Team:Calgary/Journal
Journal
Week 2: May 8 - May 12
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Week 3: May 15 - May 19
An ordered list!
- Secretion
- Syncretion
- Engineering
- Janitorial duties
- Fruit flies
- Synthesis
Team Member Classifications:
- Coworkers
- Friends
- Bilal
| Number | Meme | Description |
|---|---|---|
| 1 | Dat Boi | Ayyyyyyy |
| 2 | Doge | Wow |
| 3 | Shrek | is Love |
| 4 | Dancing Pumpkin Man | Spooky |
Week 1 (May 1 - May 5, 2017): Uploaded Sequences onto Benchling
The synthesis group focused on completing lab safety courses and laboratory training, which involved following commonly used protocols, and took inventory of our lab supplies. We also looked into the genes needed for synthesizing PHA in E. coli. After finalizing the genes, the constructs were uploaded on benchling as follows:
- Promoter T7, spacer sequence, RBS (B0034), FadE (E. coli), FadD (E. coli), PhaJ4pp, PhaC (p.aeruginosa)
- Promoter T7, spacer sequence, RBS (B0034), FadEE. coli, FadDE. coli, PhaJ (a.caviae), PhaC (p. aeruginosa)
- LacO operator, spacer sequence, promoter T7, spacer, RBS (B0034), FadE (E. coli), FadD (E. coli), PhaJ4 (p. putida), PhaC (p. aeruginosa)
- LacO operator, spacer sequence, promoter T7, spacer, RBS (B0034), FadE (E. coli), FadD (E. coli), PhaJ (a.caviae), PhaC (p. aeruginosa)
- phaCAB with T7 (constitutive) on lac I
- phaCBA with T7 (constitutive) on lac I double vector
- Hybrid promoter with number 8
- T7 (constitutive) + SS + RBS + SS + PhaC1 ap + phaJ4 (p. putida) + fadE + fadD + 10nt SS + RBS + SS + phaCAB
Week 2 (May 8 - May 12, 2017): Designing constructs and protocol for media
This week, we worked on researching protocol for media to be used for bacterial growth. Our mentors and advisers raised questions concerning the containment of the bacteria. This led our group to look into an antibiotic-free selection system; a number of methods for antibiotic free selection were brought up. We also looked into the possibility of using Bacterial Artificial Chromosome (BAC) to deliver our inserts due to concern over the large size of the insert. Ultimately, we decided that, if time allows, we will consider pursuing the antibiotic free selection method and the BAC. The lab training we participated in this week allowed us to practice protocols for performing transformations, preparing competent cells, and performing cPCR using template DNA from colonies on a streak plate. We also practiced agarose gel electrophoresis.
This week, the details of the plasmid design were discussed. We decided to use pET29b(+) as our vector because it has an inducible lacI and a T7 promoter. We also planned characterization experiments for PhaJC + FadE, and compare PhaCBA with the PhaCAB Biobrick from Tokyo 2012. We decided to code our parts on benchling with the following template: junk-ENX (prefix)-RBS-cds-SNP (suffix)-kpn1 (restriction)-junk. We finalized the three plasmid inserts with T7 promoters (____ , ____ and _____) and double-checked the spacer sequences using BLAST. We also decided to add 6x-His tags after methionine to help with characterization in the later stages of the experiment. Restriction sites were decided on for each part so that the whole final biobrick can be ligated together (3 pieces for beta-oxidation PHA sequence and 2 pieces for glucose PHA synthesis).
Week 3 (May 15 - May 19,2017): Codon optimization and ordering sequences
The synthesis group optimized codons, removed a number of restriction sites, finalized our constructs, and ordered our sequences from IDT. We also researched the chemicals required for growth media and the characterization protocols we planned to perform. We continued formulating and editing protocols for post-synthesis experiments such as chemical cell lysis, PHA extraction and purification, and Nile Red Fluorescence quantification. A table was compiled to compare the pros and cons of different procedures for processes such as PHA extraction and quantification.
Week 4 & 5 (May 22 - June 2, 2017)
This week, the entire iGEM team took a field trip to a Calgary Wastewater Plant to learn about wastewater management. This trip informed us of the applications of our project. After evaluating the economic feasibility of implementing our waste-to-plastic system for use in the municipal wastewater treatment plant, the team started to discuss other possible applications of our project (wastewater treatment, developing countries, landfills, or space). Each member of the group researched a different application of our project to present and discuss at the weekly lab meeting. The group finalized the post-synthesis protocols that were researched last week and ordered our required chemicals for these experiments.
Week 6 (June 5 - June 9, 2017): Restriction digest + electrophoresis
While waiting for the rest of the ordered gblocks™ to arrive, the synthesis group assisted the efforts of the human practices and modelling groups. We practiced coding for the wiki and researched E. Coli infections in space to evaluate its virulence and containment.
Week 7 (June 12 - June 16, 2017): Plasmid mini prep
We performed single digests on our Pet29b(+) vectors using restriction enzymes HindIII, Sal1, EcoRI, and KpnI to check that each enzyme worked. After the DNA was digested we ran the samples on 1% agarose gel. The gel showed bands representing linearized plasmids on the gel, which informed us that our restriction enzymes were functional.
Week 8 (June 19 - June 23, 2017): Run Controls and Digest gBlocks
We received streak plates of E. coli BL21(DE3) and E. coli DH5α containing pET29b(+) vectors from Dr. Wong’s lab. These stock plates were used to streak fresh plates of the E. coli. Glycerol stocks of these cells were also made to preserve them. We made O/N cultures of the E. coli DH5α and performed plasmid miniprep to obtain PET29b(+) vectors. When we tested the plasmids on the NanoDrop, the samples were contaminated and did not show high concentrations. We decided to make O/N of the E. coli DH5α and then perform plasmid miniprep again. This time, the NanoDrop results showed a successful miniprep of the pET29b(+) vectors. These pET29b(+) vectors were stored in the -20°C freezer.
Week 9 (June 26 - June 30, 2017):
We performed diagnostic testing of NotI in CutSmart™ buffer and HindIII in Fast Digest Buffer with RFP plasmids from the iGEM registry. Diagnostic testing was done because the manuals outlined digestion of NotI in Fast Digest Buffer and HindIII in CutSmart™. We wanted to see if one of the buffers would work for both restriction enzymes. We then ran the digests on 1% agarose gel. The results showed that the digest of NotI in CutSmart™ worked and that FastDigest Buffer did not work. This meant that we could use CutSmart™ buffer for both restriction enzymes. The resulting gel electrophoresis is shown below.
| DNA | Enzymes |
|---|---|
| PhaC | NotI, HindIII |
| PhaBA | HindIII, KpnI |
| pET29b(+) for PhaC | NotI, HindIII |
| pET29b(+) for PhaBA | HindIII, KpnI |
We then streaked a fresh plate of E. coli DH5α containing pET29b(+), made O/N, and performed miniprep to get more pET vectors to use for future experiments.
Week 10 (July 3 - July 7, 2017): Ligated gBlocks
This week we ligated the digested PhaC and PhaBA each into pET29b(+) vectors. After ligation we transformed our cells and incubated the plates O/N. The next day we chose four colonies from PhaC and PhaBA-transformed plates to make O/N cultures and a master plate from. We isolated the plasmid from our O/N cultures to perform a confirmation digest of the transformed cells. We performed double or single digests of the plasmids from miniprep to check that the presence of the insert within the vector and to check the directionality of the insert. The restriction enzymes and the respective sites and expected bands on the plasmids are shown below.
| Transformed Plasmids | Enzymes | Expected band size | |
|---|---|---|---|
| PhaC | For confirmation | HindIII, NotI | 5.4 kb, 1.8 kb |
| For directionality | HincII | 1.8 kb, 1.4 kb | |
| PhaBA | For confirmation | HindIII, KpnI | 5.3 kb, 2.1 kb |
| For directionality | HincII | 4.4 kb, 3.0 kb |
We ran gels of undigested and digested plasmids. When we compared the predicted Benchling digests with our results, we found that colony 1 from the transformed PhaBA matched the expected bands, suggesting that the transformation was successful because the insert was in the plasmid and the direction was correct. However, the PhaC transformants did not match or show corresponding bands with the digests performed on a transformed vector. The results are shown in the figure below.
Week 11 (July 10 -July 14, 2017):
From the confirmation digest and gel electrophoresis from last week, PhaBA colony 1 seemed to have the correct insert. Therefore, we wanted to sequence the colony to confirm that the correct insert was in the plasmid. To do so, we designed and ordered primers for pET29b(+) PhaBA and prepped our samples for sequencing. The PhaC transformant colonies 1-4 did not pass the confirmation digest screening, but to double-check for the plasmids, we isolated, digested (NotI, HindIII and HincII), and ran gel electrophoresis on the plasmids from 6 more PhaC transformant colonies (5-11) using protocols ____ and ____. The results are shown below.
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We planned out the rest of our experiments for the summer and set tasks to complete. We transformed our DH5α competent cells with each of our plasmid inserts. We ran digests to confirm that the transformants actually carried our plasmids. We also developed a protocol for sequential digest of our pET29b(+) vectors because the NotI and HindIII restriction sites are very close together on plasmid. A Beta oxidation (PhaC-J) was digested with HindIII and SalI, ligated with pET29b(+) vectors, transformed into competent DH5α cells, plated on Kan resistant plates, and left to incubate O/N at 37°C. The culturing tubes of the transformants from the Transformation protocol were kept in the 4°C fridge. No colonies were observed in the plate the next day. To troubleshoot why the cells did not grow, plasmids were isolated from the inoculation tube with the transformed culture that was kept in the 4°C fridge. Confirmation digest was performed on these plasmids. The gel electrophoresis did not work. Therefore, we decided to redigest our gblock, ligate, and transform it again. B Beta Oxidation and C Beta Oxidation were digested with HindII/SalI and SalI/KpnI, respectively following the Restriction Digest protocol. A salt solution was made to adjust for the salt concentration. After heat inactivation, the digested DNA was stored at -20°C for the weekend.
Week 12 (July 17 -July 21, 2017):
The ligated PhaC pET29b(+) and A Beta oxidation pET29b(+) were transformed into competent DH5α cells and plated on Kan LB agar plates. Colonies appeared on these plates. A master plate and O/N cultures were made to screen these transformants. The O/N culture was used to isolate plasmids from. The plasmids were digested and screened for confirmation of insert and directionality. The enzymes used are shown below along with their respective restriction sites and expected band size. The screening results showed that there was no insert.
| Transformed Plasmids | Enzymes | Expected band size | |
|---|---|---|---|
| PhaC | For confirmation | HindIII, NotI | 5.4 kb, 1.8 kb |
| For directionality | AscI, SphI | 6.1 kb, 1.1 kb | |
| A Beta Oxidation | For confirmation | HindIII, NotI | 5.4 kb, 2.2 kb |
| For directionality | Xmal | 5.0 kb, 1.5 kb, 1.1 kb |
Last week, we digested B Beta Oxidation and C Beta Oxidation and stored them. This week, we ran the digested B beta oxidation part on an low melting point (LMP) gel and recovered the part by excising it from the gel. The gel containing the B Beta oxidation insert was melted at 65°C and ligation protocol was performed. We then transformed the plasmid into competent DH5α. C Beta Oxidation was also ligated with pET29b(+) vectors and transformed into competent DH5α. The B Beta Oxidation and C Beta Oxidation transformants colonies were observed after the O/N incubation. A master plate and O/N culture of selected colonies from each plate was made. The vectors were isolated, digested for confirmation of insert, and ran 1% agarose gel.
| Transformed Plasmids | Enzymes for Insert Confirmation | Expected Band Sizes |
|---|---|---|
| B Beta Oxidation | HindIII, SalI | 5.4 kb, 2.5 kb |
| C Beta Oxidation | KpnI, SalI | 5.3 kb, 1.8 kb |
Week 13 (JULY 24 - JULY 28, 2017)
Because PhaC and A Beta oxidation did not transform with the inserts, the gBlocks were digested, ligated, and transformed this time in pET-RFP plasmids following protocol the pETRFP Digestion protocol. B Beta Oxidation colony 2 and C beta oxidation colony 3 seemed to have worked so the plasmids were isolated by miniprep and sent in for sequencing using the designed primers. The C beta oxidation sequencing results were incorrect. However, to double-check, we prepared a master plate and O/N of a few colonies from the plate of transformants. Miniprep was performed and the plasmids were digested and were run on 1% agarose gel. The results did not indicate the successful ligation of the insert. To keep the pET29b(+) E. coli DH5α fresh, a new streak plate was made. An O/N culture was made and plasmids were isolated by miniprep and stored in the -20 freezer for future use.
Week 14 (July 31 - Aug 4)
The sequencing result___. To check that the B beta oxidation transformed into DH5α on ___ was correct, an O/N was made. Miniprep was performed to isolate the plasmid. The plasmids were then digested and run on a 1.5% agarose gel at 70V for 90 minutes. The results showed that____
Week 1 (May 1-May 5, 2017)
We are all fully trained in laboratory safety! We each did 6 online courses and attended 2 seminars on lab safety and biosafety. In the lab we practiced important protocols, including media preparation, overnight culture inoculation, preparing chemically competent cells, and transforming chemically competent cells. We also did inventory, organized the lab, and began an order sheet for supplies that we will need throughout the summer. Also, we began to research ways that we will be able to use synthetic biology to extract synthesized PHB from cells, without the use of traditional chemical or mechanical lysis.
Week 2 (May 8-May 12, 2017)
We practiced more important laboratory techniques: plasmid minipreparation, preparing master plates of transformed E. coli, cPCR, and agarose gel electrophoresis. Outside the lab, we narrowed down our possible extraction methods to either an acetate-based autolysis system or a hemolysin type I secretion system. The autolysis method depends on the change in concentration of VFAs, specifically acetic acid, as the VFAs are depleted during PHB synthesis. However, we decided that there were too many unknowns and other factors at play for this system. Therefore, we decided to use the hemolysin type I secretion system. We uploaded our parts onto Benchling and began designing our plasmid, with the goal of ordering our parts early next week! Some modifications to the parts we uploaded to Benchling were made; flag tags for easier protein expression validation were added to each coding sequence and restriction sites were removed to make the parts RFC10,12,21,23,25, and 1000 compatible.
Week 3 (May 15-May 19, 2017)
This week we finished editing our secretion parts before ordering them from IDT. Our complete part (phasin-HlyA tag, HlyB, HlyD) was split into two separate gBlocks because it was too large for one. A gBlock with just the phasin-HlyA tag was ordered as well so that we could compare secretion of the endogenous E. coli secretion system to our system with upregulated HlyB and HlyD. Restriction sites for the HINDIII enzyme were added to the ends of our gBlocks so they could be easily ligated together. All stop codons were changed to TAA because it is the most effective stop codon in E. coli. Also, codons were optimized before ordering using this tool.
Week 4 (May 23- May 26, 2017)
Team members Jacob and Sam began working on the Interlab Study. To begin, chemically competent DH5𝛼 E. coli were made and stored at -80°C in glycerol stocks, PSB buffer was made and diluted several times to develop a standard absorbance curve for the study, and the plate plate reader was calibrated. Actual work on the Interlab Study was not very successful because there was little to no growth on plates of the transformed DH5𝛼, even though they were incubated at 37°C for two days. We also had a very informative tour of the Pine Creek Wastewater Treatment Plant in Calgary. Many questions were answered and the information we have gained will be used in deciding which application route our project will take.
Week 5 (May 29- June 2, 2017)
Due to the lack of growth on the Interlab Study plates last week, chemically competent DH5𝛼 E. coli were made again because there may have been issues with the first set of competent cells created. However, there was still no growth on the plates inoculated with transformed cells. On a positive note, our constructs from IDT that were ordered in week 3 arrived! We can hopefully begin work with our parts next week. Outside of the lab, lots of work was dedicated to researching the pros and cons of the four possible applications of our project (space, wastewater treatment, landfill leachate, and developing countries). The entire team will make its final decision about the application early next week.
Week 6 (June 5- June 9, 2017)
Again, Jacob and Sam’s work on the Interlab Study was unsuccessful. We tried using different protocols for making chemically competent cells and transforming those cells, but nonetheless there was still no growth after 24h incubation. This strongly suggests that there is an issue with our DH5𝛼 cells, so next week we will get new cells and try again. Due to our problematic cells, work has not yet begun with our actual secretion construct from IDT. Lalit and Kaitlin made samples for gel electrophoresis, and prepared all of the necessary supplies that will be needed on Lalit’s trip to Winston Churchill High School on Monday, June 12, 2017. During this outreach, he will present to Grade 11 students about synthetic biology, practice gel loading, and perform strawberry DNA extraction experiments with them. After consulting with experts and industry we decided that applying our project to creating PHB in space (ie. on future Mars colonies) would be the best choice with regards to a synthetic biology approach.
June 11, 2017- Geekstarter iGEM Workshop, presented by MindFuel and Alberta Innovates
Today was an iGEM workshop hosted by Geekstarter that was attended by the University of Alberta, University of Calgary, Urban Tundra High School, and University of Lethbridge iGEM teams. There were four speakers who gave ½ hour presentations on an introduction to syn bio, mathematical modeling, wiki design, and integration of art into syn bio. Later, we had the chance to speak with each presenter individually and ask questions specifically related to our project. Also, there was a collaboration period where we got to mingle with the other teams and discuss our projects with them. Overall it was a successful day where we learned a lot from the presenters, and identified possible collaborations with the other teams.
Week 7 (June 12- June 16, 2017)
On Monday, June 12 Lalit and Sam visited Winston Churchill High School to present to Grade 11 students about Synthetic Biology (which we prepared for in Week 8). We acquired new competent E. coli DH5α cells from Dr. Dong and transformed them for the interlab study. This time, there was successful transformation and colony growth of GFP cells. This supports the theory that there was something wrong with the cells that we were previously using. Therefore, for our work throughout the summer we will continue using the DH5α cells from Dr. Wong. Jacob and Sam used the new cells to successfully complete the laboratory component of the interlab study this week. Also, we performed a miniprep of psB1c3 plasmids from RFP E. coli Top10 for use with our secretion insert in the following weeks.
Week 8 (June 19- June 23, 2017)
Our parts from IDT (that arrived in Week 5) were re-suspended and were stored at -20℃, where they will remain for the rest of the summer. To prepare ourselves for work with our IDT constructs, we performed various diagnostic tests with restriction enzymes. To do this, we digested each RFP- and GFP-containing pSB1C3 with SpeI and EcoRI, and confirmed the digest on a 1% agarose gel. After some troubleshooting, we obtained two distinct bands on the gel, representing the pSB1C3 backbone and RFP/GFP insert. This indicates that these enzymes are functional and can be used on our IDT parts. We have been having difficulty creating chemically competent E. coli DH5α cells, so we are going to try a different protocol next week. Outside of the lab, Jacob and Sam completed and submitted the online portion of the Interlab Study this week. Kaitlin and Lalit attended various meetings with other members of the team.
Week 9 (June 26- June 30, 2017)
After confirming on a 1% agarose gel that the plasmids were linearized last week, we ran the samples on a 3% low melting point agarose gel to extract both the plasmid backbones and the RFP/GFP inserts. Then, to test our ligase, we ligated the GFP inserts to the RFP backbones, and the RFP inserts to the GFP backbones. As we expected, bacteria transformed with the GFP insert-RFP backbone grew green colonies and bacteria transformed with RFP insert-GFP backbone grew red colonies. This indicates that the restriction enzymes and ligase functioned properly and can be used with our precious IDT parts. Jacob and Sam tested out a new protocol for making chemically competent E. coli DH5α cells from Richard Moore, who is part of Dr. Dong’s lab here at the Foothills Hospital. They tested the cells’ competency with 100 ng/µL RFP. The transformed cells grew extremely well with numerous colonies and a high transformation efficiency. Finally, we have a protocol for making competent cells that works! Our IDT parts were digested and ligated into their corresponding backbones:
| Part/Insert | Digested with... | Backbone |
|---|---|---|
| Phasin-HlyA Tag | EcoRI, SpeI | pSB1C3 |
| Secretion Part 1 | XbaI, HindIII | pET29B |
| Secretion Part 2 | HindIII, NotI | pET29B |
We then transformed our ligated backbones and parts into our chemically competent E. coli DH5α cells.
Week 10 (July 3-July 7, 2017)
We performed a plasmid miniprep on the colonies of E. coli DH5α that were transformed with our ligated backbones/parts last week. Throughout the week we chose 8 colonies from phasin-HlyA, 8 colonies from SP1, and 2 colonies from SP2 (because there were only two transformed colonies that grew) to be digest confirmed. Each colony was run on a 1% confirmation gel. Each colony was left undigested as a control, double-digested with the same enzymes we used last week to check for the insert size, and digested with another random enzyme to check for insert directionality:
| Part/Insert | Backbone | Digested with… | Expected Band Sizes |
|---|---|---|---|
| Phasin-HlyA Tag | pSB1C3 | EcoRI, SpeI | 2.0 kb, 889 bp |
| Secretion Part 1 | pET29B | XbaI, HindIII | 5.2 kb, 2.4 kb |
| Secretion Part 1 | pET29B | HincII | 6 kb, 1.5 kb |
| Secretion Part 2 | pET29B | HindIII, NotI | 5.4 kb, 2.2 kb |
| Secretion Part 2 | pET29B | EcoRV | 6.1 kb, 1.5 kb |
By the end of the week we determined that only colony 1 of the phasin-HlyA tag had successfully received a plasmid with our part. This can be seen in the image below , where there are roughly 2 kb and 900 bp sized bands (Double digested colony 1 with EcoR1 and Spe1). Our other parts were not successfully transformed into our cells, so next week we will need to re-transform chemically competent E. coli DH5α with Secretion Part 1 and Secretion Part 2 to obtain more colonies.
Week 11 (July 10- July 14, 2017)
We submitted colony 1 phasin to be sequenced and the results of this confirmed that these cells do contain pSB1c3 with our phasin-HlyA part. We therefore transformed the psB1c3 with phasin-HlyA into BL21, which we will use for protein expression. In order to begin secretion assays with the psB1c3-phasin-HlyA that we obtained, we transformed E. coli DH5ɑ with a PHA synthesis biobrick (Part BBa_K934001) present in the iGEM 2017 distribution kit. This was done so that we do not have to wait for the Synthesis subgroup to finish their molecular cloning before we can begin testing the secretion of PHB. Outside of the lab, Jacob, Sam, and Lalit compiled all of their information from the Interlab Study and coded it into the Interlab page. On Friday, July 14 Kaitlin and some of the other iGEM team members visited the Grades 7-9 Minds in Motion summer camp on main campus. With the children, they discussed the potential implications of genetic engineering for the future and performed a strawberry DNA extraction experiment.
Week 12 (July 17-July 21, 2017)
We transformed our SPI part into pET29B, and began to work on digesting a pET29B plasmid containing an RFP insert that Rachelle made for us. This was done because HindIII and NotI were overlapping without an insert, causing our sequential digest to fail. Thus, RFP was inserted to separate the two restriction sites and permit our digestion. After a few failed attempts, we concluded that Rachelle’s ligation had failed and there was no RFP insert. Rachelle began to re-try her RFP-pET29B ligation while we digested a few more of the old colonies to make sure that the digestion error was not on our end. During this process, we had some troubles with our gel ladders, so we ran several ladders at once on a gel in order to determine which gels should be kept and used. SPI was run on a confirmation gel and the transformation looked successful based on band lengths. This was followed up by submitting the SPI for sequencing. On the wiki, Sam and Kaitlin began coding the protocols page. Furthermore, we took individual and team photos so that we could start completing the team page of the wiki.
Week 13 (July 24 - July 28)
We completed the “protocols” page of the wiki, and uploaded all of the general protocols used by everyone. We left a template on the page, so that any other group can easily upload their specific procedures in the same format as we did. We received our sequencing back for our SPI part, and confirmed that we successfully digested, ligated and transformed the SPI into DH5α. With regards to SPII, we tried multiple times to perform our sequential restriction digest of the pET-RFP backbone with HindIII and NotI, and ran into multiple roadblocks. After various troubleshooting techniques, we were finally successful upon using a DNA isolation protocol in between the two digestion steps. Following this, we excised the pET29B backbone from an LMP gel, and ligated with our digested SPII part. Ligation occurred on Friday, so the transformation was left until the following week. We also managed to create our first batch of PHB using the biobrick from the registry, and confirmed it using nile red staining.
Week 14 (July 31 - Aug 4)
We continued to work on cloning in our SPII part this week, using the same DNA isolation protocol as before in between the HindIII and NotI phases of our sequential digest. Using this technique, we successfully digested, ligated and transformed into DH5α, and had 11 colonies grow post-transformation. This was completed on Friday, so confirmation sequencing was left until the following week. Lalit performed an SDS-PAGE in an attempt to qualify phasin production in our phasin-transformed cells. Results were inconclusive, and the SDS-PAGE will be repeated at a later date. Regarding our PHB synthesis parts, Jacob transformed DH5α with a biobrick from imperial that he will compare to the part from Tokyo that he used previously. In a new vein, Sam and Kaitlin began transforming DH5α with various parts intended for a Mindfuel “Picasso” event. The goal of this event is to create art using differently fluorescing E. coli, so we transformed DH5α with CFP, YFP, a general RBS, and an arabinose induced promoter. We also began our digestions of these parts, with the ultimate intention of creating basic YFP-RBS-promoter and CFP-RBS-promoter biobricks. Finally, we created a new batch of competent DH5α cells that will hopefully last the rest of the summer. These will be tested next week to ensure their competency.
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Week 1 & 2: (May 15 - May 26)
Week 3 (May 29 - June 2)
To analyze the different mathematical models proposed in Week 1 & 2, we used the following criteria:
- Usefulness to the project
- Time required
- Skills
- Resources
Week 4: June 5 - June 16
The modelling group met with Dr. MacCullum to discuss the possible mathematical modelling methods. The group was advised that flux balance analysis and kinetic model would be the best to pursue for our project as it will inform our experiments and is feasible in the given time.OpenCobra toolbox for MATLAB was installed because it has functions for carrying out flux-balance analysis and visualizing the results.
Week 5: June 19 - June 23
This week we met with a group of postdocs working in Dr. Ian Lewis’s lab. We discussed how flux balance analysis could be helpful for our project. We also discussed flux variability analysis (FVA) in comparison to flux balance analysis. We decided that after finding optimal solutions using FBA, we can look into FVA. We were given some suggestions on some objectives we could look for in our model such as optimizing bacterial growth, optimizing PHB production using glycolysis only, and optimizing PHB production using beta-oxidation pathway only. This could be done by changing the parameters of the command optimizeCbModel(‘parameter’).
Week 6 & 7: June 26 - July 7
The modeling team had a meeting to discuss the milestones for flux balance analysis and kinetic model. We decided on the different models we plan to optimize using the flux balance analysis and kinetic modelling. We plan to study a number of models. One of the model will contain pathways for beta oxidation and PhaJ-C4 genes that can help produce PHB from medium-chain and long-chain fatty acids. The other model will contain CBA genes and PhaJ-C4, which can help produce PHB from glucose, short-chain, medium-chain, and long-chain fatty acids. The kinetic model will look into reactions that are rate-limiting in these pathways. Thus, FBA and kinetic model will work together to help optimize the production of PHB in E. coli (BL21) and modify substrate concentrations involved in the rate-limiting steps.
Week 8 & 9: July 10 - July 21
FBA: For the flux balance analysis we found an e coli model for our strain, which is BL21 (DE3). The model was found from the BiGG database. We looked into the genes and reactions it contained and the reactions that have to be added to the model. The genes of interest that the model already contained are:
- FadD
- FadE
- tolC
- LacY
- LacZ
- PhaA
- PhaB1
- PhaC1
- HlyA
- HlyB
We also used a plugin called Paint4Net to visualise the pathways/reactions in the model. A zoomed in section of the visual representation of FBA analysis resulting from calling the "draw_by_rxn" command for the coli_core_model is given in figure 1.
Kinetic: After deciding to do a kinetic model. We brainstormed what questions we would try to answer with our model. We wanted to compare PHB production via the beta oxidation pathway and the glycolysis pathway. Therefore we would create a model with phaJ and phaC and another with phaC, phaB and phaA. We also wanted to compare overexpressing fadD and fadE and see which one had a higher effect on PHB yield. The following are two other systems we hope to model. Some other questions we considered answering with our kinetic model include: What is the rate-limiting step in synthesis of PHB? What is the rate-limiting step in secretion of PHB? With those questions in mind we started searching for existing models for beta oxidation and PHB synthesis. We decided to base the PHB synthesis part of our model on the one created by the 2013 Imperial team. [1] For the beta oxidation part, since we could not find an established model we looked at the pathways for degradation of oleic acid (the main long-chain fatty acid in our media) via beta oxidation (Ren et al., 2004).
Week 10 & 11: July 24 - Aug 4
FBA: This week we looked into having a visual representation of the reactions/pathways in e coli BL21. However, the graph had too many nodes and the cobra toolbox could not visualise it. Thus, we looked into plotting a part or subsystems that included reactions of interest. This was done after optimizing the model and then calling the draw_by_rxn command. The command we used to select the specific reactions of interest such as the citric acid cycle was: fluxReactions = Model.rxns(ismember(Model.subSystems,'Citric Acid Cycle'));
Week 12: Aug 7 - Aug 11
Week 1: May 1 - May 5
In the planning phases of our project, our team envisioned a wastewater treatment application to our engineered bacteria; however, much of the planning work done before the start of May was focused on the synthetic biology and engineering aspects of our project; hence, the human practices team had not met at all until Wednesday, May 2nd. On May 2nd, our team began holding human practices meetings. These meetings, held throughout the summer, focused on both high-level policy aspects of human practices and the education and public engagement aspect of human practices. At our first human practices meeting, the human practices team brainstormed different ways to get involved with the community. We made a list of bioplastic production companies to contact regarding the input they would have for our team. We sent emails to TELUS Spark, Minds in Motion, TedX, and the wastewater treatment plant in order to secure possible meetings with these organizations. We also considered different aspects of policy to look into if we had chosen the wastewater treatment application: the safety of bioplastics, the impact of our engineered bacteria on the ecosystem, and the different applications to which our bioplastic is suited (medical implants, 3D printing, etc.) We reviewed the 2016 UofC Calgary team’s human practices efforts in order to understand possible avenues which we could take for our human practices efforts this summer.
Week 2: May 8 - May 12
We continued to follow up with contacts at the Pine Creek wastewater treatment plant. On May 9th, Michaela met with Magdalena Pop (Magda) from GeekStarter to discuss logistics for a workshop which GeekStarter was planning on holding at the University of Calgary for all of the Alberta iGEM teams. The list of requirements for the workshop were provided by Magda and were listed below:
- Room booking (lecture theatre for expert presentations and small rooms for breakout sessions)
- Catering (lunch, served buffet-style in the HRIC atrium)
- Printed signs and providing directions to the different rooms during the event (needed to know the number of signs and directions of arrows needed)
- Accommodating the final number of attendants (provided by Magda by May 23), as well as any dietary restrictions for the 50-70 planned attendees
- Help with snacks and coffee for the afternoon (which was not provided through catering company)
- No hotel rates for out-of-town guests could be arranged (as the number of attendees was not yet known)
Week 3: May 15 - May 19
We looked into sponsorship opportunities with Genome Alberta, based on past teams from Calgary receiving funding from the aforementioned organization. On May 18th, Michaela scheduled a meeting with Magda for May 30th at 2:00 PM (MST) in order to review and finalize the plan for the June 11th GeekStarter workshop. Michaela also contacted Lamiley Ludderodt , a caterer affiliated with the University of Calgary, regarding the catering for the workshop. Our team began to read into microbead policies, as we were aware that microbeads were being phased out of cosmetic products in Canada, and the option of biodegradable microbeads was not widely known to the public. We wondered how increased public awareness of the possibility of biodegradable microbeads would impact government action on what is seen in Canada as a serious ecological issue. A summary of our findings can be found on our human practices page, as well as under Week 6 of this journal.
References
Rose, C., Parker, A., Jefferson, B., & Cartmell, E. (2015). The Characterization of Feces and Urine: A Review of the Literature to Inform Advanced Treatment Technology. Critical Reviews In Environmental Science And Technology, 45(17), 1827-1879. http://dx.doi.org/10.1080/10643389.2014.1000761
