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. We examined many organisms capable of producing PHB.

• Pseudomonas putida
• Pseudomonas aeruginosa
• Aeromonas caviae
• Ralstonia eutropha

We also looked at different genes for the pathways (beta-oxidation and glycolysis) of interest. After finalizing the genes, the constructs were uploaded on benchling as follows:

• Promoter T7, spacer sequence, RBS (B0034LINK), FadE_{E. coli}, FadD_{E. coli}, PhaJ4_{P. putida}, PhaC_{P. aeruginosa}
• Promoter T7, spacer sequence, RBS (B0034LINK), FadE_{E. coli}, FadD_{E. 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_{R. eutropha} with T7 (constitutive) on lac I
• phaCBA_{R. eutropha} with T7 (constitutive) on lac I double vector
• Hybrid promoter with number 8
• T7 (constitutive), spacer sequence + RBS + spacer sequence(B0034LINK) + PhaC1 ap + phaJ4 _{P. putida}+ fadE_{E. coli} + fadD_{E. coli} + 10 nucleotide spacer sequence + RBS (B0034) + spacer sequence + phaCAB_{R. eutropha}

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 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 (phaC1_{P. aeruginosa}J4_{P. putida} and phaCBA_{R. eutropha}) 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.LINK

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 overnight 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 re-make overnights 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^tm 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.

After we tested that the restriction enzymes are functional, we digested our PhaC and PhaBA gblocks from IDT and our pET29b(+) plasmid following the Restriction Digest protocol with the enzymes listed below and ligated the digested parts following the Ligation of DNA Inserts to Plasmid Backbone protocol.

DNA	Enzymes
PhaC	NotI, HindIII
PhaBA	HindIII, KpnI
pET29b(+) for PhaC	NotI, HindIII
pET29b(+) for PhaBA	HindIII, KpnI

We then streaked a fresh plate of E. coli DH5α containing pET29b(+), made overnight cultures, 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 overnight. The next day we chose four colonies from PhaC and PhaBA-transformed plates to make overnight cultures and a master plate from. We isolated the plasmid from our overnight 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.

Transformed Plasmids		Enzymes	Expected band size
PhaC	For confirmation	HindIII, NotI	5.4 kb, 1.8 kb
	For directionality	HincII	1.8 kb, 1.4 kb
PhaBA	For confirmation	HindIII, KpnI	5.3 kb, 2.1 kb
	For directionality	HincII	4.4 kb, 3.0 kb

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 pET29b(+)-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 pET29b(+)-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, E. coli with pET29b(+)-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 pET29b(+)-phaC transformant colonies (5-11) using with restriction enzyme digest. The results are shown below.

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. PhaC-J insert from IDT was digested with HindIII and SalI, ligated with pET29b(+) vectors, transformed into competent DH5α cells, plated on Kan resistant plates, and left to incubate overnight 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. Our fadE and fadD DNA inserts from IDT 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 pET29b(+)-phaC and pET29b(+)-phaCJ were transformed into competent DH5α cells and plated on Kan LB agar plates. Colonies appeared on these plates. A master plate and overnight cultures were made to screen these transformants. The overnight 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.

Transformed Plasmids		Enzymes	Expected band size
PhaC	For confirmation	HindIII, NotI	5.4 kb, 1.8 kb
	For directionality	AscI, SphI	6.1 kb, 1.1 kb
A Beta Oxidation	For confirmation	HindIII, NotI	5.4 kb, 2.2 kb
	For directionality	Xmal	5.0 kb, 1.5 kb, 1.1 kb

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 overnight incubation. A master plate and overnight cultures of selected colonies from each plate was made. The vectors were isolated, digested for confirmation of insert, and ran 1% agarose gel.

Transformed Plasmids	Enzymes for Insert Confirmation	Expected Band Sizes
B Beta Oxidation	HindIII, SalI	5.4 kb, 2.5 kb
C Beta Oxidation	KpnI, SalI	5.3 kb, 1.8 kb

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 overnight cultures 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 overnight culture was made and plasmids were isolated by miniprep and stored in the -20 freezer for future use.

Week 14 (July 31 - Aug 4)

This week, we digested, ligated, transformed PhaC and A BetaOx,into competent DH5α using pET29b-RFP. However, transformation of A and Pha C did not work. Therefore, A beta oxidation and PhaC were digested, ligated, and transformed again. However, transformants did not grow. We ran control experiments with the enzymes to check their functionality. The results showed that the enzymes were functional. Ligation was performed again using quick ligase for the A beta oxidation insert and the PhaC insert and then transformed again. However, transformation did not work. Because the LB tube was clear we rationalized that there was no cell growth from the 1 hour incubation in the shaker after transforming. The cells in plain LB from the transformation the previous day were spun down and resuspended and plated and incubated again overnight.

Made overnight cultures of B beta oxidation in pUCIDT-Kan. Miniprepped B beta oxidation part in pET29b and the other in pUCIDT.

Overnight cultures of B Beta oxidation in pUCIDT-Kan were prepared for confirmation digest where it was digested with the enzymes below.

Tube 1	Tube 2
pUCIDT + B beta tube 1 0.5 uL DNA 1 uL HindIII-HF 1 uL SalI-HF 1 uL 10x CS buffer 6.5 uL ddH2O	pUCIDT + B beta tube 2 4 uL DNA 1 uL HindIII-HF 1 uL SalI-HF 1 uL 10x CS buffer 3 uL ddH2O

We ran the digested DNA on 1.5% agarose gel at 70V for 90 minutes and the results are shown below.

C beta oxidation was digested from gblocks and ligated with digested pET29b vectors using quick ligase and transformed into DH5α. Five overnight cultures of the C-beta-Oxidation plate from August 2nd plate were made; colonies 1 to 5 were used. Colonies 1 to 4 had no cell growth, but we performed plasmid miniprep on colony 5 and resuspended the plasmids in TE buffer. Nanodrop was performed and the concentration was 154 ng/uL. The C beta oxidation plasmid was digested with KpnI and SalI and then run on a gel at 100V for 40 minutes. The results are shown below; the ligation did not work properly. Therefore, we will be digesting this part from gBlocks once again to ligate them into the proper vectors.

Week 15 (AUG 8 - AUG 13)

A beta oxidation and PhaC

The transformed A beta oxidation and PhaC plates had film of mold on the agar. Therefore, A beta oxidation and PhaC were digested again from their gBlock. More backbone digested out of the pET29b-RFP vectors was also needed. This time, an ethanol precipitation step was used in the sequential digest of pET29b-RFP using HindIII and NotI. The two samples of digested pET29b-RFP vectors were run on a 1% agarose gel. 3uL of sample 1 and 5uL of sample were loaded into the wells after mixing with loading dye and ddH2O. The samples were run at 100V for 30 minutes. No RFP insert bands were not observed on the gel. We thought that this was due to the low concentration of DNA loaded. Therefore, we ran more on 1% LMP gel; 6uL of sample 1 and 8uL of sample 2. However, there was still no band observed. The remainder volume of the two samples of digested pET29b-RFP vectors were stored in the -20 degree freezer.

We thought that the reason why no bands were seen may have been due to improper digestion and decided to digest more pET29b-RFP vectors to isolate the backbone for ligations. More overnight cultures of DH5α pET29b-RFP were made. The vectors were isolated by miniprep, digested sequentially, and then run on LMP gel. Bands indicated that the digestions did not seem to work. Sam performed miniprep of more pET29b-RFP vectors for overnight cultures in three separate tubes labelled A, P, and S. However, only the tube labelled S recovered bands. The vector was digested and run on a 1% LMP gel, but the bands did not show the correct size of predicted fragments. We decided to wait until next week to continue experiments to isolate the pET29b backbone out of the pET29b-RFF.

B beta oxidation

B Beta oxidation in pUCIDT-Kan was digested with HindIII and SalI and run on a 1.5% LMP agarose gel. We observed the expected band at 2.5kb.

The 2.5kb band was excised from the 1% LMP gel and was ligated to the pET29b vector that was digested with HindIII and SalI. The ligation mixture was used to transform 2 tubes of competent DH5a cells because there was an excess of ligation mixture. One transformation used 45uL of the ligation mixture and the other used a lower volume of 25uL. The 2 tubes of transformed competent DH5a cells were plated on LB-Kan plates and left to grow overnight at 37 degrees. Bacterial colonies were observed on the transformed plates. The 2 plates of E. coli transformed with B beta oxidation were used to make master plates and overnight cultures for confirmation digest next week; 3 overnight cultures of the cells transformed with the higher voume of ligation mixture and 8 overnight of the cells transformed with the lower volume of ligation mixture.

C Beta oxidation

Our ligation of C beta oxidation to the pET29b vectors did not work last week. Therefore, we digested the C beta oxidation gBlock and the pET29b vectors with KpnI and SalI once again. We ligated the insert and the vector and transformed competent DH5a cells with the ligation mixture and plated 100uL of the mixture on an LB-Kan plate. The transformation was successful because colonies were seen on the plate. Four colonies were used from the transformation plate to make overnight cultures and a master plate. We planned to use the overnight cultures for confirmation digests for the following week.

Week 16 (AUG 14 - AUG 18)

A beta oxidation and PhaC

Because the gel confirmations for the isolation of the digested pET29b backbone did not work last week, we ran the pET29b-RFP digested vectors from August 10th on a 1% LMP gel to confirm the success of the digestion. We ran the remaining digested vector samples stored in the freezer from last week loading dye; 10uL of sample 1 and 4.5 uL of a sample 2. The bands were observed this time. There was one band at 5.2kb and another blurry band around the hundred bp location for the RFP insert digested out of the vector. The gel was excised to isolate the backbone at 5.2 kb.

We successfully isolated the pET29b backbone needed for ligations to our inserts. An in-gel ligation was performed with the isolated pET29b vectors and the digested A beta oxidation and PhaC inserts. Competent DH5α cells were transformed with the ligated vectors containing A beta oxidation and PhaC. The transformation for A beta oxidation and PhaC did not work. We spun down the transformed cells in the plain LB stored in the fridge from the previous day and resuspended them in 50uL LB Kan and plated the cells. However, the plates were not placed in the incubator after plating when we checked on them the next day. There were no colonies seen on the plates. Therefore, we placed the plates in the incubator for another day. Bacterial colonies were observed on the plate when we observed them the next day. We made a master plate of the 8 of the transformed colonies for both A beta oxidation and PhaC and made overnight cultures for restriction digest confirmation the next day. We screened 8 A beta oxidation colonies the following day by performing miniprep on overnight cultures.

This week, we also performed PCR on PhaC gBlock to amplify the sequence to produce more PhaC gblocks in case we need to digest more. The PCR did not work. We attempted PCR again with Taq polymerase instead of pfu and diluted the primers. The results showed a band the size of the template, but the gel bands appeared smeared.

B Beta oxidation

We performed plasmid miniprep on the B beta oxidation overnight cultures made at the end of last week. A double digest was performed on the isolated B beta oxidation vector using HindIII and SalI.

C Beta oxidation

We performed plasmid miniprep on the transformed overnight cultures made a the end of last week. The plasmids were digested using SalI and KpnaI.

Confirmation Digest of A beta oxidation, Pha C, and C beta oxidation

The digested products from A beta oxidation, PhaC, B beta oxidation, and C beta oxidation were run on a 1% agarose gel.

Transformed Plasmids	Enzymes for Insert Confirmation	Expected Band Sizes
A beta oxidation	NotI, HindIII	5.4kb, 2.2kb
PhaC	NotI, HindIII	5.4kb, 1.8kb
B Beta oxidation	HindIII, SalI	5.4 kb, 2.5 kb
C beta oxidation	SalI, KpnI	5.3kb, 1.8kb/td>

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Figure Captions

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Figure Captions

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Figure Captions

We were having troubles cloning in PhaC into a vector. We theorized that this was due to the close positions of the restriction sites. We decided to try blunt ending PhaC into the vector containing PhaBA to ligate them together into the PhaBA vector instead of trying to get PhaC into its individual vector.

Week 17 (AUG 21 - AUG 27)

We were having troubles cloning in PhaC into a vector. We theorized that this was due to the close positions of the restriction sites. We decided to try blunt ending PhaC into the vector containing PhaBA. To prepare the samples for the blunt ending, 2 samples of pET29b were digested using HindIII; the 2 microcentrifuge tubes were labelled 1 and 2. After digestion, the enzyme mix, dNTPs and blunting buffer were added to each of the two tubes. The mixture was heat inactivated at 70 degrees for 10 minutes. DNA cleanup was performed on the mixture in the two tubes. The DNA was resuspended in elution buffer. We performed a nanodrop on the DNA but the concentration was negative, which indicated that the DNA cleanup did not work.

The gel electrophoresis results from last week confirmed the successful transformation of DH5a with the ligated vector and inserts; A beta oxidation, B beta oxidation, and C beta oxidation. Overnight colonies of the cells transformed with these complete ligated inserts and vectors were made from the master plate. Overnight cultures of PhaC-pET29b colony 2, A beta oxidation-pET29b colony 1 and 6, and C beta oxidation-pET29b were made; two sets of cultures were made for PhaC-pET29b colony 2, A beta oxidation-pET29b colony 1, and C beta oxidation-pET29b colony 4. Miniprep was performed on the overnight cultures.

One set of isolated plasmids were resuspended in ddH2O and sent for sequencing confirmation (PhaC-pET29b colony 2, A beta oxidation-pET29b colony 1 and 6, and C beta oxidation-pET29b colony 4). The other set was resuspended in TE buffer for digestion confirmation (PhaC-pET29b colony 2, A beta oxidation-pET29b colony 1, and C beta oxidation-pET29b colony 4) and ligation with other parts (C beta oxidation ligation with A beta oxidation).

XhoI and HindIII were used to digest PhaC-pET29b colony 2, A beta oxidation-pET29b colony 1, and C beta oxidation-pET29b colony 4. The samples were mixed with DNA loading dye and loaded into the wells of a 1% agarose gel. The gel was run at 80V for 40 minutes.

The results for PhaC did not seem to work because there were four bands observed. C beta oxidation digested with HindIII and XhoI seemed to work but no bands were observed on the gel for C beta oxidation digested with SalI and KpnI.

We decided to do another digestion confirmation. PhaC-pET29b colony 2 was digested with NotI and HindIII, A beta oxidation was digested with XhoI and HindIII, and C beta oxidation colony 4 was digested with SalI and KpnI. The digested samples were run on a 1% LMP gel together with undigested PhaC as a control. Two bands were observed on the gel.

Because the results from the digestions were not conclusive, we made more overnight cultures of A beta oxidation colony 1 and PhaC colony 2, and C beta oxidation colony 4 for plasmid miniprep and digestion confirmation experiments. The digested samples were run on 1% LMP. The bands seemed very smeared for the lanes with loaded DNA. We searched online for reasons why the bands appeared this way. We concluded that the DNA may have been degraded due to the continuous freeze thaw that the samples went through. We decided to modify our experiments so that plasmid miniprep and digestion confirmation happen on the same day so that the DNA is kept in good shape.

We decided to run another digestion confirmation this time alongside undigested ligated insert+vectors and undigested pET29b as controls. No bands were observed for many of the samples.We decided to make 2 sets of overnight cultures of the PhaC-pET29b colony 2, A beta oxidation-pET29b colony 5, and C beta oxidation-pET29b colony 4, and B beta oxidation colony 5. The A beta oxidation part was successfully transformed into E. coli BL21(DE3). This allowed us to begin preliminary experiments to test if our bacteria is capable of PHB production while we carried on ligation experiments. We discussed some experimental conditions for our tests.

Week 18 (AUG 28 - AUG 31)

We labelled the tubes with numbers seen in the table. The plasmids were isolated from these plasmids and samples from the isolated plasmids were digested with different enzymes. Some samples were left undigested.

Tube	Content
1	PhaC-pET29b colony 2
2	PhaC-pET29b colony 2
3	pET29b(+)-fadD colony 4
4	pET29b(+)-fadD colony 4
5	pET29b(+)-phaCJ colony 1
6	pET29b(+)-phaCJ colony 1
7	pET29b(+)-fadE colony 5
8	pET29b-fadE colony 5

Samples of the plasmids prepared from the overnight culture were digested with enzymes and run on a gel. Undigested samples were also run alongside the digested plasmids to serve as controls. Samples of plasmids from overnight culture tubes 1, 6, 4, and pET29b (from freezer stock) were digested with XhoI and HindIII. Samples from tube 7 and 4 were digested with SalI and KpnI. Undigested samples from tubes 1, 6, and 4 were run.

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Figure Captions

PHB-production experiments

Calculations on M9 minimal media were done for the PHB production experiments. M9 salts were mixed with supplementation chemicals for the inoculation of PHB-producing bacteria. Stock solutions of acetic acid, propionic acid, and butyric acid were made for the VFA mixture. We required 5g/L of VFA within our final solution. The ratio from literature was a 1:2:1 ratio for propionic, acetic, and butyric acid. The final amount of the xg/L stock solution we made from 99% acids was 2.86 mL of acetic stock solution, 2.07mL of butyric stock, and 2.01mL of propionic stock.

We prepared 12 overnight cultures of E. coli BL21(DE3) transformed A beta oxidation for 24 hour growth. The bacteria in these tubes were added to M9 media with different chemical supplements.

____!!!!Link to protocols page for A beta experiment!!!___

After the flasks were inoculated with respective chemicals, they underwent 16-24 hours of inoculation following the 24 hour overnight growth of the cells. The next day, chemical extraction of the contents of flasks took place. The protocol followed was from the experiment performed by the secretion team when they were testing out past iGEM parts that were capable of producing PHB. After extraction, a white powder was collected at the bottom of the tubes. The results are shown in the picture below.

Week 19 (Sep 4 - Sep 8)

We made overnight cultures of A+B beta oxidation colonies 1-6 and pET29b(+)-phaCBA colonies 1-10 from the master plate that was streaked last week. We performed plasmid miniprep on these cells the next day. phaCBA was then digested using NotI and KpnI. We only digested pET29b(+)-phaCJ FadE (A+B) beta oxidation colonies 3 and 5 with NotI and SalI; the other colonies did not show pure samples from the nanodrop. The plasmids were digested at 37 degrees in a water bath for 1.5 hours and then heat inactivated for 20 minutes immediately after. We ran the samples on the gel. We transformed pSB1C3 into E. coli DH5a and made overnight cultures of the E. coli.

We sent pET29b(+)-phaCBA for sequencing because the gel bands confirmed the successful transformation of the part. The sequencing results showed that colony 9 was successfully ligated and cloned into E. coli DH5a. Only one band was seen for A+B beta oxidation so we decided to repeat the procedure again. We made overnights of the A+B beta oxidation colonies 1-14 and performed the same procedures for plasmid isolation and digestion confirmation.

We also began putting our parts into the pSB1C3 backbone for the iGEM registry. We made overnight cultures of the E. coli transformed with pSB1C3, pET29b(+)-phaCJ, and pET29b(+)-phaCBA. We performed miniprep on the cultures and then digested the plasmids using XbaI. We then ligated them together and transformed them into DH5a.

Week 24 (Oct 9 - Oct 15)

To quantify the amount of PHB made by our phaCBA construct, we made 9 overnight cultures of E. coli BL21(DE3) that contained the pET29b(+) phaCBA plasmid in 10mL of LB Kan broth. As a negative control, we also made 3 overnight cultures of E. coli BL21(DE3) that contained the pET29b(+) vector each in 10mL of LB Kan broth. The OD was then measured using 1mL of the overnight cultures before they were inoculated in flasks that contained the synthetic poop supernatant for 24 hours. The next day, we performed the chemical PHB extraction protocol on the cultures in the inoculation flasks. After the contents of the falcon tubes were dried, we weighed the falcon tubes and compared the weight with the initial weight of the falcon tubes. Our results are shown in the table below.

Table 1. Recorded OD₆₀₀ of the overnight cultures before inoculating the media containing VFAs, OD₆₀₀ of cells after growing in media for ~24 hours, then centrifuged and resuspended in (1x) PBS for extraction. Initial weight of 50 ml falcon tubes and final weights of tube containing PHB in grams was recorded.

Week 25 (Oct 16 - Oct 22)

The PHB extraction experiments have shown us that we are able to produce PHB with our parts. We decided to perform SDS PAGE experiments to verify that our proteins are being expressed. We performed SDS page on proteins samples that we extracted from overnight cultures of E. coli BL21(DE3) containing pET29b(+) vector, pET29b(+) vector with phaCBA insert, and pET29b(+) vector with phaCJ. We made 3 overnight cultures of E. coli containing for the three different vectors. We incubated them at 37 All twas induced with a different amount of IPTG. The results of the SDS-PAGE are shown below.

The expected sizes of the proteins produced by our constructs are as follows:

• PhaC: 65.1kDa
• PhaB: 27.2kDa
• PhaA: 41.4kDa
• PhaC: 63.4 kDa
• PhaJ: 17.7 kDa

The results showed that PhaJ was being expressed for our pET29b(+)-phaCJ construct in the insoluble phase. After trouble-shooting our experiment, we decided that the cells were not fully lysed because our insoluble proteins samples were stringy, indicating that the cell membrane was still intact for some cells. Therefore, we decided to modify the SDS-PAGE protocol by adding an sonication step after the 15 minutes incubation of cells resuspended in STET buffer and lysozyme.

We performed another SDS-PAGE with another batch of overnight cultures containing the same vectors. We sonicated the samples five times; we sonicated for 5 seconds and cooled the sample for 5 seconds on ice each time. In addition, we also decided to resuspend the insoluble protein after the soluble portion was separated in 100uL of Tris-glycine running buffer instead of the 50uL outlined on the original SDS-PAGE protocol. The results of this SDS-PAGE experiment are shown below.


Figure_:

Week 26 (Oct 23 - Oct 29)

Our SDS-PAGE results showed that the cells were still not completely lysed following the original protocol due to the stringy state of the protein samples during loading. The protein PhaJ was also being expressed in the insoluble proteins, likely due to the inclusion bodies from culturing at high temperatures. Therefore, this week we decided to lower the temperature that the subculture grows in to 28°C instead of 37°C after IPTG induction. However, our shaker temperature remained at room temperature when we checked on the cultures. Therefore, we still stored these cells after centrifuging and removal of the liquid in the -20°C freezer. We decided to make overnight cultures again and ensured that the temperature was maintained at 28°C. We then prepared the proteins from both of the samples from this week (subcultures incubated at room temperature 28°C). We made changes to the original protocol for SDS-PAGE by resuspending the frozen cell pellets in 500μL of STET buffer to reduce bubbles when sonicating and adding a sonication step. In addition, we resuspended the insoluble proteins in a higher volume of Tris-glycine running buffer; 100μL and 200μL for room temperature and 28°C samples, respectively. The gels were run for 50 minutes at 30mA.

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 (see protocols here)

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 (protocols are here).

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) and two parts of the hemolysin membrane transport protein unit (HlyB : Part:BBa_K2018027 and 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
• Restriction sites were removed from Part:BBa_K2018029 to make the part 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). SP1 contains Phasin-HlyA tag + HlyD and SP2 contains HlyB. 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. Before ordering, further modifications were made to the original parts we uploaded onto Benchling:

• All stop codons were changed to TAA because it is the most effective stop codon in E. coli.
• Codons were optimized for protein expression in E. coli 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. More information about this can be found on our Silver Human Practices page

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 Silver Human Practices 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. Outcomes of this outreach can be found on our Engagement page.

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. Richard Moore 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. Moore. Jacob and Sam used the new cells to successfully complete the laboratory component of the Interlab study this week.

Also, we isolated pSB1C3 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 EcoRI-HF, 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. The new protocol can be seen on our Experiments page.

Our IDT parts were digested and ligated into their corresponding backbones (pET29B or pSB1C3), then transformed into chemically competent E. coli DH5α cells.

Part/Insert	Digested with...	Backbone
Phasin-HlyA Tag	EcoRI-HF, SpeI	pSB1C3
Secretion Part 1	XbaI, HindIII-HF	pET29B
Secretion Part 2	HindIII-HF, NotI-HF	pET29B

Week 10 (July 3-July 7, 2017)

We isolated the plasmids of E. coli DH5α transformants from our ligated backbones/parts from Week 9. 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% agarose gel for confirmation. 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:

Part/Insert	Backbone	Digested with…	Expected Band Sizes
Phasin-HlyA Tag	pSB1C3	EcoRI-HF, SpeI	2.0 kb, 889 bp
Secretion Part 1	pET29B	XbaI, HindIII-HF	5.2 kb, 2.4 kb
Secretion Part 1	pET29B	HincII	6 kb, 1.5 kb
Secretion Part 2	pET29B	HindIII-HF, NotI-HF	5.4 kb, 2.2 kb
Secretion Part 2	pET29B	EcoRV	6.1 kb, 1.5 kb

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 of SP1 and SP2 into E. coli DH5α had failed.

Week 11 (July 10- July 14, 2017)

Colony 1 Phasin-HlyA Tag was sequenced and confirmed that these cells do in fact contain pSB1C3-Phasin-HlyA Tag. Therefore, it 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. The plans/worksheets from this workshop can be found on our Engagement page.

Week 12 (July 17-July 21, 2017)

Our SP1 gBlock was re-digested with XbaI 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 when there is no insert, making our double digests of SP2 with NotI and HindIII-HF very difficult. 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 Experiments 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 Experiments page of the wiki was competed, and we uploaded all of the general protocols used by all subgroups so far.

Our sequencing results of SP1-colony 3 showed that we had successfully transformed pET29B-SP1 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 a low melting-point agarose gel, and ligated with our SP2 part that had been digested with HindIII-HF and NotI. We also managed to create our first batch of PHB using a the PHB synthesis biobrick (PhaCAB: Part:BBa_K934001) that we transformed into DH5α in Week 11. The PHB was extracted with chloroform and poured into a sheet (Figure 3). These protocols can be found here.