom should we contact for an expert opinion if we choose this issue?
4. What is the environmental/social/political/economic impact of our engineered bacteria as a solution to this problem?
5. Is synthetic biology the best solution to solving this problem?

We made a table comparing the above aspects of the different applications. This table is shown below:

On May 31st, we had a Skype meeting with the UNBC iGEM team to discuss project details. The notes taken during that video call are summarized below:

UNBC Project details (chassis, etc.)

• Using a Level 1 Staph strain as a proof of concept for Level 2 Staph aureus
• Phage delivery system for spreading their plasmids in their bacterial population

Newsletter

• UNBC “definitely interested” in newsletter and were interested in the July issue

Collaboration opportunities:

• Their team: mostly biomedical/biochemistry
• May need help in wiki and engineering (mostly wiki since their project is very biomedical)
• They said they have a very advanced analytical lab (may be able to help us with characterizing our PHB product later on)