Team:Calgary/Journal
Journal
Week 1: May 1 - May 5
Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt.
Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea.
Week 2: May 8 - May 12
Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei.
Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.
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 |
Lorem ipsum dolor sit amet, pro menandri efficiendi in, eam ne omnium sapientem definitionem, choro scriptorem cum in. Duo choro placerat ne, ex ullum aliquip disputando per. Mutat laudem sea eu, ad quas labores theophrastus vim. Erant exerci laoreet vel ex, te his mucius consulatu consequat. Mea no option erroribus, nominati scribentur has et, pro at dicat dicit constituam. Affert doctus nam ut, novum homero indoctum vel in, elit regione virtute per te. Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.
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 what 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. Because our cells are so problematic, work has not yet begun with our actual secretion construct from IDT. Lalit and Kaitlin prepared samples for gel electrophoresis and all of the necessary supplies that will be needed on Lalit’s trip to Winston Churchill High School on Monday, June 12, 2017, where he will present to Grade 11 Students about synthetic biology, practice gel loading, and perform strawberry DNA extraction experiments with them. After long discussions and consultations with experts and industry we decided that applying our project to creating PHB in space, ie. on future Mars colonizations, would be the best choice and would make the most sense for our 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 specific 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 found out about some 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 E.coli DH5α cells from Dr. Dong and transformed them for the interlab study and finally, there was successful transformation and colony growth of GFP cells after overnight incubation. This supports the theory that there was in fact something wrong with the cells that we were previously using. Therefore, for our work throughout the summer we will continue using these 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 psb1c3 with RFP insert and psb1c3 with GFP insert 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, by the end of week. This indicates that these enzymes are functional and can be used on our IDT parts. Also, 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 extracted GFP inserts to the extracted RFP backbones and the extracted RFP inserts were ligated to the extracted GFP backbones. As we expected, bacteria transformed with the GFP inserts-RFP backbone grew green colonies and bacteria transformed with RFP inserts-GFP backbone grew red colonies. This indicates that the restriction enzymes and ligase were functioning properly and they 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 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 |
Then we transformed our ligated backbones and parts into our chemically competent E. coli DH5α cells.
Week 10 (July 3-July 7, 2017)
We performed plasmid miniprep on the colonies of E. coli DH5α cells transformed with our ligated backbones/parts last week for digest confirmation on a 1% agarose gel. 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 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, as can be seen in the image below , where there is 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, which could possibly have our insert properly ligated into the pET29B backbone.
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. So, we transformed the psB1c3 with phasin-HlyA into BL21, which we will use for protein expression. Also, in order to begin do 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 and troubleshooting PHB production 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 DH5 Alpha. 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 pET backbone from an LMP gel, and ligated with our digested SPII part. Ligation occurred on friday, and thus 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 DH5 Alpha, 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 DH5 Alpha with a biobrick from imperial that he will use to compare to the part from Tokyo that he used previously. In a new vein, Sam and Kaitlin began transforming DH5 Alpha 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 DH5 Alpha 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 DH5 Alpha cells that will hopefully last the rest of the summer. These will be tested next week to ensure their competency.
Lorem ipsum dolor sit amet, pro menandri efficiendi in, eam ne omnium sapientem definitionem, choro scriptorem cum in. Duo choro placerat ne, ex ullum aliquip disputando per. Mutat laudem sea eu, ad quas labores theophrastus vim. Erant exerci laoreet vel ex, te his mucius consulatu consequat. Mea no option erroribus, nominati scribentur has et, pro at dicat dicit constituam. Affert doctus nam ut, novum homero indoctum vel in, elit regione virtute per te. Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.
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’).
Lorem ipsum dolor sit amet, pro menandri efficiendi in, eam ne omnium sapientem definitionem, choro scriptorem cum in. Duo choro placerat ne, ex ullum aliquip disputando per. Mutat laudem sea eu, ad quas labores theophrastus vim. Erant exerci laoreet vel ex, te his mucius consulatu consequat. Mea no option erroribus, nominati scribentur has et, pro at dicat dicit constituam. Affert doctus nam ut, novum homero indoctum vel in, elit regione virtute per te. Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.
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
