iGEM 2019 — Team Calgary

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# Team:Calgary/Notebook

[![](https://static.igem.org/mediawiki/2019/f/ff/T--Calgary--logowhite.png)](https://2019.igem.org/Team%3ACalgary)

* Project

  + [Description](https://2019.igem.org/Team%3ACalgary/Description)
  + [Wet Lab Overview](https://2019.igem.org/Team%3ACalgary/WetLab)
  + [Chlorophyll Extraction](https://2019.igem.org/Team%3ACalgary/ChlorophyllExtraction)
  + [Protein Emulsification](https://2019.igem.org/Team%3ACalgary/EmulsifiedBindingProteinProcess)
  + [Repurposing Chlorophyll](https://2019.igem.org/Team%3ACalgary/RepurposingChlorophyll)
  + [Anti-Fungal](https://2019.igem.org/Team%3ACalgary/Anti-Fungal)
  + [Results](https://2019.igem.org/Team%3ACalgary/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ACalgary/Demonstrate)
  + [Judging](https://2019.igem.org/Team%3ACalgary/Judging)
  + [Awards](https://2019.igem.org/Team%3ACalgary/Awards)
* Human Centered Design

  + [Integrated Human Practices](https://2019.igem.org/Team%3ACalgary/Human_Practices)
  + [Mean Green Machine](https://2019.igem.org/Team%3ACalgary/Modelling/StandardizedSeedGrading)
  + [Sunny Days](https://2019.igem.org/Team%3ACalgary/SunnyDays)
  + [Entrepreneurship](https://2019.igem.org/Team%3ACalgary/Entrepreneurship)
  + [Education and Public Engagement](https://2019.igem.org/Team%3ACalgary/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3ACalgary/Collaborations)
  + [Safety](https://2019.igem.org/Team%3ACalgary/Safety)
* Measurement and Model

  + [Overview](https://2019.igem.org/Team%3ACalgary/Model)
  + [ICARUS](https://2019.igem.org/Team%3ACalgary/Model/ICARUS)
  + [Emulsion Construction Prediction](https://2019.igem.org/Team%3ACalgary/Model/EmulsionPrediction)
  + [Directed Protein Modification](https://2019.igem.org/Team%3ACalgary/Model/DirectedProteinModification)
  + [In Silico Emulsion System Verification](https://2019.igem.org/Team%3ACalgary/Model/InSilicoEmulsionSystemVerification)
  + [Measurement](https://2019.igem.org/Team%3ACalgary/Measurement)
  + [Software Tools](https://2019.igem.org/Team%3ACalgary/Software)
  + [BOTS](https://2019.igem.org/Team%3ACalgary/BOT)
  + [iGAM](https://2019.igem.org/Team%3ACalgary/iGAM)
  + [Results](https://2019.igem.org/Team%3ACalgary/Results)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACalgary/Parts)
  + [Collection](https://2019.igem.org/Team%3ACalgary/Part_Collection)
  + [Improve](https://2019.igem.org/Team%3ACalgary/Improve)
  + [Characterize](https://2019.igem.org/Team%3ACalgary/Contribution)
  + [Validate](https://2019.igem.org/Team%3ACalgary/Validate)
* Journal

  + [Protocols](https://2019.igem.org/Team%3ACalgary/Experiments)
  + [Appendix](https://2019.igem.org/Team%3ACalgary/Appendix)
  + [Journal](https://2019.igem.org/Team%3ACalgary/Notebook)
* Team

  + [Members](https://2019.igem.org/Team%3ACalgary/Team)
  + [Attributions](https://2019.igem.org/Team%3ACalgary/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ACalgary/Sponsors)

## Notebook / ENTRIES

## + Press for Options

## Notebook

# Entries

##

# May 6 — May 10

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

##

## Weekly Summary

This week we were finally divided into our subgroups. We were assigned to extracting the chlorophyll from the binding proteins. We looked at the chlorophyll degradation pathway and decided to concentrate our chlorophyll derivatives to pheophorbide a. The team looked at ways to obtain the compound profiles using spectrophotometry. We also decided to pursue protein purification instead of secretion.

##

# May 13 — May 17

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week we decided on a pathway to stick to and looked at more papers supporting enzyme activities. Based on these we chose to use E. coli as our chassis since Chlorophyllase, Pheophytinase, Chlorophyll b reductase, and 7-Hydroxymethyl chlorophyll a reductase have been expressed in them. We started aggregating parts for our gene circuit using parts from the registry and sequences in the papers. We also found more uses for pheophorbide a in terms of photodynamic therapy.

##

# May 20 — May 24

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

We realigned our pathway and decided on using DsbA as our signal sequence. Team also figured the general G-block structure, along with our parts. Additionally, we gathered literature on reaction buffers, and solvents and absorbances required for HPLC. Protein sequences were blasted and constructs were modified.

##

# May 27 — May 31

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week the team worked on more pymol modelling, reducing the IDT score, organizing OneNote, and working on enzyme assays. Jake also gave his tentative approval (waiting for IDT score to decrease).

##

# June 3 - June 7

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Team had an HP-heavy week, reaching out/talking to Dr. Moore, LSI hub, Dr. Gijs from Genome Alberta, Box meeting for Telus Spark. Parts needed were finalized and ordered. Team started doing lab work (plasmid minipreps and restriction digests). Team got the BL21 cells donated by Dr. Mayi's friend.

##

# June 10 - June 14

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Team made a stock of chemically competent DH5-alpha cells which were found to not be very competent. Another batch of chemically competent cells were created. Our subgroup digested and ligated our 4 enzyme parts.

##

# June 15 - June 19

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week our sequencing results for PPH and HCAR in pSB1C3 part came back positive. More digests, ligations, and transformations for DsBA and ES signal peptides in 2:1 and 3:1 vector: insert ratios were made. The team also had a phone call with Dr. Ronald Moore, a doctor and photobiology expert from the University of Alberta to discuss pheophorbide and photodynamic therapy.

##

# June 24 - June 28

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Digests and ligations, and transformations for SGR and CBR 1:1, ES 1:1, and DsbA 3:1 using EcoRI-HF and PstI and pSB1C3 plasmid were made. A Golden Gate reaction using gel-purified CBR, HCAR, and PPH PCR products was done. The team also started to incorporate DMSO in PCR reactions. We also aided the chlorophyll extraction subgroup in making serial chlorophyll dilutions to aid in their characterization of chlorophyll. The team went to CanolaPalooza, a Canola Convention in Lacombe, Alberta where we met our HP contacts Ward Toma and Dr. Veronique Barthet who work with canola oil.

##

# July 2 - July 5

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week the team attempted to clone all the parts (pSB1A3 + SGR, PPH, CBR, or HCAR) using golden gate assembly into pSB1A3 instead of pSB1C3 due to insufficient plasmid stocks. More ES and DSBA digests, ligations and transformations were done. More cPCR reactions were done for SGR, CBR, ES and DSBA were made. SGR PCR-product from the previous week was also gel-purified. After consulting with our research assistant, we will be adding negative and positive controls as part of troubleshooting our colony PCR reactions in the future.

![](https://static.igem.org/mediawiki/2019/3/3a/T--Calgary--1julyw1.jpeg)

![](https://static.igem.org/mediawiki/2019/9/91/T--Calgary--2julyw1.jpeg)

![](https://static.igem.org/mediawiki/2019/f/fa/T--Calgary--3julyw1.jpeg) **Figure 3. 1% agarose gel showing colony PCR of CBR.**  Gel ran at 100V for 30 min. Expected length of band is 1723bp.

##

# July 8 - July 12

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Did cPCR of golden gate satellite colonies and normal SGR and CBR colonies. Sequencing for ES signal sequence, CBR, and SGR came back negative but DSBA came back positive. Our Golden Gate colony PCRs had multiple incorrect bands. We did more digestions, ligations, and transformations for CBR, SGR, and ES.

![](https://static.igem.org/mediawiki/2019/c/c1/T--Calgary--1julyw2.jpeg) **Figure 1. 1% agarose gel showing colony PCR of SGR , CBR, and HCAR.**  Gel ran at 100V for 30 min. Ladder: 1kb+ gene ruler. Expected band sizes are 2545bp for SGR, 2065bp for HCAR, and 1723bp for CBR.

##

# July 15 - July 19

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

We did a huge masterplate for ES, SGR and CBR and they ended up having quite a bit of RFP contamination. This week, on Dr. Marija's suggestion, we increased the elongation time from 2 min to 2.5 min. The full constructs that we ordered (HCAR, SGR, CBR, PPH) arrived, so me and Micha began the process of digesting and ligating.

![](https://static.igem.org/mediawiki/2019/4/41/T--Calgary--1july1w3.png) **Figure 1.1% agarose gel showing colony PCR of standard and pelleted SGR and CBR.**  Gel ran at 100V for 30 min. Expected lengths of bands are 1545 bp for SGR, 1993bp for PPH, 2065bp for HCAR, and 1723bp for CBR. Ladder - Gene Ruler 1kb+.

##

# July 22 - July 26

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week was dedicated to troubleshooting results and protocols. The team started ethanol and salt precipitating DNA after the digests with double the amount of DNA. A wash step was also added to the protocols after precipitation, with the vacufuge step reduced to 15 minutes maximum. Additionally, SOC protocols were added for use in place of standard LB for transformations. Each PCR step was adjusted to increase activity and specificity based on our constructs and ingredients. Since DNA amounts were doubled for digests, the team did a PCR spree on the full constructs. More pSBIA3 backbone was digested since those last week were not, causing the failed transformations. We also talked to Dr. John Baker to discuss the SynBio discussion that will be organized in September.

![](https://static.igem.org/mediawiki/2019/3/37/T--Calgary--1july1w4.jpeg) **Figure 1. 1% agarose gel showing colony PCR of PPH and PCR of PPH and HCAR.**  Gel ran at 100V for 30 min. Expected band sizes are 2545bp for SGR, 2065bp for HCAR, and 1723bp for CBR. GS Stands for glycerol stock control which is 2100bp.

![](https://static.igem.org/mediawiki/2019/e/ef/T--Calgary--2july1w4.jpeg) **Figure 2. 1% agarose gel showing colony PCR of HCAR.**  Gel ran at 100V for 30 min. Expected band size is 2065bp. GS Stands for glycerol stock control which is 2100bp.

![](https://static.igem.org/mediawiki/2019/d/d4/T--Calgary--3julyw4.jpeg) **Figure 3. 1% agarose gel showing colony PCR of PPH.**  Gel ran at 100V for 30 min. Expected band size is 1993bp. GS Stands for glycerol stock control which is 2100bp.

![](https://static.igem.org/mediawiki/2019/9/96/T--Calgary--5julyw4.jpeg) **Figure 4. 1% agarose gel showing PCR of HCAR, PPH, CBR, and SGR.**  Gel ran at 100V for 30 min. Expected lengths of bands are 1545 bp for SGR, 1993bp for PPH, 2065bp for HCAR, and 1723bp for CBR. Ladder - NEB 1kb+

##

# July 22 - July 26

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week was dedicated to troubleshooting results and protocols. The team started ethanol and salt precipitating DNA after the digests with double the amount of DNA. A wash step was also added to the protocols after precipitation, with the vacufuge step reduced to 15 minutes maximum. Additionally, SOC protocols were added for use in place of standard LB for transformations. Each PCR step was adjusted to increase activity and specificity based on our constructs and ingredients. Since DNA amounts were doubled for digests, the team did a PCR spree on the full constructs. More pSBIA3 backbone was digested since those last week were not, causing the failed transformations. We also talked to Dr. John Baker to discuss the SynBio discussion that will be organized in September.

##

# July 29 - July 31

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Gel-purified PPH and HCAR from July 26, 2019. More transformations were made for CBR and SGR. As part of the troubleshooting process, we realized that the unusual concentrations and absorbance values from gel-purified CBR and SGR was due to resuspension of DNA with elution buffer instead of water. Colony PCR reactions for CBR and SGR were redone since the first one had short band sizes due to different temperature settings. More digests, precipitations, and ligations were made for SGR and CBR full constructs.

##

# July 29 - July 31

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

PPH and HCAR sequencing results verified its successful cloning into DH5-α cells. We then attempted transforming PPH and HCAR into BL21 cells and redid digests, ethanol precipitations, and ligations for SGR and PCR. Team had a lot of meetings about pheophorbide application.

##

# August 5 - August 9

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week the team did transformations and colony PCR on CBR and SGR (into DH5α). Colony PCR showed no bands initially but when redone, had the wrong band sizes which we suspect was due to increasing the annealing temperature to 68°C from the usual 57°C. After another colony PCR set to 57°C annealing temperature, we saw bands but still with varying sizes. We prepared more proteins (PPH, HCAR, and native BL21 pSBIA3 proteins).

![](https://static.igem.org/mediawiki/2019/3/3c/T--Calgary--1week2aug.jpeg)**Figure 2. cPCR for SGR and CBR** Ladder used: NEB 1kb+ ladder. expected band size for SGR is 2545bp and for 1723bp for CBR.

![](https://static.igem.org/mediawiki/2019/d/d3/T--Calgary--2week2aug.jpeg)**Figure 2. cPCR for SGR and CBR** Ladder used: NEB 1kb+ ladder. expected band size for SGR is 2545bp and for 1723bp for CBR.

![](https://static.igem.org/mediawiki/2019/a/a8/T--Calgary--3week2aug.jpeg)**Figure 3. cPCR for SGR and CBR** Ladder used: NEB 1kb+ ladder. expected band size for SGR is 2545bp and for 1723bp for CBR.

##

# August 5 - August 9

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Subcultures for pSB1A3, HCAR, and PPH were induced with IPTG and protein purifed, then ran on 10% SDS-PAGE Gel. The band sizes correspond to PPH and HCAR protein sizes which confirm our first replicate for PPH and HCAR purification. Team also obtained pheophorbide a from Sta. Cruz Biotech and potato dextrose plates, fungal cultures (Sclerotinia sclerotorium and Pestialotopsis microspora), and corn meal broths for culturing from Ms. Fran Cusack, a laboratory technician at the University of Calgary.

![](https://static.igem.org/mediawiki/2019/f/fc/T--Calgary--3week3aug.jpeg)**Figure 1. BL21 pSB1A3 protein purification fractions.** 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Leftmost lane is Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB).

![](https://static.igem.org/mediawiki/2019/b/b3/T--Calgary--1week3aug.jpeg)**Figure 2.PPH protein purification fractions.**10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Leftmost lane is Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB). PPH expected band size is 55.8kDa.

![](https://static.igem.org/mediawiki/2019/a/ac/T--Calgary--2week3aug.jpeg)**Figure 3. HCAR protein purification fractions.** 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Leftmost lane is Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB). HCAR expected band size is 58kDa.

![](https://static.igem.org/mediawiki/2019/9/9c/T--Calgary--week3aug.jpeg)**10% SDS-PAGE HCAR and PPH Purification Confirmation.** 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), pSB1A3 (plasmid control in BL21) - elution fraction 1, and pSB1A3 (plasmid control in BL21) - elution fraction 2, PPH - post loading fraction, HCAR - elution fraction 1, HCAR elution fraction 2, PPH - elution fraction 1, PPH elution fraction 2. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa.

##

# August 19 - August 23

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

PCR and cPCR was done for CBR and SGR. CBR PCR was not successful, and CBR and SGR were still not successfully cloned in. PPH and HCAR from August 14,2019 and August 21, 2019 were column purified, and ran on 10% SDS-page gel. The gel confirms purification of PPH for the second time, but not of HCAR. The team also talked to mycologist Dr. Heather Addy, who referred us to plant pathologists and molecular biologists working with Sclerotinia sclerotorium.

![](https://static.igem.org/mediawiki/2019/e/e5/T--Calgary--fCPCRPLSAUG.jpeg)**Figure 1. cPCR for SGR and CBR** Ladder used: NEB 1kb+ ladder. Expected band size for SGR is 2545bp and for 1723bp for CBR.

![](https://static.igem.org/mediawiki/2019/0/08/T--Calgary--pcr1week4aug.jpeg)**Figure 2. PCR products of SGR and CBR** Ladder used: NEB 1kb+ ladder. Expected band size for SGR is 2545bp and for 1723bp for CBR.

##

# August 26 - August 31

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week the team column purified, and ammonium sulphate precipitated more PPH and HCAR proteins. More digests, ethanol/salt DNA precipitation and ligations were done for CBR and SGR. We decided to double the vector ratio in SGR digests since the fragment is larger than the vector. Team faced difficulties in visualizing the gel because of the new nucleic acid stain SafeRed. Minipreps were made for colonies with bright bands. HCAR and PPH were also ammonium sulphate precipitated. Chlorophyll was prepared using 80% acetone and spinach.

![](https://static.igem.org/mediawiki/2019/f/f2/T--Calgary--cpcrweek5aug.jpeg)**Figure 1. cPCR for SGR and CBR** cPCR redone. Ladder used was NEB 1kb+. Expected band size for SGR is 2545bp and for 1723bp for CBR.

![](https://static.igem.org/mediawiki/2019/5/5c/T--Calgary--cpcr2week5aug.jpeg)**Figure 2. cPCR for SGR and CBR** cPCR redone. Ladder used was NEB 1kb+. Expected band size for SGR is 2545bp and for 1723bp for CBR.

![](https://static.igem.org/mediawiki/2019/2/21/T--Calgary--proteingelweek5aug.jpeg)**Figure 3. 10% SDS-PAGE HCAR and PPH Purification Confirmation.** 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), pSB1A3 (plasmid control in BL21) -post loading sample, elution 1, elution 2, HCAR post-loading sample, elution fraction 1, 2, PPH post-loading sample, elution fraction 1, and 2. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa.

##

# September 3 — September 6

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Sequencing results for SGR 3:1 Pelleted were negative. More digests and ligations for CBR and SGR 3:1 insert: vector were made. PPH and HCAR were column purified again. Chlorophyll a and b was extracted from spinach using 80% acetone, then acidified with 30 mM to make HCl. Team did a collaboration with SoundBio iGEM for mINTERLAB.

##

# September 9 — September 15

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

More transformations and cPCR were done for SGR. The first thin layer chromatography experiment was run using flexible cellulose TLC plates with 100% hexane as solvent. However, the first plate did not run well because the chamber was not saturated enough. We were unsure of what the orange color seen on the solvent fron on top of chlorophyll #3 and acidified chlorophyll #3 and #2 were. This might be due to pheophytin or chlorophyll all the way to the solvent front. Chris acidified chlorophyll sample to create pheophytin, and absorbance values corresponding to chlorophyll wavelengths decrease and pheophytin wavelengths increased. Pheophorbide did not run on the cellulose plates with 100% hexane. Team also attempted to use the big TLC chamber and run cellulose sheets on 100% hexane but the chamber was not saturated enough so the solvent kept evaporating from the plate. Another SDS-PAGE gel was run, and showed fourth confirmation of PPH and third confirmation of HCAR purification. Pheophorbide a was solubilized in 25% acetone. Dark treatments with pheophorbide paper disc tests across different concentrations on fungal cultures were started. More fungal cultures for light treatments (1400 lumens, white LED) were made with double disks at 1.5 cm away from the center of the fungi to the disk center, with concentrations 0mg/ml, 5 mg/ml, 25mg/ml, and 35mg/ml. The double disks were done to account for the possibility of pheophorbide diffusion into the agar plates.

![](https://static.igem.org/mediawiki/2019/3/3a/T--Calgary--firstTLC.jpeg) **Chlorophyll and Pheophytin TLC visualizaiton.**  Samples were eluted by Thin Layer Chromatography using 100% hexane on flexible cellulose plates.

![](https://static.igem.org/mediawiki/2019/f/f0/T--Calgary--proteingel1week2sep.jpeg)**Figure 2. 10% SDS-PAGE HCAR and PPH Purification Confirmation.** 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), PPH - Post-loading sample,elution fraction 1, PPH elution fraction 2, HCAR - post-loading sample, elution fraction 1, HCAR elution fraction 2, pSB1A3 (plasmid control in BL21) - post-loading sample, elution fraction 1. HCAR is 58 kDa and PPH is 55.8kDa.

![](https://static.igem.org/mediawiki/2019/3/34/T--Calgary--TLC1week2sep.jpeg)**Figure 3. Pheophorbide a, pheophytin, and chlorophyll visualizaiton.** Samples were eluted by Thin Layer Chromatography using 80:20 acetone: hexane, and methanol as solvent on flexible cellulose plates.

![](https://static.igem.org/mediawiki/2019/3/3a/T--Calgary--disc1week2sep.jpeg)**Figure 4. Pheophorbide fungal disc testing layout.**Discs were place 1.5cm away from the center of fungal culture. Discs were submerged in 1 mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, and 35 mg/ml Pheophorbide a. Control solution was submerged in 25% acetone without pheophorbide a.

![](https://static.igem.org/mediawiki/2019/4/4a/T--Calgary--fungi2sepweek2.png)**Figure 5.Pheophorbide fungal disc testing for  *Sclerotinia sclerotiorum*  and  *Pestalotiopsis microspora* (day 2).**Discs were place 1.5cm away from the center of fungal culture. Discs were submerged in 1 mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, and 35 mg/ml Pheophorbide a. Control solution was submerged in 25% acetone without pheophorbide a.

![](https://static.igem.org/mediawiki/2019/1/1a/T--Calgary--fungi1sepweek2.png)**Figure 6. Pheophorbide fungal disc testing for  *Sclerotinia sclerotiorum*  and  *Pestalotiopsis microspora* layout (day 3).**Discs were place 1.5cm away from the center of fungal culture. Discs were submerged in 1 mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, and 35 mg/ml Pheophorbide a. Control solution was submerged in 25% acetone without pheophorbide a.

##

# September 16 — September 22

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week we collected fungal data and troubleshooted TLC experiments. We tried using 100% acetone and 80:20 acetone:hexane as our solvent which eluted our compounds better than 100% hexane did, but were not separated distinctly. We also did colony PCR for CBR and SGR, and prepared some overnights of CBR colonies.

![](https://static.igem.org/mediawiki/2019/0/03/T--Calgary--fungi1sepweek3.png)**Figure 1.Pheophorbide fungal disc testing for  *Sclerotinia sclerotiorum*  and  *Pestalotiopsis microspora* (day 5).**Discs were place 1.5cm away from the center of fungal culture. Discs were submerged in 1 mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, and 35 mg/ml Pheophorbide a. Control solution was submerged in 25% acetone without pheophorbide a.

![](https://static.igem.org/mediawiki/2019/8/87/T--Calgary--fungi2sepweek3.png)**Figure 2. Pheophorbide fungal disc testing for  *Sclerotinia sclerotiorum*  and  *Pestalotiopsis microspora* (day 6).**Discs were place 1.5cm away from the center of fungal culture. Discs were submerged in 1 mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, and 35 mg/ml Pheophorbide a. Control solution was submerged in 25% acetone without pheophorbide a.

![](https://static.igem.org/mediawiki/2019/f/f6/T--Calgary--TLC1week3sep.jpeg)**Figure 3. Pheophorbide, pheophytin, and chlorophyl TLC.** Samples were eluted by Thin Layer Chromatography using 100% acetone as solvent on flexible cellulose plates.

![](https://static.igem.org/mediawiki/2019/9/9e/T--Calgary--TLC2week3sep.jpeg)**Figure 4. Pheophorbide, pheophytin, and chlorophyl TLC.** Samples were eluted by Thin Layer Chromatography using 80:20 acetone: hexane and 100% acetone as solvent on flexible cellulose plates.

##

# September 23 — September 29

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Team decided to take the week off.

##

# October 1 — October 6

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

More CBR and SGR digests, ligations, and transformations (without salt/ethanol precipitation to conserve DNA) in 2:1 ratios into DH-5α were made. The plasmid minipreps were then sent for sequencing, which returned negative. PPH from elution fraction 2 which was long stored in the -20C fridge without being resuspended in glycerol , or ammonium sulfate precipitated was tested for pheophytin reactions using the kinetics function in NanoDrop machine under wavelength of 409nm. However, there was no change observed across 40 minutes, potentially indicating non-functional proteins. Team tried using 100% methanol and 80:20 Acetone:Hexane as a solvent in cellulose plates for Thin Layer Chromatography. Pheophytin was still not eluting fully from the spotting line, so we tried using silica plates for the first time to eliminate the possibility of high adsorption of the compounds to the cellulose plate. Using the silica plate, we were able to visualize the fluroescence of chlorophyll, pheophytin, and pheophorbide for the first time. We ran our first pheophytinase reactions using the aforementioned non-functional proteins with pheophorbide a without reaction buffer with 100% methanol as the solvent but there was little to no red fluorescence.

![](https://static.igem.org/mediawiki/2019/3/35/T--Calgary--Oct2TLC.jpeg)**Figure 1. Pheophorbide a, pheophytin, chlorophyll sample visualization.** Samples were eluted by Thin Layer Chromatography using 80:20 acetone: hexane, and methanol as solvent on flexible cellulose plates.

![](https://static.igem.org/mediawiki/2019/9/9e/T--Calgary--OctWEEK1tlc.jpeg)**Figure 2. Pheophorbide a, pheophytin, pheophytinase reactions, chlorophyll visualization.** Samples were eluted by Thin Layer Chromatography using methanol as solvent on a silica plate.

![](https://static.igem.org/mediawiki/2019/5/55/T--Calgary--OctWEEK1tlc1.jpeg)**Figure 3. Pheophorbide a, pheophytin, chlorophyll visualization.**Samples were eluted by Thin Layer Chromatography using methanol as solvent on a flexible cellulose plate.

![](https://static.igem.org/mediawiki/2019/e/e8/T--Calgary--OctWEEK1tlc2.jpeg)**Figure 4. Pheophorbide a, pheophytin, and pheophytinase reaction.** Samples were eluted by Thin Layer Chromatography using methanol as solvent on a silica plate.

##

# October 7 — October 13

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

Team did more digests, ligations, transformations, cPCR, and minipreps for SGR and CBR. pSBIA3, PPH, and HCAR proteins were ammonium sulfate precipitated, but the salt was not fully dissolved so the supernatants from the three elutions were aggregated in a falcon tube. We were unsure wether the proteins would still be functional, but ran the solutions on silica plates using methanol as a solvent. Since pheophorbide and pheophytin were still not easily distinguishable from each other, a solvent mixture of 70:30 methanol:hexane was used. More subcultures were made, induced, protein-purified, and ammonium-sulfate precipitated. New pheophytin stock was made using chlorophyll from fresh spinach, and neutralized to pH 8.0 to ensure pheophytinase will not denature.

![](https://static.igem.org/mediawiki/2019/b/b1/T--Calgary--cpcrLabelledweek2.jpeg) **Figure 1.1% agarose gel showing colony PCR of standard and pelleted SGR and CBR.**  Gel ran at 100V for 30 min. Expected lengths of bands are 2545 bp for SGR and 1732 for CBR. Row 1 : Lane 1: Ladder - NEB 1kb+ | Lanes 2-4: Standard SGR| Lanes 6-12: Pelleted SGR. Row 2: Lane 1: Ladder -NEB 1kb+ | Lane 2-5 : Standard CBR | Lane 6-13: Pelleted CBR.

![](https://static.igem.org/mediawiki/2019/5/5d/T--Calgary--Oct2TLC4.jpeg) **Figure 2. Pheophytin sample visualization.** Samples were eluted by Thin Layer Chromatography using methanol:hexane (70:30) solvent on a silica plate.

![](https://static.igem.org/mediawiki/2019/a/ad/T--Calgary--Oct2TLC3.jpeg) **Figure 3.Old pheophytinase elution fraction (non-ammonium sulfate precipitated) reaction, pheophytin (acidified chlorophyll stock) and pheophorbide a visualization.** Samples were eluted by Thin Layer Chromatography using methanol:hexane (70:30) solvent on a silica plate.

![](https://static.igem.org/mediawiki/2019/a/a8/T--Calgary--OCT_Week2TLC.jpeg) **Figure 4. Failed ammonium sulfate-precipitated pheophytinase reaction, pheophytin, and pheophorbide a visualization.**  Samples were eluted by Thin Layer Chromatography using methanol:hexane (70:30) solvent on a silica plate.

![](https://static.igem.org/mediawiki/2019/d/de/T--Calgary--Oct2TLC2.jpeg) **Figure 5. Pheophytin, pheophorbide a, chlorophyll a+ b visualization.**  Samples were eluted by Thin Layer Chromatography using methanol:hexane (70:30) solvent on a silica plate.

##

# October 14 — October 20

## [Complete PDF](https://static.igem.org/mediawiki/2019/3/32/T--Calgary--Pheophorbidejournal.pdf)

---

## Weekly Summary

This week we sent CBR and SGR minipreprs for sequencing which came back negative. We also ran our two sets of PPH, HCAR, and pSB1A3 column-purified elution fractions in 10% SDS-PAGE gel which showed our bands of interest. A western blot was also made which showed antibody fluorescence for HCAR, and bands without fluorescence for PPH. We started adding pheophorbide a + pheophytin mixtures to our TLC plates as a control. Our reactions still do not show any signs of pheophorbide production. We suspect that the ammonium-sulfate precipitations either precipitated or denatured our proteins, so we prepared cell lysate and non-ammonium sulfate precipitated elution fractions for our pheophytinase reactions. The reactions were left at 37°C for a day then run on silica plates with 70:30 methanol:hexane. We saw pheophorbide a production by pheophytinase for the first time, which was more prominent in the cell lysates than the elution fractions.

![](https://static.igem.org/mediawiki/2019/f/ff/T--Calgary--TLC_PB_1.jpeg)![](https://static.igem.org/mediawiki/2019/3/38/T--Calgary--TLC_PB_1_dark.jpeg) **Figure 1. Pheophytinase reaction visualization Replicate #1.** Samples were eluted using a methanol:hexane (70:30) solvent system on a silica plate. Lanes (read left to right) contain 24 hour reactions using Nickel-NTA purified PPH elution fraction 3, 2, 1, whole cell lysate (containing recombinant PPH) fraction 2, 1, pheophytin + pheophorbide + reaction buffer solution, pheophorbide a without reaction buffer. Reaction buffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. This is one of three replicates. The left (8A) figure is the raw image taken using an LG G6 Camera whereas the right (8B) is the same image with modified brightness:contrast ratio using the imaging software FIJI.After troubleshooting, Pb + Pn + rxn buffer lane was found to not have pheophorbide a in it which is why the neon-pink fluorescence curve was not observed.

![](https://static.igem.org/mediawiki/2019/9/9a/T--Calgary--TLC_PB_3.jpeg)![](https://static.igem.org/mediawiki/2019/9/95/T--Calgary--TLC_PB_3_dark.jpeg) **Figure 2. Pheophytinase reaction visualization Replicate #2.**  Samples were eluted using a methanol:hexane (70:30) solvent system on a silica plate. Lanes (read left to right) contain 24 hour reactions using Nickel-NTA purified PPH elution fraction 3, 2, 1, whole cell lysate (containing recombinant PPH) fraction 2, 1, pheophytin + reaction buffer solution, pheophytin + pheophorbide a + reaction buffer. Reaction buffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. This is one of three replicates. The left (8A) figure is the raw image taken using an LG G6 Camera whereas the right (8B) is the same image with modified brightness:contrast ratio using the imaging software FIJI.

![](https://static.igem.org/mediawiki/2019/0/00/T--Calgary--TLC_PB_2.jpeg)![](https://static.igem.org/mediawiki/2019/0/00/T--Calgary--TLC_PB_2_1.jpeg) **Figure 3. Pheophytinase reaction visualization Replicate # 3.** Samples were eluted using a methanol:hexane (70:30) solvent system on a silica plate. Lanes (read left to right) contain 24 hour reactions using Nickel-NTA purified PPH elution fraction 3, 2, 1, whole cell lysate (containing recombinant PPH) fraction 2, 1, pheophytin + reaction buffer solution, pheophytin + pheophorbide a + reaction buffer. Reaction buffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. This is one of three replicates. The left (8A) figure is the raw image taken using an LG G6 Camera whereas the right (8B) is the same image with modified brightness:contrast ratio using the imaging software FIJI.

![](https://static.igem.org/mediawiki/2019/9/93/T--Calgary--SDSpagePPHHCAR.jpeg) **10% SDS-PAGE HCAR and PPH Purification Confirmation.**  10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), PPH elution fractions 3,2,1 HCAR elution fractions 3, 2, 1 pSB1A3 (plasmid control in BL21) - elution fraction 2, and 1. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa

##

# May 20 — May 24

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week our subgroup discussed various chassis, plasmids, parts, and signal peptide sequences to ultimately come to a finalized decision on the technical aspects of our project and construct. At the start of the week, we decided to use Bacillus subtillis for our chassis, but then after researching the advantages and disadvantages of this chassis compared to E. coli, we ultimately decided to use E. coli strain BL21 (DE3) for our chassis. In regards to cloning, we will utilize the Golden Gate Assembly technique to put our construct together. In all, there will be four parts (promoter + RBS; signal peptide; 6XHIS + 6GIX; double terminator) that will be assembled for our construct. Additionally, we determined a part to improve for our gold medal requirements; the Golden Gate Flipper BBa\_K1467400 by changing the construct to include a constitutive promoter, a stronger RBS site, and a bidirectional terminator. This part will be used to make pSB1C3 golden gate compatible. Finally, we worked with our modelling team to create PDB files of protein models which contain the various signal peptides with the 6XHIS tag and the 6GIX protein. The files will be used by our modelling team to figure out any problems that may arise during protein folding, and the protein interactions in the emulsion.

##

# May 27 — May 31

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week, our subgroup finalized our parts design after recieving confirmation via protein modelling that our chosen tags shouldn't negatively impact protein structure stability. We registered with IDT and we will order our parts as gBlocks from them as soon as they contact us. In addition, we ordered the Golden Gate Flipper (BBa\_K1467400) from the iGEM registry. In the lab, we prepared for cloning work by creating chemically competent E. coli DH5-alpha cells. Outside of the lab, members of our subgroup focused on many aspects of our human practices work including several visits to Webber Academy to give an educational talk about synthetic biology, work on our synbio education package, and entreprenuership activities. We also pursued various funding opportunities and began to create a sponsorship package for the team.

##

# June 3 — June 7

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

We spent the first half of this week conducting a very thorough lab clean-up and taking inventory of our supplies. In the middle of the week, the wet lab ordered all of our parts from IDT. To our surprise, half of them arrived within the week! We will begin work to clone them into our donor plasmid (pSB1C3) next week. Outside of the lab, we continued various human practices activites, including meeting with canola farmers and working on our synthetic biology training package.

##

# June 10 — June 14

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

We recieved all of our parts from IDT throughout the course of this week, and began the process of cloning each part into our donor plasmid (pSB1C3) before use in Golden Gate reactions. Initial confirmation of cloning success was obtained via colony PCR reactions. By the end of the week, we were able to sequence confirm one of the signal peptides (PhoA signal peptide) in pSB1C3. We also PCR-amplified all of the IDT GBlocks in order to increase the amount of DNA that we have to work with. Cloning efforts will continue next week. Members of our subgroup also focused efforts on the Mindfuel Seed Fund application and preparation for our Faculty Talk presentation.

##

# June 17 — June 21

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

After optimization of our colony PCR protocol early in the week, we continued to clone all of our parts into the donor vector (pSB1C3) and the Golden Gate RFP Flipper into the destination vector (pSB1A3). We screened these ligations using colony PCR. Attempts at digest confirmations throughout the week were not successful, so we resorted to DNA sequencing. We were able to sequence confirm one more signal peptide in pSB1C3 (MalE), and we are waiting on the sequencing results for others. Aside from cloning, we gel-purified the IDT GBlocks that were PCR-amplified last week. We also began experiments to validate models that will be used to determine the chlorophyll concentration in canola oil. We also focused on preparations for our Faculty Presentation that will occur early next week.

##

# June 24 — June 28

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

Early in the week, we recieved positive sequencing results for several signal peptides (OmpA, TorA, and DsbA) and the 6XHis-6GIX coding sequence in pSB1C3, as well as our improved Golden Gate RFP Flipper in pSB1A3 (our destination vector). Once we confirmed the destination vector, we were able to proceed with our first Golden Gate reactions. We used a combination of PCR products and parts in donor vectors for these reactions in order to create all six of our constructs (one for each different signal peptide). After transformation, we had some white colonies that putatively indicate successful Golden Gate reactions. Further analysis will be performed on these colonies. Efforts continued in cloning the other IDT parts into pSB1C3. We also recieved our 'no signal peptide' control construct from IDT and began cloning it into pSB1A3. We were observing some satellite colony growth on our LB agar plates containing ampicillin, so we spent some time on optimization to reduce the issue. We also performed a ligation control for the EcoRI/PstI digests, which showed no growth and therefore helped us determine that in the absence of a digested IDT GBlock, the digested linearized backbone will not religate closed. More replicates for the experiments validating the chlorophyll concentration in oil models were performed this week.

Our team gave a presentation to faculty members and other researchers at our university this week, and we recieved value feedback about our project and experimental plans. In addition, we attended CanolaPALOOZA in Lacombe, Alberta. This event brings together many members of the canola farming and canola oil industries, and therefore gave us a valuable opportunity to conduct some human practices work.

##

# July 1 — July 5

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

Throughout the week, we worked towards confirming all of our constructs via colony PCR. This analysis was followed by digest confirmations, but once again these did not work correctly. Colonies that appeared to contain the correct construct were sent for sequencing, and we are awaiting results. In the meantime, we began transforming the putative assembled constructs into BL21 (DE3), which is our expression chassis. We repeated the Golden Gate reactions for the constructs that have not yet been successfully assembled. We were still having difficulties with satellite colony growth on our LB agar with amplicillin plates, so we tested different antibiotic concentrations. We also tried to mitigate this issue by making streak plates of the promising colonies. Our team also ran a bottle drive this week to fundraise for our travel expenses.

##

# July 8 — July 12

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

Since we were having difficulties with ampicillin for use with pSB1A3, we started this week by attempting to switch our destination vector to pSB1K3. Unfortunately, none of the ligation transformations showed any growth, so we quickly abandoned this idea and stuck with streaking instead. We received sequencing results that showed that our 'no signal peptide' control construct had successfully been cloned into pSB1A3. Efforts to transform this part into E. coli BL21 (DE3) began this week. The sequencing reaction for our Golden Gate-assembled construct sent last week unfortunately failed. We PCR-amplified all of our GBlocks from IDT to create DNA stockpiles for performing Golden Gate, and then repeated all of the Golden Gate reactions again. In addition, we repeated our colony PCR screening for all of the colonies that are white in appearance, indicating that the assembled construct has replaced the RFP flipper in the destination vector. Constructs containing the signal peptides from DsbA, MalE, OmpA, PhoA, and TorA showed positive cPCR results and were sent for sequencing. The construct containing the 'YcbK' signal peptide was cPCR-confirmed later in the week and will be sent for sequencing next week. We also began the process of transforming all of the constructs containing signal peptides into E. coli BL21 (DE3). Members of our subgroup also took part in education and outreach work this week by beginning to plan an informational 'Bacteria Night' event.

##

# July 15 — July 19

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

By the end of this week, we were able to assemble all of our constructs using Golden Gate Assembly. And we were able to sequence confirm all of our constructs and transform them into E. coli BL21 (DE3). We began preparing the necessary reagents for SDS-PAGE and became acquainted with many new protocols. We cultured and induced E. coli BL21 (DE3) that were untransformed or transformed with our 'no signal peptide' construct, then processed these samples and ran whole cell lysate fractions on SDS-PAGE gels. Unfortunately, we were unable to produce an SDS-PAGE gel that was able to be effectively run our samples. We will continue to experiment with different SDS-PAGE gel protocols in the weeks to come.

##

# July 22 — July 28

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

At the beginning of the week, we got evidence that our 6GIX protein was successfully produced both in induced and uninduced cultures. However, when we attempted to replicate our results we ran into troubles regarding the staining and destaining of the gels. After multiple trials of the SDS-PAGE gel we ultimately discovered that the coomassie gel staining solution using Coomassie R-250 is the best at staining the gel, and will be using this stain for future gels.

##

# July 29 — August 3

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week we continued trying to replicate our gel results from last week which had shown successful expression of the 6GIX protein. However, even after using the correct staining compound, the gels did not convey a significant amount of protein was being produced. We also ran samples from the periplasmic extractions from the signal peptide constructs, but again did not see any significant results. To attempt to figure out the source of our troubles regarding protein expression, we obtained and used new tubes of IPTG when inducing, and discarded the old stocks of IPTG. Additionally, we also started trying to create an emulsion system, but did not obtain any promising results. Next week, we will continue troubleshooting our protein expression and emulsion protocols.

##

# August 5 — August 10

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week we began our first attempt at protein purification using a Ni-NTA column. We also modified different components of our current protein expression method to figure out where we have been going wrong with protein expression. We tested two different IPTG samples (from another lab and our stocks) and also ran a positive control (a construct from our RA, Marija's old lab) to test our protein expression protocol's validity. We also detected contamination in our BL21 glycerol stocks, so made a new glycerol stock after confirming with cPCR.

##

# August 12 — August 18

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week we focused on protein purification of the no signal peptide construct and pSB1A3 empty vector control as well as preparing the 'registry flipper' BBa\_K1467400 for comparison with the flipper we created. Initially, we had thought the problem for our protein purification had been our IPTG sample, but after comparing samples with our IPTG and Marija's IPTG,we realized they were both functional. From this, we decided to experiment on the amount of IPTG added to induce the samples and compared samples with 1mM and 2mM IPTG. However, we still did not see a band of the correct size for our protein. Thus, next week we will be sending the No-SP BL21 glycerol stock for sequencing to check for any mutations. In regards to the 'registry flipper', the week was spent following the procedures to get the flipper into pSB1A3 in order to compare it with the flipper we have been using.

##

# August 19 — August 24

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week we continued to troubleshoot our protein purification protocol, and sent a sample of No signal peptide construct from a new BL21 streak plate to sequencing. In regards to protein purification, we subcultured and induced 1mM IPTG for No SP old plate, No SP new streak plate, and pSB1A3 empty vector control. However, we tested the OD and processed samples at different induction times including after 8 hours, 16 hours, and 24 hours to see if there was an optimum induction period for protein expression. Our efforts in protein expression halted on Thursday, when our sequencing results returned showing mutations which caused a frameshift interfering with protein production. We finally discovered the source of all our issues with protei010n production and purification! Now the plan is to send the original miniprepped sample of No SP construct, No SP in DH5a, and No SP in new BL21 cells to sequencing to see if the mutations are present at all stages of our experimental process and go from there.

##

# August 26 — August 30

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

We finally gained more evidence of successful protein expression after discovering the mutation and troubleshooting accordingly, and were able to create more replicates. We are still in the process of obtaining results for the periplasmic isolations from the signal peptide constructs, and will continue to work towards these in the weeks to come. We also started preparing for the comparison of our improved RFP flipper to the registry flipper (pSB1A3-BBa\_K1467400) by attempting to clone the parts into the same type of plasmid (pSB1A3 or pSB1C3).

##

# September 2 — September 8

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

We received our engineered version of 6GIX (called ModGIX) and set up ligations into pSB1C3 and pSB1A3. We continued making more replicates of our protein purification samples, and did periplasmic extractions from our signal peptide cultures. Additionally, we attempted to gather data for our comparison between the improved flipper and the registry flipper, but the streak plates grew too slowly, so we will attempt this again next week.

##

# September 9 — September 15

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week we continued creating more replicates for the periplasmic extractions from the signal peptide construct cultures. As was evident in previous weeks, the construct with MalE seems to be the most promising in secreting our protein. Additionally, we continued attempting to ligate the ModGIX construct into pSB1A3, and purified more 6GIX protein from the no signal peptide construct.

##

# September 16 — September 22

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

This week we sent our miniprep of the ModGIX-pSB1A3 to sequencing, and discovered that it was actually our no signal peptide construct. We also processed cultures of the no signal peptide and control, which will be purified next week. Besides this, we began preparing for one of our events, Bacteria Night by making a streak plate with the colored bacteria that we have.

##

# September 30 — October 6

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

We attempted our first western blot for the 6GIX protein from the periplasmic extractions and the no signal peptide cultures, but will need to troubleshoot in the weeks to come. Additionally, we continued purifying more 6GIX protein and continued gathering replicates for the periplasmic extractions from the signal peptide constructs. Finally, we took the time to restock lab consumables and clean the lab.

##

# October 7 — October 13

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

Similar to previous weeks, we continued purifying more protein to be used in the emulsion experiments, as well as created more replicates of our periplasmic extractions. Though we received confirmation of the presence of the 6GIX protein from the no signal peptide cultures, there was no confirmation that the ModGIX protein was being produced. We will continue to troubleshoot protein expression of ModGIX in the future. We also miniprepped the RFP-flipper parts from the registry, pSB1C3-BBa\_K1467100, pSB1C3-BBa\_K1467200, pSB1C3-BBa\_K1467300, and pSB1C3-BBa\_K1467400 in preparation for further characterization of these parts.

##

# October 14 — October 21

## [Complete PDF](https://static.igem.org/mediawiki/2019/8/86/T--Calgary--6GIXjournal.pdf)

---

## Weekly Summary

The last week before wiki freeze, we primarily focused on parts characterization, and purifying more 6GIX protein to obtain more results. We also completed a western blot which confirmed the presence of the 6GIX protein. Finally, we spent our time writing up wiki content.

##

# May 6 — May 10

---

## Weekly Summary

We had the task of modelling chlorophyll degregation to its derivatives allowing us to know the respective concentrations over time and which pathways will be more favored in the process. This is useful for knowing which enzymes to overexpress to obtain the desired derivative after chlorophyll a and b have been extracted from the canola oil.

For a separate model, we were given the task to simulate an environment with a chlorophyll binding protein. The end goal is to feed homologies from the protein previously discovered (called 6GIX) into our simulations and measure the respective fitnesses (assuming 6GIX is incompetent of binding to ligands dissolved in oil). Fitness would be measured by its binding affinity to chlorophyll that is dissolved in the surrounding oil and its ability to maintain a steady state (resist denaturation). So we hit the ground running with GROMACS - a computational chemistry software that simulates molecule interactions with great detail. It is of vital importance that we are given access to ARC - the supercomputer hosted at the University of Calgary which will drastically speed the times of our simulations. We started using a Linux machine to install the massive software - we chose the "quick and dirty" installation (which still took 1 hour).

We started our first simulation with the goal of observing our protein in water for one nanosecond (the basic tutorial was followed here: http://www.mdtutorials.com/gmx/lysozyme/index.html). The environment was captured in a box the size of 6GIX with only a 1nm gap measured off the maxima of 6GIX in all directions. From there we placed the protein topology with the four chlorophyll ligands removed as a .pdb file and added a respective amount of solvate (water). 6GIX has a -10e charge without its ligands so to ensure charge balance 10 sodium ions were added to the scene. These steps did not take long to produce for our linux desktop however the next steps of getting the protein to its minimum energy state and creating equilibrium for temperature and pressure took around three days to simulate. The last step of the simulation which would produce molecular dynamics of the system for one nanosecond was unable to finish before we decided to rerun this simulation of ARC.

While ARC was processing out job we looked into instaling VMD (Visual Molecular Dynamics), a software that takes the simulation output from GROMACS and creates a graphical representation of it. From here we can visualize molecular positions in timesteps less than a nanosecond and make use of scripting to measure fitnesses of the protein - how this measurement will be done is still being looked into. We discussed our next simulations and decided on a few intermediary models before we simulate the most crucial one that will be most applicable to our project. We would be wasting computing if we went straight to the most advanced simulation without knowing whether the protien in question can even be partially in oil in the first place (let alone take chlorophyll out of oil). Our next intermediary simulation will be immersing the protein in oleic acid (which composes 61% of canola oil) to see how GROMACS simulates and handles denaturation (6GIX is insoluble in lipids).

Chlorophyll degradation kinetic models began to be developed. Literature shows that only a first order rate law mechanism has been proposed for chlorophyll degradation studies. This model has been previously use to characterize degradation for in-vivo experiments in coleslaw, olives, pickles, and other foodstuff. Model pathway considers 2 parallel pathways, (1. chlorophyll -> chlorophyllide -> pheophorbide) & (2. chlorophyll -> pheophytin -> pheophorbide). No particullar consideration was given to the enzymes or any of the "hydroxymethyl" intermediates in the chlorophyllide B pathway. Given explanation for this particular model seems to be that A & B pathways share the chlorophyllase and Mg-dechalatase enzymes, so the chemical groups can be lumped into one (model does not consider cofactor concentration, cycling, or conversion between chlorophyll A and B). Experimental data from papers seems to fit model fairly well, however this may be due to large experiment periods. Matlab scripts for the first order rate law were written to hopefully measure the validity of the model with in-vitro data.

##

# May 13 — May 17

---

## Weekly Summary

Work on chlorophyll degradation models continued. Continued literature review did not reveal any new kinetic mechanisms in the suggested pathway - however a small amount of studies do outline thermal and pH effects, as well as propose models for this. Possible reasons that new mechanisms have not/rarely been considered is because of natural species concentration in vivo, long reaction time, and a lack of relevance in industrial processing. Mechanisms have to be proposed then, for each intermediate step and parameters have to be obtained from experimental results from best fit data.

Enzymes between each step began to be studies, to determine the best potential mechanism. Currently, michaelis-menten model would seem to be a possibility for degradation of both chlorophyll A and B to chlorophylide A, however it does not take into consideration selectivity for either - (paper shows that the B pathway is selectively favorable for some reason). Other potential mechanism is ping-pong, but there is no evidence to suggest that the enzyme is tranformed between complexes for any reason (yet). Ping-pong might be a good choice for any of the "mirror-steps" but, there is no evidence to suggest enzyme transformation - more reading into mechanisms must be considered.

Chlorophyll degradation subgroup and model workflow were redefined. Instead of looking to optimize parts sequence to maximize amount of each enzyme produced, enviornmental conditions should be optimized first.
Initially, kinetic data will be produced for experiments between each step (chlorophyll B to chlorophyllide B via chlorophyllase enzyme). Once trials are performed and kinetic parameter, experiments with the entire systme will be done at once - so cofactor concnetrations, pH, and temperature will be optimised for this experiment with the kinetic models. An inducible expression model defining the rates of production for each enzyme can also be built, but was deemed to be possibly too dificult, and not a priority. Scirpting work was continued, with an emphasis on the model framework itself. Essentially, enzyme mechanisms, parallel rate law systems, fitting and optimization algorithms are being structured independently so when experimental data is available, model framework is set up and ready to go.

##

# May 20 — May 24

---

## Weekly Summary

###### Protein modelling:

We finished the simulation of 6GIX solvated in oil and GROMACS blew up, giving us fatal errors due to too large of molecule movement. This is not anything interesting because we thought that 6GIX would denature in oil. We found packmol which is a program that offers a more intuitive interface for setting up MD (Molecular Dynamic) environments to use in GROMACS. Packmol takes some time to export these environments, and currently we are building an environment where 6GIX is half solvated in oleic acid and half solvated in water. Another environment (which is the most analogous to the applicability of our project idea) is being built as well, however we are struggling getting the data structure for chlorophyll and the number of molecules needed within the area specified that would constitute a liquid phase. This model would encapsulate 6GIX in a sphere of water with oil outside of this sphere which would contain dissolved chlorophyll. It is in this simulation where we would hope to see chlorophyll move across the surfactant layer and bind with 6GIX in the water. We also are looking to measuring the fitness of 6GIX by evaluating the free binding affinity which can presumably be measured computationally. The main issues looking forward are how the binding affinity is affected for later ligands after one ligand attatches - the optimal number of ligands 6GIX can contain is unknown at this point. Another issue is simulating 6GIX with a histone tag, which is needed for the protein extraction after E.Coli produces its complex. To add a histone tag to a .pdb file might best be accomplished using the software pymol.

###### Degradation Modelling:

Degradation pathway was redefined to sequential steps: Chlorophyll B -> 7-hydroxymethyl Chlorophyll a -> Chlorophyll a -> pheophytin a -> pheophorbide a. Respective enzymes for each reaction are: Chlorophyll b reductase, 7-hydroxymethyl chlorophyll a reductase -> Mg-dechalatase -> pheophytinase.

New workflow: continue to set up scripts indipendent of experiments -> using MM mechanism for all, will be developing ratio tests for the 2-step assays - have not looked at temperature, pH, or cofactor dependence yet. For temp, initially will be using arrhenius correlation - some literature has shown adding a cp\*pH term fits pH, but this is done on fruit over large periods of time - dependent deviations may not be measurable.

###### Circuit optimization

Began learning the focused circuit construct. Using previous years' work as a learning platform.

Packmol finished the build for 6GIX in the centre of a surfactant layer of oil and water. On the oil side, 6GIX was completely disintegrated and there was one subunit remaining on the water side, however it looked very denatured. This tells us 6GIX cannot even remain partially in oil. The next environment will be 6GIX immersed in water with a surfactant layer with oil on one side of the cube. This is to see if the surfactant between the oil and water will have an effect on the stability of 6GIX.

##

# May 27 — May 31

---

## Weekly Summary

###### Protein modelling:

We were asked to model 6GIX with some modification made by the wet lab. They added HIS tags to some ends of the subunits which will be needed for purification in the production of the protein. We just simulated molecular dynamics for one nanosecond in gromacs and obtained RMSD (root mean squared deviation) data of the backbone struture. We graphed this data compared to the unmodified 6GIX and saw some deviations, however out of the five different modification, none of the deviations were large enough to consider denaturing. Thus the wet lab went ahead and ordered their parts since the added tags did not denature to protein to any significatn extent.

###### Degradation:

Scripts incorporating independent temperature effects were finished. No limits on them though - temperature effects use arrhenius on kcat which may not be accurate either - need to determine what is the best way forward for this set up. Single variable variation function and testing scripts were also completed.

###### Circuit optimization:

Looking at possible pseudo-reaction schemes for cirucit kinetics. Still learning SYNBAD

##

# June 3 - June 7

---

## Weekly Summary

###### Frost Modelling (Sunny Days)

PCA and initial neural networks completed

Created outline for testing and validation.

Explored MLP's for multivariate regression on time series.

###### Economic Modelling (Italian Wedding Soup)

Learned Khan Acad. basics

###### Monaco

Learning Synbad

Finished differential descriptions of current reaction scheme.

###### Pickles/coleslaw:

Working on deriving temperature/pH effects using gibbs differentials

Began to look at 2 variable variation to obtian surface response plot (3D graph)

Looking at different mechanism design (struggling with this)

###### Catalina:

Looking at emulsion modelling - what type of tools are used, equilibrium types, application in industry, current research, etc...

###### protein modelling:

Started gathering RMSDs, videos, and summaries to inform wetlab based on the 6gix modifications presented last week.
Generated Summmary graphs to compare stability between 6gix and tagged proteins.

##

# June 10 - June 14

---

## Weekly Summary

###### Protein Modelling

Finished Oleic acid model this week now it is just working with energy minimization in biphasic systems. Learned commands and theory around accounting for periodic boundary conditions.
Completed electrostatic interaction modelling and homology structural prediction for 5DQR.
This was then utilized to build the tag they will be using.

###### Italian Wedding Soup

###### Marinara:

\*New project started - spreadsheet analysis of existing market , will include sensitivity analysis to inform minimum effectiveness of separation system to net 0

###### Pickles/Coleslaw:

Contnued work on temp & pH effect derivations

Meeting with degradation subgroup: Use ordered double substrate mechanisms and random release for reactions (reductases use substrate second, cofactor first) (mg dech & phytinase use substrate first, cofactor 2nd)

Subgroup is working on determining possible release (ask LV).

There is a chemical that will be used for cycling - micha will send papers

Finished double variation scripts - surface plots can be obtained now.

Need to figure out how to set up scripts to work on 2 step, not total cumulative

###### Sunny Days

PCA and Neural nets are under way and undergoing optimization

Explored RNN's for multivariate regression on time series.

###### ketCHUp

###### Monaco

Monaco, after intense work, figuring out dozens of differential equations and multitudes of parameters, was deemed nearly impossibly difficult.

can only be pursued effectively if wet lab does experiments fast and has quantifiable results by August 1st.

will be doing literature review until further notice - possible feedback regulation mechanism could be interesting to increase productive lifetime

SYNBAD is deemed an awful tool to run these on.

###### Salad Dressings:

Identified polderman equation as possible set up for settler design

Henschke's method could be useful, but it does not include surfactant mechanics AND needs specialised equipment

Started reading into surface response method (a stochastic approach) for separation kinetics - need to read more - relatively simple but need viscometer and electrophoresis thing

Met with Dr. Nashat Nassar - agreed that LLE experiments would be good to perform

Need to build ternary (maybe quartary?)phase diagram

Mentioned either McCabe-thiel or hunter-nash method for analysis would be appropriate (didn't say anything about kremser)

Modelling approach could be to consider emulsion as a homogenous solvent, build ternary phase diagram using optimized formulation from surface response (NEEDS MORE RESEARCH)

For formulation experiments, phase diagrams might need to be constructed for the emulsion composition (could be an alternative to surface response)

Equilibrium of surfactants can be described by winsor types (1,2,3,4). There seem to be different kinetic formulations for each type. Need to consider the transition between winsor types (might be infered from phase diagram - tricritical point seems to be conditions for winsor type 3).

##

# June 17 - June 21

---

## Weekly Summary

###### Protein Modelling:

Continued Work on generating models for the biphasic systems.
Generated dataset of pair distance over the simulation for the 6gix tetramer
Used this with functional principal component analysis to understand latent variables in the proteins movement

###### Italian Wedding Soup/Marinara:

Continued to look into emulsion theory. Realised that all well-characterized deterministic models need parameters measured by equipment that we don't have available. Also found that there is alot of work in emulsion formulation optimization for the food industry. More research is required.

###### Pickles/Coleslaw:

Kinetics scripts were reworked to test multiple enzyme:substrate ratios in parallel

###### Sunny Days/KetCHUp

RNN weather model completed.
Hybrid PNMM model completed.
Sunny Days writeup begins.

###### Salad Dressings (SCALE UP):

Looked at hunter-nash, and McCabe thiel. Hunter-nash will not be applicable due to composition of system - dealing at 30 ppm - 100 ppm on average. Idea of setting up a ternary phase diagram for emulsion-oil-chlorophyll will not work becaue equilibrium relationship of our scale will most likely be linear (TPDs work better at larger compositions ~ >10%. This implies that dilute McCabe-thiel OR kremser might work for conventional LLE, but seems like a rugged method for including emulsions.

Process mechanism for scale up is currently gravity decanter. How does this operate? temp? formulation? is everything mixed at the same time? Needs deeper consideration.

##

# June 24 - June 28

---

## Weekly Summary

###### Protein Modelling:

Created an emulsion bubble simulation with 6gix tetramer inside a water bubble all surrounded by Oleic acid.

###### Italian Wedding Soup/Marinara:

###### Pickles/Coleslaw:

Complete but can be improved
Sunny Days/KetCHUp
ketCHUp abandoned
Sunny Days Validated
Nearly completed writing

###### Salad Dressings:

Met with Dr. Giovanniantonio natale about modelling colloids - didn't seem to optimistic about approaching potential models due to (previously mentioned) surfactant problem. Experimental fit models may be assessed by assessing different formualtions of Span:Tween ratio and S:W:O ratios - try to measure concentration variance in real time. We probably don't have the equipment to do so. Conclusion: To PROPERLY study mass transfer mechanism, need to understand the effect of surfactant. Theoretical model will likely be difficult to develop.

Important takeaways: effect of surfactant on mechanism - formulation and composition properties - effect are "understood" through analysis of winsor types. Look into statistical approach - something similar to surface response. ALSO need to start testing these effects. Will look into the methodology for emulsion preparation.

Obtained tween 80 from a nearby lab, courtesy of marija. Seb will be practicing making emulsions in the lav until Span 80/Tween 80 arrive. -

##

# July 2 - July 5

---

## Weekly Summary

###### Protein Modelling:

Started collecting instantaneous root mean square fluxuation data for 500 discrete time points for a single 6gix monomer.
Began generating a massive model to demonstrate the aggregative properties of the 6gix monomers. The initial attempt failed after 80 hours of computation. This was then trouble shooted and an aggregation model was established for both 1 and 5 ns time intervals totalling almost an entire week of computation time.

###### Italian Wedding Soup/Marinara:

Found paper that outlines economics of farmers

###### Scale-up:

Created McCabe-Thiele Script for Liquid-Liquid Extractions at low concentrations. Will derive a kremser equation as well to analytically determine stage number. Goal is to consider alternative process operations to syn-bio for comparative assesment (we want syn-bio approach to be clearly better).

More practice with emulsions. testing different formulations and observing size (emulsion structure). Began to look into possible neural net approach - different types of hybrid nets are used in similar studies, but existing approaches have illogical mehtods - like using 1 net to predict different ampiphile effects, and not including HBL or OHBLD measurement. Jake, Rylan, and Andrew mentioned that a classification approach using SVM and/or k-means clustering might be able to interporlate rather than using a neural net.

Began to look into clay filtration - clay bleacing via slurry tank seems to be industry standard. Identified Montromillonite k10 to be a good adsorbent from literature. However, kinetic and equilibrium models for this clay (for chlorophyll extraction) is not immediatly obvious. Needs more research. Need this information to build clay model (comparative assesment).

Met with Dr. Hector de la hoz sigler - chemical kinetics and bioprocess professor. Said that he thinks that current degradation scheme is not scalable. Too many enzymes, and will likely run into cycling issues with specificity of sodium dithionate. I asked him about my immmobilization idea: if enzymes operate as classical heterogenous catalyst, they may likely be re-used easily. Convert chlorophyll into something more aquoues-soluble or something benficial to oil pigmentation or stability. Hector said that you run into the same problem wth recycling regardless. Also gave advice on assays for double-substrate enzymes and method of analysis (hold 1 var constant, obtain K1, do the same to other, obtain K2). Lineweaver burk plot used to be used for obtianing Km, but it is better to do non-linear regression (Sum of squares etc..). Reccomended "Bioprocess Engineering - basic concepts" book.

###### Sunny Days:

Continued editing of the paper

##

# July 8 - July 12

---

## Weekly Summary

###### Protein Modelling:

Started implementing Expectation Maximation clustering through the use of Gaussian mixture models to identify the amino acids that attribute the most variance to the model.

###### Italian Wedding Soup/Marinara:

Met with UofA to incorporate our economic models together since they are also affecting farmers
Established Requirements of economic model
three prong approach -
-supply demand curves and shifts
-Current Process (acid activated clays)
-Our Process (protein)
Created Supply/Demand Plot for Farmers

Found cost of clays/shipping as well as many other factors for the project

###### Scale-up:

Created more scale-up matlab scripts to further kinetic models, included temperature kinetics via arrhenius expression included deactivation kinetics.

Emulsion work continued as standard. Developed methodology to run highly parallelized experiments by operating on dilution lines.

###### Sunny Days:

More editing and wiki writeup first draft.
Got ok to go to publisher from Dr. Mayi and Dr. LV

##

# July 15 - July 19

---

## Weekly Summary

###### Protein Modelling:

Completed Genetic Algorithm to generate 6gix modifications based on in silico targeted selection. This was used to find suitable replacements to the 12 amino acids that attributed the most variance in the dynamics of the protein as found in the Expectation Maximization clustering based on the functional principal component analysis results.

###### Economic Modelling:

Found that the cost of direct synthesis is 160 000x the worlds money supply/tonne protein
Found the cost of growing the cells in terms of reagents

###### Scale-up:

Started writing scripts for adsorption column. Other adsorbents like silica and zeolites would be operated using a packed column - would have to include TSA or PSA to operate adsorption process continously. Began to look into these models on aspen adsorption. Also, began to read more into adsopriton isotherms and order montmorillonite clay from sigma.

Met with Dr. Jinghuang Hu - biochemical engineering and bioprocesses professor. Mentioned same idea as hector - current degradation pathway is not cost effecient. Mentioned a possible immobilization technique using lignin nanoparticles instead of hydrogel or fillament set up - forwarded the paper to seb. Immobilize enzymes on nanoparticle surface, then can dilute into a more soluble solvent (similar to previous idea). The study employs this approach for butanol butyruc acidm and butyl butyrate. Dr. Hu offered any resources he can provide us with are at our disposal. Will look more into nanoparticle immobilization.

###### Phase Diagrams:

Span 80 and tween 80 came in! Also created a dilution phase diagram so we know how much water to add to be able to run many tests, this tool will be used to run experiments. Began to set up emulsion tests with this composition AND tested a quick and dirty way to simulate green oil. Added crude chl-etoh stock to oil in a measured approach to get ~30ppm of chl, and boiled off ethanol. Seemed to be relatively successful.

Created a prototype SVM and neural net model for ternary phase diagrams through changing temperature in order to extrapolate and interpolate the phase behaviour of systems in temperature.

###### Weather Modelling:

Models and paper successfully submited to the publisher Alberta Academic Review.

##

# July 22 - July 26

---

## Weekly Summary

###### Clay Filtration:

We filtered chlorophyllated oil through acid activated clay. These pictures show the before/after filtration. it filters well but these conditions cause a 20% mass loss in canola oil (yikes.)

###### Phase Diagrams:

Created an improved data visualization system to enable faster and more versatile viewing of generated ternary phase diagrams.
Also, improved the modifiability of the system.

###### Protein Modelling

Ran 6 Pheophorbide simulations for the day to obtain models demonstrating its equilibrated dynamics within water.
Then used these simulations and their lambda values to generate Gibbs Free Energy of Solvation.

###### Weather Modelling

Waiting for updates from the publisher on the Peer Review Process

###### Economic Modelling:

##

# July 29 - August 2

---

## Weekly Summary

###### Clay Filtration:

Filtration experiments continued. Experimental approach: equilibrium experiments to determine an approproate adsorption model. From literature, langmuir and freundlich are most likely. Three temperatures are observed, 25, 60, 100 degrees celsius. Different weights are observed, 0 (control), 5, 10, 15, 20 wt%. Absorbance (and deltaW) is measured before and after. Qe and Qt values are obtained from mass balances, and then input into fitting scripts. Best fitting parameters are calculated from different models, and then refit to data set. The same will be done for kinetic tests, but on 5 minute intervals to capture intial adsorbance rate. Equilibrium and kinetic models are combined, hopefully fit parametrically through arrhenius (still need to look at the derivations to see if the mechanism makes sense) and obtain a general model.

Currently still need to improve the measurement technique for chlorophyll. A standard curve can be built and used, but then material balance for the model might not work.

###### Phase Diagrams:

Worked on SVM, K-nearest-neighbours, and MLP primary stage phase diagram interpolation.
Used the first batch of real experimental results to get MLP secondary stage phase diagram interpolation.

Worked on data visualization.

###### Protein Modelling:

###### Weather Modelling:

Much progress was made on the Wiki writeup.

###### Economic Modelling:

Economics Hard, No real progress.

##

# August 5 — August 9

---

## Weekly Summary

###### Clay Filtration:

Standard dilution curves were built for comparative extraction of chlorophyll. Results were taken at absorbances 649, 664, 670

Explain T1, t2, t3, then explain what langmuir and freundlich are, not in detail but enough to know what is happening
T1 fits langmuir and freundlich well - T2 (60) and T3 (100) don't - might be due to change in physical characteristic of pore site - equivalence in in binding energy is no longer valid. There is a model that described sigmoid-like adsorption, assumptions are different. Refered to as langmuir s-type or sigmoid type.

Performed a second round of experiments on T1 with 0.5%, 1%, 2%, 2.5% and 5% loadings at T1. Isotherm results are given below. Still need an effective way to describe oil loss mathematically as a function. It seems that the chlorophyll absorbance data fits the langmur isotherm at this temp well. Still need to include the quality of fitting parameters in the scripts.

Experimental results also have begun to show the relationship between %removal and temperature. Higher temperature, as predicted, allows improved removal of chlorophyll, but also causes a lower loss in oil. Thus, higher temperature operaiton has a higher oil recovery ratio. However it is more expensive to operate at a higher temperature - will attempt to adress this with economic trade off. A possible approach would be co-optimization of clay and protein bleaching. Still need to formulate continuum models. Possible approach would be to build solid/liquid separation into a black box model, and allow it to operate continously. Perform heat exchanger calculations off of slurry output.

###### Phase Diagrams:

SVM hyperparameter (gamma and cost) optimization algorithms implemented in addition to the K optimizations for K nearest neighbours. Improved results seen.

###### Protein Modelling:

###### Weather Modelling:

###### Economic Modelling:

Started working on Stonks, the NN to predict canola futures. This is mostly an educational exercise as I wait for the lab to give data back on efficiency of the proteins. I'm using a MultiLayer Perceptron MLP to start off the project, then an Recursive Neural Network RNN. Later I plan to work integrate PCA to identify what causes the most dramatic shifts in the futures.

Also started looking into utility costs. Utilities can extrapolate the total cost of the operation. utilities generally are 15% of the cost of operation. using [Towler,\_Gavin;\_Sinnott,\_Ray\_K.]\_Chemical\_Engineer(z-lib.org).pdf

##

# August 12 — August 16

---

## Weekly Summary

###### Clay Filtration:

To do:
Continued filtration experiments - doing validation and experiments on lower loadings. Currently only T1 is working well.
Wi

###### Phase Diagrams:

Continued working on data visualization techniques.

###### Protein Modelling:

###### Weather Modelling:

More wiki writing was conducted.

###### Economic Modelling:

Created a script based on multiple assumptions that calculates the needed recycling capablities to have a decent return on investment. If we recycle less than 99.8% of protein there will not be a return on investment whilst there is a return on investment just shy of 5 years with a recycle factor of 99.91%

##

# August 19 - August 23

---

## Weekly Summary

###### Weather Modelling:

A weather model began being constructed for predicting minimum temperature a week in advance, in order to provide more accurate and detailed frost predictions in terms of duration and severity, as a tool for influencing farmers as to when to swath their canola crops in the face of killing frosts.

###### Phase Modelling:

More phase diagrams were created.
Confidence evaluation was explored.

##

# August 26 - August 30

---

## Weekly Summary

###### Weather Modelling:

The swathing model was deemed not worth pursuing.

###### Phase Modelling:

A confidence function was designed and implemented, to inform future data locations.
Area analysis functions were created to visualize phase area vs temperature.

##

# May 6 — May 10

---

## Weekly Summary

The 2019 Calgary iGEM team kicked off the summer by reviewing the medal requirements for this year's iGEM competition. We made sure all of our organizational tools (Mendeley, Benchling, etc.) were in place for the summer, and began chipping away at the mandatory laboratory safety courses which are required to work in University of Calgary labs. We also began literature review of a few potential project topics.
The dry lab explored the feasability of a bioreactor CAD tool by reading through literature and talking with Dr. Dave Anderson, a bioinformatics professor at the University of Calgary. We also proposed developing a paper discovery engine that would suggest papers with contrasting ideas. This was received well by our team, so we decided to explore it further in following weeks.
Monday , 05-06 Notes
- Continued Literature review among the two possible projects, a bioreactor CAD tool, and a contradictory paper finder
-Team meeting with the entire group.
-Contacted multiple individuals in industry for HP listed below with their role.
-
Dale Schuurmans NLP proffessor @uAlberta
James Wright NLP and Game Theory at Ualberta
Robert Holte Machine Learning @uAlberta
AltaML Machine Learning Company
Richard Sutton Research Scientist @DeepMind
Nick Savidov Rural Bioreactor Guru
Aquatera Landfill Bioreactor users
Enerkem Company that heavily uses bioreactors
Ed Dziengielewski Landfill Manager - Peace Regional Landfill
Deloitte Consulting Company
Russ Greiner NLP, ML, Bioinformatics Proff @uAlberta
Amii Too AI Research Group
Darwin AI AI Company
Derrick Rancourt Director @ Center for Genome Engineering
Milo Koretsky Virtual Bioreactor Controller
Marc Strous Leader of the Energy Bioengineering Group (Works with bioreactors)
Dr Moshirpour AI help at UofC
Reg Ouellette NLP help at successful start-up
Adam Meyers Linguistics/CS prof at NYU, created Termolator
meyers@cs.nyu.edu
-
-Developed ideas and relationship with Dr. Nick Savidov at lethbridge college towards bioreactor tool
-looked into NLP and looked into algorithms
- Worked heavily on the modelling portion of our project so far 4 models have been selected for further inquiry
- Modelling of Degredation of Chlorophyll into its derivatives
- Thermodynamic modelling of chlorophyl a and b
-modelling proteins using SWISS server
-Using a genetic algorithm for binding protein selection
-Put in Work on Opentron application
-theorized bioreactor module for opentron machine
-Bioreactor idea, abandon the CAD (cannot compete with AutoCAD) and cannot compete with companies that sell them
-Bioreactors are not inaccessible to farmers as they are very ingenious, but they don't have access to the high-tech versions of them through CAD or pre-built as they need cheap bulk, unlike Pharmaceutical companies that need quality.
-Replace by just creating a spreadsheet to help farmers create their own bioreactors to be nutrient efficient. Modeling integrated into the spreadsheet/app/website.
-wood chip bioreactors very expensive and not useful
-Paper Discovery, Highly useful in the world of science as science is progressing faster than a person can keep up. Projects like CRAFT have been successful to summarize and figure out the current paradigm of say oncology.
-Our project can certainly pursue comparisons/Contradictions between research papers.
-Ethical Issue: Concentrates science into a dogma and new contradicting ideas that go against dogma will be discarded.
- Toured the Life Sciences Innovation Hub
-Met with stats prof (Andrew)
BioReact - Johnson Su bioreactors seem to be the most popular composting for farmers
-Meeting with Anderson (modeling)
-Thoughts of splitting team into NLP and automation
-Experimented with NLTK
-Explored different lemmatizers
-Discussed how one defines "constrast" - ultimately decided that we can't look for direct contrast, we should instead look at distance in similarity
-Update iGEM Calgary to include last years information and show the current team
-Worked on method to convert research papers to .txt based documents - PDF to txt done, text just needs a word-splitter
-Project Summaries
-Abandoned Bioreactors in meeting.
-Lots of learning done
-NLTK by software team
- Updated igemCalgary.ca to reflect current team and added 2018 project summary
Bioreactor idea highly invested into cumulating into meeting friday to decide on the project - Bioreactors as a main project abandoned.
Started learning NLTK for paperTrail

##

# May 13 — May 17

---

## Weekly Summary

-Termolater
-creates terminology and can be used with pubMed with ~ 80% accuracy,
-creates relationships between words
-sent email to creator (Adam Meyers), willing to talk
-recently updated
-Meeting with Dr Moshirpour
-Project feasible
-http://spark.apache.org/, http://spark.apache.org/mllib/
-Lab automation
-Cloning, Protein purification, Chromatography - already done and difficult
-Incubator Camera + App
-Feasible and inexpensive Camera, Algorithm, App https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5191158/
-^needs to be built for our Incubators
-Track Protein Concentration in cells.
-Feasible with GFP tracking, https://www.cell.com/cell-reports/pdf/S2211-1247(15)01363-7.pdf
-Track Protein Concentration in medium
-Needs centrifuge, much harder to do, maybe we can find a way for a project
-nanodrop
-PCR
-app that texts you when the PCR is nearly done, currently no method for notification
-Gel automation
-uses ingredients to mix a gel for you at the press of a button
-assistant
-to ask questions like dilute this by 10x how much do i need
-keep track of protocols and holds your hand along the way
-Enzyme characterization assay
-Wait to see if we get opentron
-Reducing lab waste
-autoclavable glass instead of plastic
-Other project paths
-Filter (remove all proteins) and bioreactor for wet lab

###### NLP

Looking for ways to make project more SynBio-y
Did more research into machine learning and creating NLP API's.
Did more research into using term extraction API's.

###### Wiki

Started Journals
Completed basic structure for Journals, HP, Protocols
integrated journals to the actual Wiki
Utrecht has a very good SEng in the website

##

# May 20 — May 24

---

## Weekly Summary

-NLP
-Met with Dr. Far to discuss NLP and approaches to tackle determining relationships between keywords.
-Worked on creating a graphing application to connect research papers based on shared keywords.
-cGEM website completed
-NLP Termolator works
-NLP currently working on the graph
-Chrome Extension 100% doable
-Economic model with frost detection
-NLP direction not useful based on prof opinions

##

# May 27 — May 31

---

## Weekly Summary

-Met with Dr. Zahra Shakeri Hossein Abad to discuss NLP approaches and possible projects.
Focus on Paper
-Weather Scraper
-Machine Learning: Created Neural Networks for Multivariate Regression of future weather data.

###### Wiki Updates

-

###### Telus SPARK project

-

###### GC Richness Tool

-Found one online that claims to do it

##

# June 3 - June 7

---

## Weekly Summary

Innovate Calgary Summaries completed

###### Telus Spark

-Created frame for build, will order stuff and contact Spark for info and direction
-3D Model for 3D Printing job using "TinkerCAD"
-Successfully built block for Telus Spark

###### GC Richness Tool

###### Canola Heat Units (ketCHUp)

-Started

###### Wiki

##

# June 10 - June 14

---

## Weekly Summary

###### Mean Green Machine

-HP/requirements analysis for 2 weeks before physical and actual work

###### Telus Spark

-Wiring and buttons testing
- Blueberry expands with stepper motor
-Reprint of block

###### Encore

-Successfully ran file
-Started work on GUI and UX
-Using Django
Wiki
?

##

# June 17 - June 21

---

## Weekly Summary

###### Mean Green Machine:

-HP
-Explored Foreground Smear Shape Extraction with OpenCV.

###### Telus Spark (Cerberus):

-Successful reprint of the block -> will now be able to press buttons
-Plan to remove buttons to make it magnet activated for tactile feedback
-Illustration for our project

###### Encore:

-Worked on the GUI

###### Wiki:

?

##

# June 24 - June 28

---

## Weekly Summary

###### Green Seed Standardization:

-Given the go-ahea to start work
-Refined project requirements with Canolapalooza

###### Codon Optimization:

-Kept working on GUI and Script integration

###### Wiki:

?

###### Telus SPARK:

-Slower progress

##

# July 2 - July 5

---

## Weekly Summary

###### Green Seed Standardization:

Meeting with Dr Murari for hardware specs and camera/hardware constraints and avenues.
Exploring object extraction methods and colour analysis methods.
Created and printed first seed holder. The PLA formed in such a way that the divots were too shallow to use.
Generated another seed holder with more breathing room but it ultimately failed due to being processed with too low of a fill percentage

###### Codon Optimization:

###### Wiki:

?

###### Telus SPARK:

##

# July 8 - July 12

---

## Weekly Summary

###### Green Seed Standardization:

Found cameras for project - raspberry PI
Use backwards lens to make macro lens
Printing continued on seed holder which were ultimately unsuccessful as others using printers stopped the print.
A successful print of the seed holder was also stolen from the printer this week :(
Refined the colour analysis software.

###### Codon Optimization:

Managed to make codon harmony run with new classes
but now it doesnt work because multithreaded sad
Due to multiprocessing requirements of a linux machine, this project will be put on hold until i get a linux machine

###### Wiki:

Started creating team descriptions

###### Telus SPARK:

Plan to work on it more now

##

# July 15 - July 19

---

## Weekly Summary

###### Green Seed Standardization:

Successfully printed the rack for the seeds, camera is in.

###### Codon Optimization:

Attempted working on campus. exceeded disk quota to be able to run it.
Got Mike's Computer with Ubuntu(Linux)
Downloading Python, update Ubuntu, Install Pip to install python packages
Installed codon-harmony

###### Wiki:

First Official wiki meeting with entire dry lab about style.design

##

# July 22 - July 26

---

## Weekly Summary

###### Green Seed Standardization:

Discovered seed rack could be optimized further to aid in the use of the tool.
Generated new model that would be able to meet these needs.
Built the box prototype, and tested the

###### Codon Optimization:

GREAT SUCCESS ACTUALLY PASSES TESTS
Next step: Make my changes run without issue, pass arguments through a script, and then submit a pull request
PULL REQUEST SENT IN, decreases code coverage whatever that means. It is extremely easy for django to use it so it should be fine
Code coverage went down so pull request was rejected.
Attempt 2.0
Failed. Did not pass tests
Code owner (Brian Weitzner) provided really good suggestion on dealing with dictionaries
Didnt work due to it not being compatible with tox (previous versions of python)
Redesigned it.
Passes tests again. Attempt #3 to pull request.
Failed Catastrophically

###### Wiki:

##

# July 29 - August 2

---

## Weekly Summary

###### Green Seed Standardization:

Construction on the lightbox began.
DIscovered that the camera was no good & began searching for a better one.
Finished the light box lid.
Began printing camera holder.
The seed analysis software was reformatted.
Some of the seed data was analyzed.

Many attempts to get the software to work on the pi were tried.
IMG\_0342.JPG
IMG\_0348.JPG
IMG\_0349.JPG

###### Codon Optimization:

Continued failure,
Pull Request passes tests. Now I wait for Brian to accept the changes I made. Once that happens. I will be able to codon\_harmony.runner(my\_dict) and have it work easily.

Brian hates the aesthetic.

Proposes solutions that are incompatible with tox (py version 3.6 gets angry)

When told he said to "Just bump the requirements up to python 3.7" Which i don't know how to do. So I'm in a bit of a pickle

I've also been working on the look of the website.

I also tested the software, (unoptimized) on sequences. I ran it on modGIX, and it worked very well, no issue.

I then tested the it on the old protein sequence (Chris') and compared it to IDT.

IDT gave a score of 21.1 to codon\_harmony rather than 23.8 for pure optimization, which is greatly disappointing, even if it beat pure optimization.

Repeats were worth 11.4 points, hairpins 4, and high GC content 5.7 points. So I believe optimizing for repeats may be the best solution. Codon-optimizer from UBC was really good at that.
image.png
image.png
image.png
image.png

###### Wiki:

Everyone spent time working on the wiki content (Weather model, GC richness, and Protein modelling, Scale-Up)

##

# August 5 — August 9

---

## Weekly Summary

###### Green Seed Standardization:

Lots of progress made on wiki content.

Box works, but alternate options for the camera are being looked at for higher image focus.
Working on the UI for usability with a standard computer.
image.png
image.png

###### Codon Optimization:

Lots of progress made on the wiki content

###### Wiki:

Cannibalized Nottingham Wiki Scripts as they had functionalities we want

Rylan started working on coding Phase diagrams so we have pages up and ready.

##

# August 12 — August 16

---

## Weekly Summary

###### Green Seed Standardization:

The GUI tool gained more features, namely the ability to select specific seeds and look at their data.

The modified flood fill algorithm used by the program was visualized.

###### Codon Optimization:

After debating over how to implement the improved algorithm, we hit a stopping point where we didn't know hpw to make an effective algorithm do everything. Just finding the repeats couldve been a extremely difficult task requiring monstruous machinery. So Andrew found a solution that made everything much simpler. Leave it up to random chance. So the idea is to implement a genetic algorithm that exsentially acts like the synthesis score except we create populations of dna sequences, which we then score, giving the fitness value. We then keep the best ones and mutate those until we have a strong candidate.

An eventuality is to have the genetic algorithm crossbreed the best ones to create some of the offspring. some of them will die but some may be even better.

A lot of progress of made on the genetic algorithm, the majority of the fitness scoring systems have been put into place. We currently can generate a population based on an initial sequence, mutate those sequences, and check the fitness for the population of sequences for all parameters except for repeats and host-codon usage as those have more dependencies. We can also select the top 20% of the population and kill off the others.

###### Wiki:

Prahbu made a side bar

Rylan made a whole layout with functional definitions

##

# August 19 - August 23

---

## Weekly Summary

###### Green Seed Standardization:

Methods of validation were discussed. Namely, comparing the tool grading with grading conducted by the local Canadian Grain Commission building.

Wiki writing and images were worked on.

##

# August 26 - August 30

---

## Weekly Summary

###### Codon Optimization:

Abandoned the integration of system into codon-harmony due to wasted time. will directly import into website.
Genetic Algorithm successfully runs, does not optimize correctly but that can be changed by changing the weights of each function.

Currently attempting to multi-process system for speed as it is slow.

###### Green Seed Standardization:

Talked to Sean Mason about functional design. for the project and explained a system for crushing seeds as well as validating our current thoughts. he will also be useful as he offered his Solidworks services.
Decided design considerations and looks for the project as well as assign groups.


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Calgary/Experiments

[![](https://static.igem.org/mediawiki/2019/f/ff/T--Calgary--logowhite.png)](https://2019.igem.org/Team%3ACalgary)

* Project

  + [Description](https://2019.igem.org/Team%3ACalgary/Description)
  + [Wet Lab Overview](https://2019.igem.org/Team%3ACalgary/WetLab)
  + [Chlorophyll Extraction](https://2019.igem.org/Team%3ACalgary/ChlorophyllExtraction)
  + [Protein Emulsification](https://2019.igem.org/Team%3ACalgary/EmulsifiedBindingProteinProcess)
  + [Repurposing Chlorophyll](https://2019.igem.org/Team%3ACalgary/RepurposingChlorophyll)
  + [Anti-Fungal](https://2019.igem.org/Team%3ACalgary/Anti-Fungal)
  + [Results](https://2019.igem.org/Team%3ACalgary/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ACalgary/Demonstrate)
  + [Judging](https://2019.igem.org/Team%3ACalgary/Judging)
  + [Awards](https://2019.igem.org/Team%3ACalgary/Awards)
* Human Centered Design

  + [Integrated Human Practices](https://2019.igem.org/Team%3ACalgary/Human_Practices)
  + [Mean Green Machine](https://2019.igem.org/Team%3ACalgary/Modelling/StandardizedSeedGrading)
  + [Sunny Days](https://2019.igem.org/Team%3ACalgary/SunnyDays)
  + [Entrepreneurship](https://2019.igem.org/Team%3ACalgary/Entrepreneurship)
  + [Education and Public Engagement](https://2019.igem.org/Team%3ACalgary/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3ACalgary/Collaborations)
  + [Safety](https://2019.igem.org/Team%3ACalgary/Safety)
* Measurement and Model

  + [Overview](https://2019.igem.org/Team%3ACalgary/Model)
  + [ICARUS](https://2019.igem.org/Team%3ACalgary/Model/ICARUS)
  + [Emulsion Construction Prediction](https://2019.igem.org/Team%3ACalgary/Model/EmulsionPrediction)
  + [Directed Protein Modification](https://2019.igem.org/Team%3ACalgary/Model/DirectedProteinModification)
  + [In Silico Emulsion System Verification](https://2019.igem.org/Team%3ACalgary/Model/InSilicoEmulsionSystemVerification)
  + [Measurement](https://2019.igem.org/Team%3ACalgary/Measurement)
  + [Software Tools](https://2019.igem.org/Team%3ACalgary/Software)
  + [BOTS](https://2019.igem.org/Team%3ACalgary/BOT)
  + [iGAM](https://2019.igem.org/Team%3ACalgary/iGAM)
  + [Results](https://2019.igem.org/Team%3ACalgary/Results)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACalgary/Parts)
  + [Collection](https://2019.igem.org/Team%3ACalgary/Part_Collection)
  + [Improve](https://2019.igem.org/Team%3ACalgary/Improve)
  + [Characterize](https://2019.igem.org/Team%3ACalgary/Contribution)
  + [Validate](https://2019.igem.org/Team%3ACalgary/Validate)
* Journal

  + [Protocols](https://2019.igem.org/Team%3ACalgary/Experiments)
  + [Appendix](https://2019.igem.org/Team%3ACalgary/Appendix)
  + [Journal](https://2019.igem.org/Team%3ACalgary/Notebook)
* Team

  + [Members](https://2019.igem.org/Team%3ACalgary/Team)
  + [Attributions](https://2019.igem.org/Team%3ACalgary/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ACalgary/Sponsors)

## Notebook

# Protocols

# General Laboratory Protocols

---

LB Agar Plates

1. Mix into 1L of dH2O:

* 10g of Tryptone (1%)
* 5g of yeast extract (0.5%)
* 10g NaCl (1%)
* 15g Agar (1.5%)

2. Autoclave and cool
3. Add necessary antibiotics and mix thoroughly

* 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
* 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
* 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)

4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.

LB Liquid Broth

1. Mix into 1L of dH2O:

* 10g of Tryptone (1%)
* 5g of yeast extract (0.5%)
* 10g NaCl (1%)

2. Autoclave and cool
3. Add necessary antibiotics and mix thoroughly

* 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
* 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
* 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)

4. Aliquot as required

SOB Media

1. Per litter, add:

* 950 mL deionized H2O, 20g tryptone, 5g yeast extract, 0.5g NaCl.

2. Shake until dissolved. Add 10 mL of 250 mM KCl solution:

* 250 mM KCl solution: dissolve 1.86g KCl in 100mL of deionized H2O.

3. Adjust pH of medium to 7.0 with 5N NaOH (±0.2 mL).
4. Adjust volume of solution to 1.0L with deionized H2O.
5. Autoclave.
6. Add 5mL of sterile 2M MgCl2 solution

* 2M MgCl2 solution: dissolve 19g MgCl2 in 90 mL deionized H2O.

7. Adjust volume of solution to 100mL with deionized H2O. Sterilize by autoclaving.

Reference: Green, Michael R., and Joseph Sambrook. Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, 2012.

SOC Media

1. Autoclave SOB medium. Cool to 60 ̊C.
2. Add 20 mL Sterile 1 M Glucose

* Dissolve 18g glucose in 90 mL of deionized H2O

* Adjust volume of solution to 100mL with deionized H2O

* Sterilize by passing it through a 0.22μm filter

Reference: Green, Michael R., and Joseph Sambrook. Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, 2012.

Rehydration of Registry DNA

1. Add 10μl of ddH2O to the desired well of the distribution kit plate
2. Pipette up and down 3-5 times (until solution becomes red)
3. Incubate at room temperature for 10 minutes
4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

Chemically Competent  *E. coli*  cells

1. Culture O/N in 2ml LB at 28°C, shaking
2. Subculture (1:50) by adding 1ml O/N culture to 50ml LB with 10mM MgSO4 (500μl of 1M MgSO4) and 1mM KCl (50μl of 1M KCl)
3. Shake at 28°C to OD600 = 0.3 to 0.4
4. Chill on ice for at least 10 minutes
5. Put into 50ml pre-chilled Falcon tube and centrifuge at 2500g for 8 minutes at 4°C (3450rpm in Allegra X-12 centrifuge)
6. Resuspend in 10ml ice-cold 100mM CaCl2, gently mix on ice, then ice for at least 10 minutes
7. Centrifuge at 2500g for 8 minutes at 4°C
8. Resuspend in 500μl 100mM CaCl2 with 10% glycerol on ice, then incubate on ice for 10 minutes
9. Aliquot 50μl into pre-chilled 1.5ml microcentrifuge tubes on ice. Store at -80°C

Bacterial Transformations

1. Add ≤ 5μl (≤ 1/10 of the cell aliquot amount) of the DNA sample to a chemically competent cell aliquot. Mix by pipetting gently, then incubate on ice for 30 to 45 minutes.
2. Heat shock at 42°C for 1 minute
3. Incubate on ice for 5 minutes
4. Add 250μl of plain LB or SOC media aseptically, then incubate for 30 to 90 minutes at 37°C, shaking

* If resistance is Kan, must incubate for *at least* 1 hour

5. Plate 100μl (if big plates) or 50μl (if small or half plates) aseptically
6. Incubate plate at 37°C overnight or until growth is observed

\*If transformation fails:

* Spin down transformed cells for 5 minutes to pellet
* Resuspend pellet in 100μl of media
* Plate and incubate

Plasmid Miniprep

P1 (Resuspension buffer) at 4°C on ice

* 50mM Tris•HCl (pH = 8), 10mM EDTA, 100μg/ml RNase A

P2 (Lysis buffer) at room temperature

* 200mM NaOH, 1% SDS

P3 (Precipitation buffer) at room temperature

* 3M potassium acetate (pH = 5.5)

1. Grow 2 to 6ml culture O/N
2. Transfer 2ml (at a time) to 2ml microcentrifuge tube(s) and pellet at 14000rpm for 5 minutes. Discard supernatant and repeat as necessary
3. Resuspend in 300μl ice-cold P1
4. Add 300μl P2, gently invert 3x. Quickly add 300μl P3 and invert 3x
5. Spin at 14000rpm for 10 minutes at room temperature
6. Retain supernatant in 1.5ml microcentrifuge tube
7. Add 650μl of 100% isopropanol, gently invert, and incubate for 10 minutes at room temperature
8. Spin at 14000rpm for 10 minutes, then discard supernatant
9. Wash pellet with 500μl of cold 70% ethanol
10. Spin at 14000rpm for 5 minutes
11. Discard supernatant. Carefully tap tube to remove remaining ethanol. Dry pellet in vacufuge
12. Resuspend pellet in 20μl of sterile ddH2O or TE buffer. Nanodrop to determine concentration. Store at -20°C

Glycerol Stock

1. Add 500μl of 50% glycerol to a 1.5ml tube aseptically
2. Add 500μl of 50% of overnight culture to the tube aseptically. Mix gently
3. Store at -80°C

Agarose Gel Electrophoresis

1. Mix 100ml of 1X TAE buffer with 1g of regular or LMP agarose (for a 1% gel). For a small gel, mix 30ml of 1X TAE with 0.3g agarose.
2. Microwave covered for about 1.5 minutes or until agarose is dissolved
3. Cool and add 4μl SYBR safe. For a small gel, add 1.5μl SYBR safe.
4. Set up gel tray and balance properly. Pour and cast gel. LMP gels should be cast in the fridge.
5. Run sample at 100V (regular gel) or 80V (LMP gel) until sample is ¾ of the way down the gel.

Restriction Digest

1. To a microcentrifuge tube, add:

* Required amount of DNA to be digested
* 1/10 final total volume of appropriate 10X buffer
* 1μl of each restriction enzyme (diluted)
* ddH2O to final volume

2. Incubate at 37°C for 30 minutes to 3 hours
3. Heat inactivate restriction enzymes at 82°C for 20 minutes
4. For digest confirmations, run on regular agarose gel. For subsequent ligation, run on LMP gel and excise or gel-extract. Otherwise, store at -20°C

DNA Excision from Low Melting Point Gel

1. For a 1% gel, add 0.3g low melting point agarose to 30mL TAE buffer in a 250mL Erlenmeyer flask and microwave (covered) until agarose is fully dissolved
2. Allow flask to cool until warm to the touch before adding 1.5μL RedSafe nucleic acid staining solution. Gently swirl to mix
3. Pour agarose into assembled gel casting tray in the fridge. Remove any bubbles with a pipette tip and place comb in gel
4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer
5. Load samples of DNA containing 6X loading dye
6. Run gel at 80V for 30 minutes or until loading dye is 2/3 way down the gel
7. Remove the gel from casting tray and place it on a UV-light viewer. While using a UV-protecting face mask and shield, turn on the UV light and use razor blade to excise out the desired DNA bands (as quickly as possible as UV-light damages the DNA). Place each band into individual 1.5mL microcentrifuge tubes
8. Melt the gel at 65°C for 5 minutes
9. Measure the volume of the excised gel slice. Add an equal volume of ddH2O to the tube and mix well. Determine the concentration of DNA and write on tube.
10. Use in ligation or store at -20°C

\*Gel-excised DNA samples may need to be re-heated to 65°C once again before ligation to desolidify

Ethanol/Salt DNA Precipitation

1. To DNA sample, add:

* 1/10 volume of 3M sodium acetate

* 2 to 3 volumes of 100% ethanol

2. Freeze at -80°C for 45 minutes
3. Spin at max for 30 minutes at 4°C
4. Discard supernatant Spin 5 minutes at 14,000g.
5. Wash with 200-500 uL 70% ethanol
6. Remove supernatant.
7. Dry pellet in the vacufuge for 10-15 minutes.
8. Resuspend in required amount of ddH2O and vortex gently.
9. NanoDrop and record concentration

Ligation of Digested DNA

1. To a microcentrifuge tube, add:

* Digested vector DNA (in appropriate ratio)
* Digested insert DNA (in appropriate ratio)
* 1/10 of total final volume of aliquoted 10X T4 DNA ligase buffer
* 1μl of T4 DNA ligase (1U/μl)
* ddH2O to final volume

2. Incubate at room temperature for 2 hours
3. Transform chemically competent cells with the ligation product.
4. Leave remaining ligation product at room temperature overnight, and transform again the following day.
5. Store remaining product at -20°C

Golden Gate Assembly

1. To a microcentrifuge tube, add:

* Insert and vector DNA samples (PCR products or in donor vectors) in 1:1 or 2:1 insert:vector ratio
* 1μl of BsaI-HF v2 (2U/μl)
* 1μl of T4 DNA ligase (2U/μl)(diluted)
* 2μl of T4 DNA ligase buffer
* ddH2O to final volume

2. Place in thermocycler and select or set up the appropriate program as follows:

* Repeat 25x:

1. Digestion -> 42°C for 2 minutes
2. Annealing -> 16°C for 5 minutes

* Final digestion step -> 60°C for 10 minutes
* Heat inactivation step -> 80°C for 10 minutes

PCR

1. Combine in a 0.2ml microcentrifuge tube:

* 5μl of NEB 10X standard Taq buffer (final concentration = 1X)
* 0.25μl of NEB Taq (final concentration = 1.25U/50μl)
* 1μl of 10μM forward primer (final concentration = 0.2μM)
* 1μl of 10μM reverse primer (final concentration = 0.2μM)
* 1pg to 1ng of template plasmid DNA (final concentration < 1000ng/μl)
* 1μl of 10mM Kapa dNTPs (final concentration = 200μM)
* ddH2O to 50μl

2. Vortex 2 to 3 seconds to mix, then centrifuge briefly to settle
3. Place in thermocycler and select or set up the appropriate program as follows:

* Initial denaturation -> 95°C for 30 seconds
* Repeat 25 to 30x:

1. Denaturation -> 95°C for 15 to 30 seconds
2. Annealing -> Tm - 5°C (45 to 68°C) for 15 to 60 seconds
3. Extension -> 68°C for 1 minute per kilobase

* Final extension -> 68°C for 5 minutes
* Hold at 4°C

Colony PCR

1. Create cPCR mastermix as follows:

* 20μL 10X Taq Buffer
* 4μL 10μM VF2 primer (or other forward primer)
* 4μL 10μM VR primer (or other reverse primer)
* 4μL 10mM dNTPs
* 1μL Taq Polymerase
* 127μL ddH2O

\*Each mastermix aliquot is enough to run 10 cPCR reactions/screen 10 colonies

1. Add 4μL of ddH2O to each PCR tube or well (of a 96-well plate)
2. Using pipette tips or sterile toothpicks, touch an individual labelled colony, swirl in the ddH2O, and then streak on masterplate. Repeat for as many colonies as desired.
3. Add 16μL of cPCR mastermix to each tube/well
4. Place in thermocycler and select or set up the appropriate program as follows:

* Initial denaturation → 95°C for 5 minutes
* Repeat 30x:

1. Denaturation → 95°C for 30 seconds
2. Annealing → Tm - 5°C (For VF2/VR primers, 53°C) for 30 seconds
3. Extension → 68°C for 1 minute per kilobase

* Final extension → 68°C for 5 minutes
* Hold at 4°C

Inducing with IPTG

1. Add 50uL of 1.0M IPTG to the subcultures with optical densities between 0.4 to 0.6.
2. Place in the shaking incubator for 8 hours at 37°C or 24 hours at 25°C

SDS-Page

## Preparing Protein Samples

1. Mix an aliquot of the protein sample with an appropriate amount of 4x SDS-PAGE loading buffer (200mM Tris-Cl pH 6.8, 8% SDS, 40% glycerol, 0.4% bromophenol blue) to get a final concentration of 1x SDS-PAGE loading buffer

* Add 1% β-Mercaptoethanol if required

2. Boil samples for 2 minutes and spin down

## Bacterial Transformation

## Running Gels

3. Set up SDS-PAGE apparatus with the comb-side of the gels facing the inner chamber
4. Fill inner chamber completely with 1x SDS-PAGE running buffer (1x Tris-Glycine, 0.1% SDS), and outer chamber halfway

* 1x SDS-PAGE running buffer can be reused approximately 4-5 times

5. Load standards and samples
6. Run for 10 minutes at 100V, followed by 30 minutes at 180V

## Staining Gels

7. Remove gel from plates and place in a box with enough Coomassie Blue staining solution (0.25% Coomassie R-250, 40% methanol, 10% glacial acetic acid) to cover the gel. Shake at room temperature for 45 minutes
8. Destain the gel by replacing the Coomassie Blue staining solution with destaining solution (40% methanol, 10% glacial acetic acid) and shaking at room temperature for 10-30 minutes
9. Repeat destaining step as necessary until distinct bands can be visualized
10. The gel can be stored in dH2O at room temperature for a few weeks

Spinach Chlorophyll Extraction

1. Measure 20mL of 80% acetone and add 1g fresh spinach leaves without stems. Grind until crushed thoroughly.
2. Centrifuge the solution with the crushed spinach at 3750 rpm, -10°C for 9 minutes.
3. Transfer the supernatant to a falcon tube and cover with foil to protect against photosensitivity.

Pheophytinase Reactions

1. Prepare cell lysate and eluted proteins (refer to Ni-NTA Column Chromatography protocol).
2. If using **cell lysate**: In a centrifuge tube, add 100uL cell lysate, 100uL pheophytin solution, 10uL of 20mM EDTA, and 50 uL of the pheophytinase reaction buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100).
3. If using  **elution fractions**: add 100uL elution fraction, 100uL pheophytin solution, 10uL of 20mM EDTA, and 50uL of the pheophtyinase reaction buffer.
4. Place in the incubator or heating block at 35°C and leave overnight or as needed.

Ni-NTA Column Chromatography

1. Make 2-6ml overnights
2. In the morning, subculture 2:50 in 50ml or 100ml LB + appropriate antibiotic. Incubate at 37°C, shaking until OD600 reaches 0.3 - 0.6 (0.5 is optimal)
3. Induce with 100mM IPTG (50μl of 1M IPTG into 50ml, 100μl of 1M IPTG into 100ml) and incubate for 8-12 hours at 37°C or 16 hours at 30°C
4. Centrifuge on max for 5 minutes and discard supernatant (x2 if 100ml culture). Freeze pellet
5. Conduct 3x 20 minute freeze-thaw cycles for the pellet at -80°C and 37°C
6. Resuspend in 10 ml Resuspension/Equilibration buffer (1X PBS pH 7.4, 300mM NaCl, 10mM imidazole)
7. Treat with 1mg/ml lysozyme (1ml of 10mg/ml lysozyme with 1% Tween80 into 10ml resuspension) for 30 minutes shaking at 37°C
8. Transfer to 15ml falcon tube
9. Sonicate (3x of 30A, 5 sec on, 5 sec off, 2 minutes total)
10. At this point, you can take a 500μl sample to recover the soluble and insoluble whole cell lysate fraction if you so choose.
11. Freeze sonicated samples for 20 minutes or overnight

1. Take out required number of Ni-NTA columns and let them reach room temperature
2. Take off top cap off and remove bottom plug. Place into a 50ml falcon tube and centrifuge at 700g for 5 minutes. Discard flowthrough
3. Put 10ml of Resuspension buffer into the column and incubate for 10 minutes. Centrifuge at 700g for 5 minutes. Discard flowthrough and replace the bottom plug
4. Load the 10ml of sonicated sample supernatant onto the columns and replace top cap. Incubate at 37°C, shaking for 30-60 minutes or longer.
5. Label new 50ml falcon tubes with ‘post-loading sample’. Remove bottom plug and cap from the columns then place into the 50ml falcon tube. Centrifuge at 700g for 5 minutes and keep the flowthrough if desired.
6. Perform 3 washes with 10ml Wash buffer (1x PBS pH 7.4, 300mM NaCl, 25mM imidazole). After each wash, centrifuge at 700g for 5 minutes and collect the wash samples if desired
7. Add 4ml of Elution buffer (1x PBS pH 7.4, 300mM NaCl, 250mM imidazole) to the column, and incubate for at least 5 minutes. Centrifuge at 700g for 5 minutes and label collected fraction “Elution fraction 1”
8. Repeat addition of 4ml of Elution buffer to the column, and incubate for at least 5 minutes. Centrifuge at 700g for 5 minutes and label collected fraction “Elution fraction 2”
9. Repeat addition of 4ml of Elution buffer to the column, and incubate for at least 5 minutes. Centrifuge at 700g for 5 minutes and label collected fraction “Elution fraction 3”
10. Wash column with 50mlo of MES buffer (20mM MES, 100mM NaCl). Centrifuge at 700g for 5 minutes (or until all buffer is through) and discard flowthrough
11. Wash column with 50ml of nanopure water. Spin at 700g for 5 minutes (or until all water is through) and discard flowthrough
12. Add about 30ml of 20% ethanol to the column and place back into the fridge.
13. Eluted samples can be used immediately or stored at 4°C or -20°C

Periplasmic Protein Isolation

1. Make 2ml O/Ns and incubate at 37°C, shaking
2. In the morning, subculture 2:50 in LB + appropriate antibiotic. Incubate at 37°C, shaking until OD600 reaches 0.3 - 0.6 (0.5 is optimal)
3. Induce with 100mM IPTG (50μl of 1M IPTG into 50ml) and incubate for 8-12 hours at 37°C or 16 hours at 30°C
4. Transfer to 50ml falcon tubes and centrifuge for 10 minutes at max. Discard supernatant
5. Resuspend pellet in 10ml of 30mM Tris-HCl pH 8 + 20% sucrose
6. Incubate for 10 minutes at room temperature
7. Centrifuge for 10 minutes at max. Carefully discard supernatant
8. Resuspend the pellet in 500μl of ice (fridge)-cold 5mM MgSO4 solution and transfer the suspension to a 1.5ml microcentrifuge tube
9. Incubate the suspension for 30 minutes on ice
10. Pellet the cells at 13000rpm for 10 minutes
11. Transfer supernatant (shock fluid) to a fresh 1.5ml labelled microcentrifuge tube. This sample can now be visualized using SDS-PAGE, used in column chromatography, or stored at -20°C

## If cytoplasmic preparation also desired:

1. Resuspend pellet in 800μl of 1X PBS pH 7.2
2. Add 80μl of 10mg/ml lysozyme with 1% Tween80. Incubate at 37°C for 0.5 hour
3. Sonicate a sample of this solution before use (optional)
4. Freeze samples 10-15 mins and boil 5 mins before sonication

Western Blot

1. Run SDS PAGE gel
2. Cut out PVDF membrane (also cut a corner to mark bottom and upper)
3. Activate PVDF membrane with methanol by incubating for 15 sec
4. Soak activated membrane in transfer buffer (1x Tris-glycine, 20% methanol),  to equilibrate for 5 min

1. Do not let the membrane dry out! If a white spot is visible, rewet membrane in methanol first and then equilibrate in the transfer buffer

5. Rinse gel in transfer buffer, wet filter paper and pads in the transfer buffer
6. Assemble the sandwich in the transfer cassette as follows:

1. White side of cassette (+)
2. Sponge
3. Whatmann filter paper (1 piece)
4. PVDF membrane
5. SDS Gel
6. Whatman filter paper (1 piece)
7. Sponge
8. Black side of Cassette (-)

7. Insert the cassette into the slot with black to balck and white to red
8. Cover to just over top wire with transfer buffer after adding ice pack in the slot. Put the apparatus in the ice
9. Run at 100 V for 1-3 hours
10. Remove membrane from the sandwich. Do not allow to dry out
11. Block the membrane in blocking solution (1x PBS with 5% skim milk) for 1 hour at R/T with shake (or at 4 degrees overnight without shake)
12. Pour off blocking solution. Add primary antibody in blocking solution (1x PBS with 5% skim milk, 0.1ug/mL primary antibody) and incubate for 1 hr with shake
13. Wash 3x with wash buffer (0.1% Tween in 1x PBS, a.k.a. PBST), 5 min each with shake
14. Add secondary antibody in blocking solution (1x PBS with 5% skim milk, 0.04ug/mL secondary antibody) and incubate for 1 hr
15. Wash 3x with wash buffer (0.1% Tween in 1x PBS, a.k.a. PBST), 5 min each with shake; additional wash with ddH2O with shake
16. Add ECL western blotting substrate: premix 1 mL of each solution (solutions 1 and 2 from ECL kit) in a falcon tube and add the mixed solution to the membrane by pipetting several times to make sure the membrane fully contacts with the solution. Incubate at R/T for 5 min.
17. Run protocol at BIO-RAD ChemiDocTM station using protocol as follows:

1. blots>>>Chemi>>>manually set exposure time: 10, 30, 50, ….300 second until you see the band

Pheophytin Production

1. Add HCl to chlorophyll solution until final HCl concentration is 20 mM.
2. After 2 minutes, add NaOH until 20mM final concentration in solution to neutralize the solution.

* For pheophytinase reactions, add enough NaOH and HCl until final pH is 8.00.

Thin Layer Chromatography

1. Draw a line 2.0cm away from the base of the silica plate.
2. Use P20 pipettes to drop/apply solution to be run, leaving at least 1 cm away from the next solution. Add dropwise and allow to dry before re-application. Allow the whole silica plate to dry before placing in reaction chamber.
3. Pour solvent of interest (70:30 Methanol to Hexane ratio) in the reaction chamber. Apply paper towels or filter papers on the walls of the chamber and wet with solvent of interest to saturate the chamber.
4. Place the silica plate in the chamber after 5 minutes or full saturation. Wait for solvent to travel up the plate until solvent front of desired distance is reached.
5. Mark the solvent front with a pencil. Visualize under UV light.

# Fungal Protocols

---

Fungal Culturing

1. Aseptically cut a small portion of fungal growth from an existing culture, near the edge of mycelial growth. Minimizing the amount of agar you transfer, place culture in the centre of a fresh potato dextrose agar plate.
2. Grow in dark conditions at room temperature for 4 - 5 days.

Pheophorbide Saturated Disc Application

1. Hole punch Whatman filter paper to make small discs with standard size
2. Autoclave using dry conditions
3. Prepare ~100 µL desired dilutions in 0.5 mL microcentrifuge tubes using 25% acetone
4. Mark the bottom of the culture plate with desired locations of discs
5. Using forceps/tweezers aseptically saturate a disc by submerging in pheophorbide solution
6. Apply discs central to marked spots

Pheophorbide Mycelial Inhibition Data Collection

1. Using a ruler measure from the epicentre of the fungal culture (mark this to keep it constant through all future data collection) to the edge of the furthest hyphae (edge of mycelial growth)
2. Take this measurement for all treatments or in 8 equal perpendicular directions for growth controls

* Growth control data is then used as an average of these 8 measurements

# Emulsion Protocols

---

Acid Activated Clay Experiments

1. Important note: Perform experiments in low lighting conditions or cover oil sample beakers thoroughly in aluminum foil. Chlorophyll is highly light sensitive and will degrade under regular lighting.
2. Per 10mL sample of unprocessed green oil
3. Measure desired samples of Montmorillonite K10 acid activated clay to desired adsorbent loading

* Adsorbent loading is defined as MassClay/MassFluid

4. Prepare 10mL sample of unprocessed green oil
5. Heat 10mL sample of green oil to desired temperature; check temperature after 15 minutes of heating to ensure steady state.
6. While heating, add magnetic stir bar and turn on stir plate agitation to 600 rpm
7. Slowly add clay sample to the green oil, and allow heating/stirring to continue for 90 minutes
8. Turn off stirring/heating, and immediately transfer contents to a 15ml falcon tube
9. Centrifuge oil-clay solution at 3750rpm for 10 minutes
10. Transfer yellow oil supernatant into a new 15ml falcon tube, and allow to settle for 60 minutes
11. Record the mass change of the total processed oil
12. Aliquot 250ul of yellow oil and dilute with 750ul of commercial canola oil. Mix thoroughly with stir rod
13. Place diluted processed oil sample into a clean 1ml cuvette
14. Place 1ml of diluent blank oil into a clean 1ml cuvette
15. With UV-Spectrophotometer, blank oil diluent at 670nm
16. Measure diluted processed oil sample at 670nm and record absorbance

Bovine Serum Albumin (BSA) Preparation:

1. Weigh out 1g of BSA per mL of desired solution volume
2. Prepare buffer of choice in a 15 or 50mL falcon tube
3. Slowly add a portion of the weighted BSA into buffer
4. Gently rock falcon tube until BSA crystals are completely solubilized
5. Repeat steps 3-4 until all the weighted BSA is solubilized
6. Allow to settle in 37℃ for 30 minutes
7. Filter protein-buffer solution through syringe filter
8. Collect filtered solution

Protein Emulsification within Oil-in-Water emulsions:

1. Obtain a handheld tissue homogenizer
2. Prepare stock surfactant of Span 80 and Tween 80 at a 11.25:1 volumetric ratio
3. Thoroughly mix oil and surfactant by vortex
4. Allow to settle at 37℃ for 10 minutes – assure there are no air bubbles in the oil-surfactant mixture
5. Push homogenizer plunger to the bottom of the homogenizer vial
6. Slowly pour oil-surfactant phase into the homogenizer with the plunger in
7. Slowly add the aqueous protein-containing phase into the homogenizer and allow it to settle
8. Slowly pull plunger to the top of the aqueous phase

* Note: be careful to not produce a vaccum in the solution while pulling plunger
* Note: do not break the air-liquid interface of the solution – this avoids mixing in air

9. Pushing plunger back down to the bottom of the vial to begin emulsification
10. Repeat process until solution is homogenously mixed
11. Pour homogenizer contents into a flat bottom vial
12. Place vial onto a rocking table at the slowest settings for 30 minutes
13. Allow emulsion solution to settle out overnight
14. Sample 400uL from excess oil phase into microcentrifuge tube
15. Centrifuge sample at 1000rpm for 20 minutes to separate any leftover aqueous phase
16. Sample 200uL from supernatant and dilute in 1mL of canola oil
17. Place diluted processed oil sample into a clean 1mL cuvette
18. Place 1mL of diluent blank oil into a clean 1mL cuvette
19. With UV-Spectrophotometer, blank oil diluent at 670nm
20. Measure diluted processed oil sample at 670nm and record absorbance

Project

[Description](https://2019.igem.org/Team%3ACalgary/Description)
[Wet Lab Overview](https://2019.igem.org/Team%3ACalgary/WetLab)
[Chlorophyll Extraction](https://2019.igem.org/Team%3ACalgary/ChlorophyllExtraction)
[Protein Emulsification](https://2019.igem.org/Team%3ACalgary/EmulsifiedBindingProteinProcess)
[Repurposing Chlorophyll](https://2019.igem.org/Team%3ACalgary/RepurposingChlorophyll)
[Anti-Fungal](https://2019.igem.org/Team%3ACalgary/Anti-Fungal)
[Demonstrate](https://2019.igem.org/Team%3ACalgary/Demonstrate)
[Results](https://2019.igem.org/Team%3ACalgary/Results)

Human Centered Design

[Integrated Human Practices](https://2019.igem.org/Team%3ACalgary/Human_Practices)
[Entrepreneurship](https://2019.igem.org/Team%3ACalgary/Entrepreneurship)
[Education](https://2019.igem.org/Team%3ACalgary/Public_Engagement)
[Collaborations](https://2019.igem.org/Team%3ACalgary/Collaborations)
[Safety](https://2019.igem.org/Team%3ACalgary/Safety)

Modelling

[Overview](https://2019.igem.org/Team%3ACalgary/Model)
[Measurement](https://2019.igem.org/Team%3ACalgary/Measurement)
[Software Tools](https://2019.igem.org/Team%3ACalgary/Software)
[Results](https://2019.igem.org/Team%3ACalgary/Results)

Parts

[Parts Overview](https://2019.igem.org/Team%3ACalgary/Parts)
[Collection](https://2019.igem.org/Team%3ACalgary/Part_Collection)
[Improve](https://2019.igem.org/Team%3ACalgary/Improve)
[Characterize](https://2019.igem.org/Team%3ACalgary/Contribution)
[Validate](https://2019.igem.org/Team%3ACalgary/Validate)

Journal

[Entries](https://2019.igem.org/Team%3ACalgary/Notebook)
[Protocols](https://2019.igem.org/Team%3ACalgary/Experiments)
[Judging](https://2019.igem.org/Team%3ACalgary/Judging)
[Appendix](https://2019.igem.org/Team%3ACalgary/Appendix)

Team

[Members](https://2019.igem.org/Team%3ACalgary/Team)
[Attributions](https://2019.igem.org/Team%3ACalgary/Attributions)
[Sponsors](https://2019.igem.org/Team%3ACalgary/Sponsors)

[![](https://static.igem.org/mediawiki/2019/c/cf/T--Calgary--facebookIcon.svg)](https://www.facebook.com/igemcalgary/)
[![](https://static.igem.org/mediawiki/2019/b/ba/T--Calgary--instaIcon.svg)](https://www.instagram.com/igemcalgary/?hl=en)
[![](https://static.igem.org/mediawiki/2019/0/02/T--Calgary--linkedinIcon.svg)](https://www.linkedin.com/company/igem-calgary/about/)
[![](https://static.igem.org/mediawiki/2019/3/3a/T--Calgary--twitterIcon.svg)](https://twitter.com/igemcalgary?lang=en)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Calgary/Results

[![](https://static.igem.org/mediawiki/2019/f/ff/T--Calgary--logowhite.png)](https://2019.igem.org/Team%3ACalgary)

* Project

  + [Description](https://2019.igem.org/Team%3ACalgary/Description)
  + [Wet Lab Overview](https://2019.igem.org/Team%3ACalgary/WetLab)
  + [Chlorophyll Extraction](https://2019.igem.org/Team%3ACalgary/ChlorophyllExtraction)
  + [Protein Emulsification](https://2019.igem.org/Team%3ACalgary/EmulsifiedBindingProteinProcess)
  + [Repurposing Chlorophyll](https://2019.igem.org/Team%3ACalgary/RepurposingChlorophyll)
  + [Anti-Fungal](https://2019.igem.org/Team%3ACalgary/Anti-Fungal)
  + [Results](https://2019.igem.org/Team%3ACalgary/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ACalgary/Demonstrate)
  + [Judging](https://2019.igem.org/Team%3ACalgary/Judging)
  + [Awards](https://2019.igem.org/Team%3ACalgary/Awards)
* Human Centered Design

  + [Integrated Human Practices](https://2019.igem.org/Team%3ACalgary/Human_Practices)
  + [Mean Green Machine](https://2019.igem.org/Team%3ACalgary/Modelling/StandardizedSeedGrading)
  + [Sunny Days](https://2019.igem.org/Team%3ACalgary/SunnyDays)
  + [Entrepreneurship](https://2019.igem.org/Team%3ACalgary/Entrepreneurship)
  + [Education and Public Engagement](https://2019.igem.org/Team%3ACalgary/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3ACalgary/Collaborations)
  + [Safety](https://2019.igem.org/Team%3ACalgary/Safety)
* Measurement and Model

  + [Overview](https://2019.igem.org/Team%3ACalgary/Model)
  + [ICARUS](https://2019.igem.org/Team%3ACalgary/Model/ICARUS)
  + [Emulsion Construction Prediction](https://2019.igem.org/Team%3ACalgary/Model/EmulsionPrediction)
  + [Directed Protein Modification](https://2019.igem.org/Team%3ACalgary/Model/DirectedProteinModification)
  + [In Silico Emulsion System Verification](https://2019.igem.org/Team%3ACalgary/Model/InSilicoEmulsionSystemVerification)
  + [Measurement](https://2019.igem.org/Team%3ACalgary/Measurement)
  + [Software Tools](https://2019.igem.org/Team%3ACalgary/Software)
  + [BOTS](https://2019.igem.org/Team%3ACalgary/BOT)
  + [iGAM](https://2019.igem.org/Team%3ACalgary/iGAM)
  + [Results](https://2019.igem.org/Team%3ACalgary/Results)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACalgary/Parts)
  + [Collection](https://2019.igem.org/Team%3ACalgary/Part_Collection)
  + [Improve](https://2019.igem.org/Team%3ACalgary/Improve)
  + [Characterize](https://2019.igem.org/Team%3ACalgary/Contribution)
  + [Validate](https://2019.igem.org/Team%3ACalgary/Validate)
* Journal

  + [Protocols](https://2019.igem.org/Team%3ACalgary/Experiments)
  + [Appendix](https://2019.igem.org/Team%3ACalgary/Appendix)
  + [Journal](https://2019.igem.org/Team%3ACalgary/Notebook)
* Team

  + [Members](https://2019.igem.org/Team%3ACalgary/Team)
  + [Attributions](https://2019.igem.org/Team%3ACalgary/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ACalgary/Sponsors)

# Results

# Human Centered Design

## [IHP](https://2019.igem.org/Team%3ACalgary/Human_Practices)

**Goal:** In designing our project, our team wanted to make sure that our work was addressing real-life issues. We sought out the opinions of various stakeholders, including farmers, manufacturers, and leading scientists in our project design.

**Result:** We were able to integrate human practices at all 5 stages of our project design. First, we contacted farmers and oil producers in order to understand the scope of the green seed problem. Using our discussion with stakeholders as a starting point, our team began to ideate project ideas. In order to inform our design, we spoke to lead scientists to flag potential issues and improve our biological design. In order to evaluate our project, we brought our designs back to stakeholders to make sure our project aligned with their visions for the industry. Last but not least, we continued our conversation with various faculty members and the Canadian Grain Commission to further iterate on our design.

**Future Directions:** As we continue to expand our project, we hope to maintain our relationship with stakeholders at each level to make sure our project grows in the right direction.

## [Sunny Days](https://2019.igem.org/Team%3ACalgary/SunnyDays)

**Goal:**
Since a main cause of the green seed problem is frost, farmers and the alberta canola association expressed how it could be useful to have better frost prediction.

**Result:**
We designed software that can accurately predict long term daily-lows for temperature in a local town, Vulcan.

**Future Directions:**
We want to increase accuracy, as well as increase its ability to predict frost using other parameters.

## [Mean Green Machine](https://2019.igem.org/Team%3ACalgary/Modelling/StandardizedSeedGrading)

**Goal:**
Farmers expressed displeasure with the current archaic method of grading seeds. We wanted to build an inexpensive method to standardize the grading of canola seed.

**Result:**
We designed software, that when coupled with our lightbox to standardize conditions, could with a few tweaks very accurately grade canola seeds.

**Future Directions:**
We want to increase accuracy and speed by changing our code to c++, as well as training a dataset with a convolutional neural net.

## [Entrepreneurship](https://2019.igem.org/Team%3ACalgary/Entrepreneurship)

**Goal:** After identifying the green seed problem as a major problem within the green seed industry, our team looked into ways in which our solution could be implemented into the industry.

**Result:** Through our industry outreach, we were able to find that our emulsion system could be used to supplement clays in existing chlorophyll removal infrastructure to maximize chlorophyll removal and oil yield at a lower cost.

**Future Directions:** After the 2019 Giant Jamboree, we hope to patent our Emulsified Binding Protien (EBP) process, and move towards creating a start-up company for our design.

## [Education and Public Engagement](https://2019.igem.org/Team%3ACalgary/Public_Engagement)

**Goal:** In order to strengthen the Alberta canola industry, our team aimed to use education and public engagement to enhance the skilled workforce, create proper infrastructure, and foster consumer acceptance of canola products.

**Result:** Through thorough education of new iGEM team members, discussion of synthetic biology principles with high-schoolers across the city, and engagement activities with the general public, we were able to address all three of these objectives.

**Future Directions:** We hope to expand our outreach efforts to reach even more people. We hope to continue educating future synthetic biologists through our synthetic biology education package.

# Wet Lab

## [Chlorophyll binding protein Production](https://2019.igem.org/Team%3ACalgary/ChlorophyllExtraction)

**Goal:** Our team aimed to design constructs and express and purify 6GIX, a water soluble chlorophyll-binding protein, for use in our emulsified binding protein (EBP) process.

**Result:** Our team successfully used Golden Gate assembly to create seven constructs to express the 6GIX protein. Six of these constructs contained each of the different signal peptides OmpA, MalE, TorA, YcbK, DsbA, and PhoA signal peptides. We also were able to create a construct without a signal peptide. Using the T7 promoter system and Ni-NTA chromatography, we were able to successfully induce and purify 6GIX.

![](https://static.igem.org/mediawiki/2019/4/48/T--Calgary--6GIXgel.jpeg)

Figure 1. SDS-PAGE gel showing whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.

The correct protein size was visible at 21kDA in a western blot using Anti-His MAb (from mouse) and Anti-mouse IgG conjugated with HRP antibodies.

![](https://static.igem.org/mediawiki/2019/7/7b/T--Calgary--blat.png)

Figure 2. Western blot of whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. A His-tagged positive control was also included. The marker used is the NEB colour protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (MBL) for primary antibody and Goat Anti-Mouse:HRP (Jackson ImmunoResearch Laboratories) for secondary antibody.

The 6GIX protein with PhoA, DsbA, and MalE signal peptides were successfully secreted into the periplasm of the cells, with the brightest protein band visible for secretion using the MalE signal peptide.

![](https://static.igem.org/mediawiki/2019/5/51/T--Calgary--SPgel.png)

Figure 3. SDS-PAGE gel showing periplasmic purification of 6GIX with six different signal peptides, no signal peptide, and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.

**Future Directions:** In order to further characterize the 6GIX protein, we hope to assess the binding affinity of 6GIX to chlorophyll.

## [Protein Emulsification](https://2019.igem.org/Team%3ACalgary/EmulsifiedBindingProteinProcess)

**Goal:** After obtaining a purified 6GIX protein sample, we aimed to provide a proof-of-concept for our Emulsified Binding Protein (EBP) system by showing that 6GIX is able to bind chlorophyll in an emulsion.

**Result:** Preliminary emulsion tests show that 6GIX is capable of binding chlorophyll in an emulsion.

![](https://static.igem.org/mediawiki/2019/8/8e/T--Calgary--Emulsions.png)

Figure 4. Acid-activated clay, BSA positive control, 6GIX, and buffer negative control emulsions. BSA and 6GIX were emulsified at equal concentrations. The upper phase is a pure oil phase and the lower phase is a oil in water microemulsion. Processed and unprocessed canola oil samples were also imaged for reference.

![](https://static.igem.org/mediawiki/2019/e/e1/T--Calgary--Seb.png)

Figure 5. Percent change in chlorophyll concentration in oil after emulsification with BSA and 6GIX.

6GIX is shown to have superior chlorophyll-binding capabilities to BSA.

**Future Directions:** We hope to induce and purify ModGIX, a 6GIX protein improved for stability, and compare its efficiency to 6GIX in emulsion.

## [Chlorophyll Repurposing](https://2019.igem.org/Team%3ACalgary/RepurposingChlorophyll)

**Goal:** Our team aimed to characterise CBR, HCAR, SGR, and PPH, which are four enzymes involved in the degradation pathway of chlorophyll to pheophorbide a.

**Result:** Using ICARUS, our universal spacer, we were successfully able to purify HCAR and PPH using Ni-NTA chromatography. 10% SDS-PAGE gels were stained with Coomassie blue to reveal HCAR and PPH presence in the elution fractions of a Nickel-NTA column purification.

![](https://static.igem.org/mediawiki/2019/8/8b/T--Calgary--NEB-Color-Prestained-Protein-Standard-Broad_Range-%2811%E2%80%93245-kDa%29.jpeg)![](https://static.igem.org/mediawiki/2019/3/3c/T--Calgary--HCAR-PPH-SDS-PAGE-4-USE.png)

Figure 6. 10% SDS-PAGE HCAR and PPH Purification Confirmation. 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), PPH - post loading fraction, PPH - elution fraction 1, PPH elution fraction 2, HCAR - post loading fraction, HCAR - elution fraction 1, HCAR elution fraction 2, pSB1A3 (plasmid control in BL21) - elution fraction 1, and pSB1A3 (plasmid control in BL21) - elution fraction 2. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa. This SDS-PAGE Confirmation is replicate 4/5, other replicate images are available [here.](https://2019.igem.org/Team%3ACalgary/Appendix#SDS-PAGE)

The identity of HCAR was confirmed in a western blot using Anti-His MAb (from mouse) and Anti-mouse IgG conjugated with HRP antibodies.

![](https://static.igem.org/mediawiki/2019/4/4f/T--Calgary--HCAR-Western-Blot.png)

Figure 7. Western Blot HCAR Purification Confirmation. 10% SDS-PAGE was run and transferred to a PVDF membrane. The antibodies used were Mouse Anti-HIS-tag mAb (MBL) for primary antibody and Goat Anti-Mouse:HRP (Jackson ImmunoResearch Laboratories) for secondary antibody. ECL was used to visualise. Lanes from left to right on the SDS-PAGE were ladder (Color Prestained Protein Standard, Broad Range (11–245 kDa) (NEB)), ~60 kDa protein (positive control) in two lanes, pSB1A3 (plasmid control in BL21) - elution fraction 3, HCAR - elution fraction 1, HCAR - elution fraction 2, HCAR - elution fraction 3. HCAR is 58 kDa.

Through thin layer chromatography analysis of pheophytin, pheophorbide, and reactions involving our recombinant PPH protein, we were able to qualitatively show that pheophytinase (PPH) is able to enzymatically convert pheophytin into pheophorbide.

![](https://static.igem.org/mediawiki/2019/0/00/T--Calgary--TLC_PB_2.jpeg)