Difference between revisions of "Team:BABS UNSW Australia/Notebook"
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<h4>June 8-12 2015<h4> | <h4>June 8-12 2015<h4> | ||
<ul> | <ul> | ||
| − | We | + | We were all off this week to study for exams. Instead we spent our time doing iGEM. On Monday, we had a day long meeting. We managed to decide on the final parts of our project and plan how we were going to get them done. Following this, we started designing all the DNA parts, primers, and G-blocks we would need. Rhys worked on lactococcus signal peptides and anchoring domains, Mac worked on designing primers for synechocystis plasmids, Izzy designed the Tse2 extraction primers, the pseudoknot G-blocks - all with Daniel's help. We also updated our rapidly growing DNA parts/biobricks database. |
<h5><b>Wet lab:</b></h5> | <h5><b>Wet lab:</b></h5> | ||
| − | + | We cloned and miniprepped synechocystis addgene plasmids. However, this was not enough DNA to transform synechocystis so we grew more overnight cultures and did 5x minipreps for each plasmid. We also got ready to culture mammalian cells by ordering media and sourcing them within the school. | |
</ul> | </ul> | ||
| + | |||
| + | <h4>June 11-15 2015<h4> | ||
| + | <ul> | ||
| + | |||
| + | <h5><b>Wet lab:</b></h5> | ||
| + | This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped. | ||
| + | </ul> | ||
| + | |||
<h4>Inspiration</h4> | <h4>Inspiration</h4> | ||
Revision as of 05:13, 17 July 2015
Notebook
- recombinant dehalogenases
- dynamic gene editing
- type 3 secretion systems for protein delivery
- biophotovoltaic cells
- synthetic endosymbiotic relationships for therapeutic use (endosynbio!)
- invasin/listeriolysin
- colA/minC control system
- xenophagy
- Not different enough from past projects.
- Synechocystis issues such as long doubling time, transformation and lack of expression tools. Furthermore we lack a strong validation for why we want to use Synechocystis.
- It is unclear what we can reasonably achieve with the project as in its current form. If Synecho fails or is too slow we won't have much.
- invasion: Invasin and LLO ph induced expression. Improvement on previous systems as it responds to the environment and does not produce LLO unless it needs it. This system has been named phLOW. It will work in partner with the cre/lox safety switch.
- Safety/Kill switch: Lox sites flank the invasion genes with Cre downstream and under the influence of the same promoters. This should result in the invasion genes excision and subsequent degradation after they have been expressed to invade a cell. We may need to play around with slowing down the transcription via rare codon elements or pseudoknots. If possible we aim to make the kill switch be positively selected for, one suggestion is using an auxotrophic strain and amino acid synthesis on the plasmid. Another is transferring an essential gene from the chromosome to the plasmid.
- Population control: AHL mediated population density sensor that alters gene expression to produce an antimicrobial peptide. This would result in a dynamic, oscillating population density.
A brief summary of our trials, tribulations and fleeting moments of joy.
Late November - Early December 2014
- Following a lecture recruitment hustle by Rob, team members applied and were selected for the iGEM team. We all started reading up on synthetic biology and dreaming potential projects ranging from far-fetched to science fiction to milk.
17-19 February 2015
-
The team attended a three day bootcamp run by Rob, Daniel and Chris. We learned the basics of molecular biology techniques for the iGEM competition - cloning, enzyme digests, ligations and transformations. We participated in many sessions (some lead by team members) about other aspects of the competition - wiki design, primer design, actual design design. We also started presenting and discussing project ideas, and brainstorming ways to make each of them better.
2-6 March 2015
-
The uni semester starts, and we had our first meeting on the first day. We discussed communication strategies (online labbooks, OneNote vs Evernote) and role allocation. Most importantly, we started seriously talking about various projects ideas and their respective advantages and disadvantages.
Projects discussed included:
9-13 March 2015
-
Despite our 8am Monday morning meeting, we enthusiastically discussed a narrowed down list of projects, and set down pros and cons for each. After some discussion and a preferential vote, we settled on Endosynbio! Despite lying on the sci-fi end of the spectrum, we found some seemingly achievable goals to achieve with our project and decided it was worth the risk. The three parts as we decided were:
16-20 March 2015
-
At this Monday morning meeting, we discussed a wide range of topics. We started getting organised in terms of funding, outreach, wiki, team roles and labwork. We were all still spending a lot of time reading up and dreaming up project and outreach ideas.
We had a separate design meeting (Kris, Mac, Daniel) where several ideas, including the DNA rainbow serpent, were borne (and rapidly buried).
Not satisfied with our one-hour meetings, the team (minus advisors) planned to meet all day on saturday. It was surprisingly fun, productive, and everyone left feeling optimistic.
23-27 March 2015
-
Monday morning meeting: more project discussion. What symbionts/host cell lines should we use? Synechocystis seemed to be the favourite. We also decided on the importance of a very effective kill switch.
30 March-3 April 2015
-
Another early Monday morning meeting. Prior art searches showed significant similarity of our planned project to two previous iGEM projects (Warsaw 2009-2010). The main concern of this meeting was finding ways to differentiate our project from those, and make it novel. Ideas included a focus on population control within the cell and making the host reliant on the endosymbiont.
We finished writing our first grant application, for a grant offered by NSW Trade & Innovation.
We investigated flights to Boston in September. Australia is very far from the rest of civilisation, so this was important.
13-17 April 2015
-
A difficult week for the team, as we dealt with the loss of one our team members. The project took a hiatus as we mourned Ben. Words cannot express the shock and deep sadness we all felt. Though this wiki notebook seems like a strange forum to discuss such a tragic event, to gloss over it would be impossible. His absence was deeply felt. Several team members attended his funeral this week, and this further emphasised the tragedy of his passing. He was surrounded by loving family and friends. His ambition and passion for science and helping others has driven and informed the rest of our project. Rest in peace, Ben. Words on a wiki cannot do you justice, but I feel like you would appreciate it.
20-24 April 2015
-
We had our weekly Monday morning meeting, and a separate wiki and kill switch design meeting.
NEW! Wetlab:
We started lab work: making media (LB and SOC) and pouring agar plates. Also, the distribution kit arrived.27 April-May 1 2015
-
This was an assessment-heavy week for all of us, so not much progress was made. However, we managed to write and distribute our first monthly newsletter.
Wet lab:
This week consisted of familiarising ourselves with the lab and with using the iGEM distribution kit. Four BioBrick parts related to our project were resuspended and transformed into E. coli. All successful transformants were miniprepped to isolate their plasmid DNA, which was then quantified via NanoDrop/Qubit and visualised via PCR and electrophoresis. Stocks of LB and LB agar were prepared as well as TAE buffer for future gels. Our culture of Synechocystis has also been incubating this week, with significant green growth now evident.May 4-8
-
Many things starting to progress on the outreach front - collaboration discussions with the BIOMOD team, Izzy working on SmartSparrow programming and getting feedback from high school teacher surveys, and the conception of a UNSW-hosted Aussie iGEM meet up.
We were all starting to feel a bit lost with our project due to the lack of clear goals, and many problems becoming apparent.
For example:
Wet lab:
More competent DH5a cells were prepared (19x200 ul). The previous weeks PCR products (2.7P, 2.24B, 2.3I and 4.17F) were run on a gel, no bands were detected. The 10X TAE stock was used to produce a 1X solution that did not set. This was retested the following day and did set. The failed PCR was retried and ran once again on a gel, this time it worked! New parts were cloned, they include 1.30 (strong constitutive promoter, strong RBS), 2.20L (promoter activity reporter), 4.5E (AHL inducible H2O2 production), 1.4H (apple fragrance), 3.4K (lox site for Cre recombinase) and 2.3I (AHL). Of the plated samples 4 were successful and prepared for mini-prep. These were then mini-prepped and concentrations were read using the nanodrop. They were very low. These were cultured overnight and re-prepared. The following nanodrop yielded some great concentrations. Unfortunately once again no bands were registered on the gel, probably due to the use of the incorrect elution buffer. LB media (500ml) and LB agar (500ml) were prepared and the lab space was tidied.May 11-15
-
During our Monday morning meeting we discussed the results of our mega-meeting. The key goals of our project are now:
Wet lab:
We amplified the biobricks from the mini-preps from last week with PCR. These also ran very poorly on the gel. These minipreps were sent to the Ramachotti centre for sequencing to confirm part identity, but the signals from the sanger sequencing was also very poor.May 18-22 2015
Very, very busy assessment week for all. We even called off our weekly meeting.
Wet lab:
Our first 3A assembly was attempted this week. We digested a linerized backbone, 1:3O and 2:2OL and ligated them together.
May 25-29 2015
We discovered a recently-published paper very similar to our project (Lee, Tae J., et al. "A Power-Law Dependence of Bacterial Invasion on Mammalian Host Receptors." (2015): e1004203). After briefly panicking, we decided to view this as a positive - it helped us select cell lines, and assured us our research was topical. Parts we'd ordered from the iGEM registry were quarantined as they arrived in Australia. We were worried they'd be destroyed, but eventually they were passed on to us. The details of the Aussie meet up were firmed up, with other teams agreeing on a date.
Wet lab:
The synechocystis culture was subcultured this week! We transformed the ligated plasmid from last week, but no colonies were observed after 48 hours :( . Other ordered parts arrived from the registry, were plated and prepared for minipreps.
June 1-5 2015
Lots happening. We organised details of the gathering, started planning a UNSW debate about ethics in synthetic biology, and booked accommodation for several Boston nights. We ordered more parts from the registry,
Wet lab:
Lactococcus expression vectors arrived from Addgene and were plated on ampicillin plates in preparation for minipreps
M17 media was prepared.
Gel was run with various parts, including ligation reactions, PCR-amplified parts, and minipreps of registry parts. Still troubleshooting. We are looking for a new gel tank to see if that was an issue.
June 8-12 2015
We were all off this week to study for exams. Instead we spent our time doing iGEM. On Monday, we had a day long meeting. We managed to decide on the final parts of our project and plan how we were going to get them done. Following this, we started designing all the DNA parts, primers, and G-blocks we would need. Rhys worked on lactococcus signal peptides and anchoring domains, Mac worked on designing primers for synechocystis plasmids, Izzy designed the Tse2 extraction primers, the pseudoknot G-blocks - all with Daniel's help. We also updated our rapidly growing DNA parts/biobricks database.
Wet lab:
We cloned and miniprepped synechocystis addgene plasmids. However, this was not enough DNA to transform synechocystis so we grew more overnight cultures and did 5x minipreps for each plasmid. We also got ready to culture mammalian cells by ordering media and sourcing them within the school.
June 11-15 2015
Wet lab:
This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped.
Inspiration
-
Very, very busy assessment week for all. We even called off our weekly meeting.
Wet lab:
Our first 3A assembly was attempted this week. We digested a linerized backbone, 1:3O and 2:2OL and ligated them together.May 25-29 2015
We discovered a recently-published paper very similar to our project (Lee, Tae J., et al. "A Power-Law Dependence of Bacterial Invasion on Mammalian Host Receptors." (2015): e1004203). After briefly panicking, we decided to view this as a positive - it helped us select cell lines, and assured us our research was topical. Parts we'd ordered from the iGEM registry were quarantined as they arrived in Australia. We were worried they'd be destroyed, but eventually they were passed on to us. The details of the Aussie meet up were firmed up, with other teams agreeing on a date.
Wet lab:
The synechocystis culture was subcultured this week! We transformed the ligated plasmid from last week, but no colonies were observed after 48 hours :( . Other ordered parts arrived from the registry, were plated and prepared for minipreps.
June 1-5 2015
Lots happening. We organised details of the gathering, started planning a UNSW debate about ethics in synthetic biology, and booked accommodation for several Boston nights. We ordered more parts from the registry,
Wet lab:
Lactococcus expression vectors arrived from Addgene and were plated on ampicillin plates in preparation for minipreps
M17 media was prepared.
Gel was run with various parts, including ligation reactions, PCR-amplified parts, and minipreps of registry parts. Still troubleshooting. We are looking for a new gel tank to see if that was an issue.
June 8-12 2015
We were all off this week to study for exams. Instead we spent our time doing iGEM. On Monday, we had a day long meeting. We managed to decide on the final parts of our project and plan how we were going to get them done. Following this, we started designing all the DNA parts, primers, and G-blocks we would need. Rhys worked on lactococcus signal peptides and anchoring domains, Mac worked on designing primers for synechocystis plasmids, Izzy designed the Tse2 extraction primers, the pseudoknot G-blocks - all with Daniel's help. We also updated our rapidly growing DNA parts/biobricks database.
Wet lab:
We cloned and miniprepped synechocystis addgene plasmids. However, this was not enough DNA to transform synechocystis so we grew more overnight cultures and did 5x minipreps for each plasmid. We also got ready to culture mammalian cells by ordering media and sourcing them within the school.
June 11-15 2015
Wet lab:
This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped.
Inspiration
-
We discovered a recently-published paper very similar to our project (Lee, Tae J., et al. "A Power-Law Dependence of Bacterial Invasion on Mammalian Host Receptors." (2015): e1004203). After briefly panicking, we decided to view this as a positive - it helped us select cell lines, and assured us our research was topical. Parts we'd ordered from the iGEM registry were quarantined as they arrived in Australia. We were worried they'd be destroyed, but eventually they were passed on to us. The details of the Aussie meet up were firmed up, with other teams agreeing on a date.
Wet lab:
The synechocystis culture was subcultured this week! We transformed the ligated plasmid from last week, but no colonies were observed after 48 hours :( . Other ordered parts arrived from the registry, were plated and prepared for minipreps.June 1-5 2015
Lots happening. We organised details of the gathering, started planning a UNSW debate about ethics in synthetic biology, and booked accommodation for several Boston nights. We ordered more parts from the registry,
Wet lab:
Lactococcus expression vectors arrived from Addgene and were plated on ampicillin plates in preparation for minipreps
M17 media was prepared.
Gel was run with various parts, including ligation reactions, PCR-amplified parts, and minipreps of registry parts. Still troubleshooting. We are looking for a new gel tank to see if that was an issue.
June 8-12 2015
We were all off this week to study for exams. Instead we spent our time doing iGEM. On Monday, we had a day long meeting. We managed to decide on the final parts of our project and plan how we were going to get them done. Following this, we started designing all the DNA parts, primers, and G-blocks we would need. Rhys worked on lactococcus signal peptides and anchoring domains, Mac worked on designing primers for synechocystis plasmids, Izzy designed the Tse2 extraction primers, the pseudoknot G-blocks - all with Daniel's help. We also updated our rapidly growing DNA parts/biobricks database.
Wet lab:
We cloned and miniprepped synechocystis addgene plasmids. However, this was not enough DNA to transform synechocystis so we grew more overnight cultures and did 5x minipreps for each plasmid. We also got ready to culture mammalian cells by ordering media and sourcing them within the school.
June 11-15 2015
Wet lab:
This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped.
Inspiration
-
Lots happening. We organised details of the gathering, started planning a UNSW debate about ethics in synthetic biology, and booked accommodation for several Boston nights. We ordered more parts from the registry,
Wet lab:
Lactococcus expression vectors arrived from Addgene and were plated on ampicillin plates in preparation for minipreps M17 media was prepared. Gel was run with various parts, including ligation reactions, PCR-amplified parts, and minipreps of registry parts. Still troubleshooting. We are looking for a new gel tank to see if that was an issue.June 8-12 2015
We were all off this week to study for exams. Instead we spent our time doing iGEM. On Monday, we had a day long meeting. We managed to decide on the final parts of our project and plan how we were going to get them done. Following this, we started designing all the DNA parts, primers, and G-blocks we would need. Rhys worked on lactococcus signal peptides and anchoring domains, Mac worked on designing primers for synechocystis plasmids, Izzy designed the Tse2 extraction primers, the pseudoknot G-blocks - all with Daniel's help. We also updated our rapidly growing DNA parts/biobricks database.
Wet lab:
We cloned and miniprepped synechocystis addgene plasmids. However, this was not enough DNA to transform synechocystis so we grew more overnight cultures and did 5x minipreps for each plasmid. We also got ready to culture mammalian cells by ordering media and sourcing them within the school.
June 11-15 2015
Wet lab:
This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped.
Inspiration
-
We were all off this week to study for exams. Instead we spent our time doing iGEM. On Monday, we had a day long meeting. We managed to decide on the final parts of our project and plan how we were going to get them done. Following this, we started designing all the DNA parts, primers, and G-blocks we would need. Rhys worked on lactococcus signal peptides and anchoring domains, Mac worked on designing primers for synechocystis plasmids, Izzy designed the Tse2 extraction primers, the pseudoknot G-blocks - all with Daniel's help. We also updated our rapidly growing DNA parts/biobricks database.
Wet lab:
We cloned and miniprepped synechocystis addgene plasmids. However, this was not enough DNA to transform synechocystis so we grew more overnight cultures and did 5x minipreps for each plasmid. We also got ready to culture mammalian cells by ordering media and sourcing them within the school.June 11-15 2015
Wet lab:
This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped.
Inspiration
Wet lab:
This was the first week of exams, but we still managed to make progress in the lab. Several previously digested parts were ligated to begin assembling the pHlow plasmid. Transformations were were not successful. The next round of ligations week was also not successful - there was growth on the positive control, but none on the ligated plates. For the third round of digests/ligations, we used NEB buffer instead of CutSmart buffer for the digests and transformations were successful. Kill switch design was progressed, and the two plasmid toxin-anti-toxin system was decided on. Primers for extraction of Tse2 from composite part were received, and resuspended. Synechocystis PCC6803 was transformed with Addgene plasmids over 5 days. Because of the slow growth time, we will not know if it was successful for 2-3 weeks. Because of a stuff up, cells were not in log phase when transformed. Lactococcus was subcultured on new GM17 plates but no growth was observed. We received new Biobricks (our third shipment of extra bricks) and these were plated, cloned, and miniprepped.Inspiration
You can see what others teams have done to organize their notes: