Team:Newcastle/Notebook
Alternative Roots
Notebook
NOTEBOOK
Follow the Newcastle iGEM team on their journey
Day 1 -07/06/18
Started the day with introductory talks explaining iGem and the wiki, we then went onto look at previous competition entries. Jem Stach and Phil Wright spoke about their experience in the field, research interests and what makes a good competition entry.
Day 2 - 08/06/18
The day began with a talk from Jon Marles-Wright about the importance of human practices and the impacts of our projects in the real world. This was followed by Dana Ofiteru who taught us about mathematical modeling of biological systems. In the afternoon, plant biologist Max Kapralov discussed using synthetic biology to improve the efficiency of photosynthesis. Finally, Rachel Armstrong (professor of experimental architecture) discussed the potential of microorganism in waste management systems and energy production in the built environment. Rachel also outlined political and economic issues surrounding synthetic biology.
Day 3 - 11/06/18
We started the day with a talk from Angel Goni Moreno who discussed the principles of synthetic biology and provided guidance on the implementation of logic in Biology. The Masters students who are working on the Interlab study explained their role in the project and we brainstormed ideas. Following this, the Synthetic Biology Masters students discussed their individual projects involving modelling lab work. Each of the team gave a presentation on a previous competition entry that has won an award.
Day 4- 12/06/18
We started the day with a talk from Alice banks and Colette Whitfield. Followed by brainstorming with Tom, refining the original ideas with some extra. Then the marine biologist/Engineered talked to us about boat fouling. Finally we had a talk from Vas Andriotis who focused on plant biology (corn yield). Finishing with brainstorming.
Day 5- 13/06/18
The day began with a talk from Martyn Dade-Robertson who discussed the use of biotechnology in architecture and his research into pressure sensing E.coli. In the afternoon Harold Freemann led a discussion around the use of mathematical modelling and its uses in synthetic biology. Finally Alice Banks and Colette Whitfield covered current topics in synthetic biology and the equipment available to us in the laboratory.
Day 6- 14/06/18
This was our first day in which we were given freedom to explore our ideas and gain a necessary background surrounding the topics in order to refine into 6 ideas that will be pitched to research supervisors and the guest speakers who have given presentations since the project began.
Day 7- 15/06/18
We continued to explore our ideas for the majority of the day, narrowing our proposals down from ten ideas to six. We worked more as individuals, researching specific aspects of the proposals.
Day 8- 18/06/18
Today was more orientated around the ethical implications Synthetic Biology has. We started with a discussion with Dr. Ilke Turkendag, a lecturer in Law Innovation & Society. We discussed our six proposals and outlines possible ethical/legal issues which could arise. This was followed by an interesting talk from Simon Woods and Ken Taylor, they spoke about potential stakeholders and how we could go about talking/communicating with them. They also took each of our ideas and looked at the potential impacts our projects could have on the wider public. We then finished the day preparing for our six pitches on Tuesday. The OpenTrons OT-2 robot, which the Newcastle iGEM team won, was also delivered today1.
Day 9- 19/06/18
We pitched the six ideas that had been selected to the advisers and masters students. After receiving feedback and suggestions from the advisory team we narrowed it down to two ideas to be taken forward. Further we constructed a template for both idea presentations and allocated team members to each idea. Umar is now our least favorite team member :)
Day 10/11- 21/06/18
Following on from the 2 ideas that we chose to take forward, we spent these next 2 days researching further into these proposals. During our research, we took a holistic approach when evaluating our ideas and attempted to account for topics such as human practices, ethical implications and possible art and design ideas. We incorporated this into our 2 ideas which we then pitched to the supervisory team and Master's students. We also managed to successfully setup the OT-2 robot. The smiles speak for themselves...
Day 12- 22/06/18
This day was used to carefully consider both ideas that were pitched the previous day before voting on one to take forward. We started by summarizing each idea in 140 characters which highlighted some discrepancies in individual team members understanding of the ideas. After these discrepancies were rectified, the team spent some time considering each idea before voting on one to take up as our project. In the end, it was decided that the project involving engineering microbes to colonize plant roots and attract nitrogen-fixing bacteria was the best to take forward. We spent some time thinking about the modeling/human practices/lab work that may be involved in this project.
Day 13- 25/06/18
On our first official day of project-specific work, various tasks were allocated to team members for them to research. Some background reading was conducted on the topic, the team name was confirmed and some ideas for the team logo were suggested. Also, some ideas for a hydroponics system were discussed. With a rare bit of sunshine, we decided to work outdoors.
Week commencing: 25/06/18
On the Monday, members of the team were provided an overview of the plant growth facilities at Newcastle University by
Vas Andriotis who has shown great interest in our project since its pitch and has given many resources to enhance
and develop our ideas. The remainder of the group began on the research for the several experiments that will be ran in
parallel over the Summer.
Tuesday was dedicated to further fleshing out our research by identifying species which would be suitable for our iGEM project.
Some members moved in an artistic direction and produced a handful of potential logos before the entire team voted on our two
favorites *put logos here*. Once these were decided, merchandising began development.
Development on our hydroponics system moved into full swing on the Wednesday, with our 2 engineers beginning work on the general
structure of the system which has been influenced by conversations with plant biologists within the university; working once again
with Vas in order to understand his needs from the system.
Thursday presented our team with the incredible opportunity to speak with Steven Burgess who is currently performing plant research
at the University of Illinois in order to discuss our project and identify potential for sharing our work among the wider
scientific community.
Members of the Biology and Engineering teams went to meet with Dr Montero-Calasanz; a Microbiologist whom specialises in plant-microbe
interactions. This gave a valuable opportunity to identify potential protocols which would relate to our chassis organism. The week was
rounded off by talks from Alice Banks and Collette Whitfield concerning sponsorship and outreach.
Week commencing: 02/07/18
The team focused on preparation for the wetware projects by ordering the Pseudomonas chassis, in addition to various free-living nitrogen-fixing bacteria. A variety of flavinoid chemoattractants were also ordered in order to reduce the wait time before lab work begins. The human practices and outreach team continued getting their work by in touch with potential sponsors. Some of the team members with prior experience in programming began working with the OT-2 to design a heat-shock protocol for E. coli using the python-based language within the OpenTrons API. The whole team received inductions for the Cat2 lab so that we can begin the wetware side of the project and follow all the safety protocols ans rules and regulations set out by the University for safe lab use. After receiving the lab inductions, we prepared the lab equipment for testing Arabidopsis germination so that we can begin testing the following week.
Week commencing: 09/07/18
The ball started to roll this week, we managed to get access to the labs and plant our first set of seeds in agar as well as preparing competent E. coli cells. We began the process of styling our wiki using bootstraps and designing the poster for the European Meetup. Towards the end of the week we attended a modeling workshop, ran by Synthetic Biology Masters students, this gave us an insight into modelling and tips we can apply to our modeling work. The team was also lucky enough to recieve goodiebags of Eppendorf We had previously decided that the naringenin flavonoid would most likely be the most successful, therefore looked into utilising parts from the naringenin operon previously used by TU Darmstadt 2014 iGEM team. We also Skyped Daniel Sachs, a member of the TU Darmstadt iGEM team to hear his advice on what worked well with the operon and with which promoters. Finally, we codon optimised one of the gblocks for the operon (TAL) and ordered all the segments with their corresponding RBS’s from IDT.2.
Week commencing 16/07/2018
This week was full of action. The architects in the team 3D printed a cold deck for the OpenTrons OT-2 and laser cut accompanying trays to hold test tubes. Now that we have the cold deck we can manufacture trays to hold lots of different apparatus increasing the number and complexity of experiments we can carry out on the OpenTrons. In addition, we had a meeting with Martyn Dade-Robertson from the School of Architecture, Planning & Landscape; he spoke to us about the importance of finding a context in which to situate our human practices, once we have this it will provide the design parameters necessary to progress. Finally, stencils were made for the 'Alternative Roots' logo enabling us to tag any hardware we build.
Some of the team also travelled to Munich this week for the European iGEM meetup. Those who attended throughly enjoyed
liasing with other iGEM teams as well as the event organisers and speakers. The team's poster was on display at the
meetup which gathered some interest around our project as well as many questions and suggestions for improvement which
have been duly noted by the team for further consideration. Potential collaborations were also sought after in Munich.
Lab work for the chemotaxis assays commenced with trial runs using the DH5α E. coli control in order to test
the protocol. It was noted from these attempt that the concentration of naringenin was too high as the plates
did not show signs of growth for 2 days; after which, signs of growth were minimal.
The concentration was diluted to 100µM and 50µM for future trials to see if this would overcome the issue. We also Designed forward and reverse primers for each of the four segments, for detection that the operon had been transformed.
Week commencing 23/07/2018
The long-awaited Pseudomonas fluroescens arrived this week,having come all the way from Germany,
meaning our iGEM team could start preparation for the transformation process. The team worked with
Dr Montero-Calasanz to make initial agar and liquid cultures in both LB and TS mediums and now
have enough bacteria to start the initial transformation experiments.
This week the some of the parts for the hardware arrived meaning we could get underway with the
construction of the hydroponics. First the basic circuit for the LED lights was created, the code
we used to control these lights was an adaptation of a freely available library (FastLED) which can
be found online. We made minor adjustments to suit our requirements, for example the
light intensity was increased. Our micro-controller (Arduino), was then loaded with
this edited code. This device will be responsible for monitoring and maintaining our entire
system. We faced issues with timing the lights for a 16 hour day and 8 hour night cycle.
We decided to use a ‘count’ within a loop to control when the LED’s need to be turned off.
This meant we had to calibrate for the time a single loop took and multiply that up for
16 hours. We are currently waiting for some parts to arrive so that we can begin construction of an
automated switching circuit.
The week was rounded off with a presentation to the stakeholders in the project, such as the PI's
and advisers, to provide an update on how the project has progressed thus far. The presentation
also offered a valuable opportunity to highlight areas of the project that needed to be strengthened
and was a good opportunity to develop our presentation skills prior to the Jamboree in Boston.
Week commencing 30/07/2018
This week we managed to get off campus and talk to potential stakeholders. We were lucky enough to arrange an interview with GrowModule 365. As a company, they produce shipping containers that contain hydroponics systems. There concept adopts a new approach to farming, allowing crop production in unconventional places. The visit allowed us to see how traditional farming is being challenged and gain an insight into hydroponics systems. We gained lots of useful information from the meeting; we learned which wavelengths are most effective for increased yields, the alternative growing mediums available, the markets perception to innovative farming and which control parameters are most important. Paul Brown (Director) gave us lots of useful contacts within the industry and seemed very passionate about our project, however he did mention some areas of the market may be sceptical when it comes to eating produce that has been grown using genetically engineered microbes.
Week commencing 06/08/2018
This week we started to work on a video for the opening of the presentation in Boston. We wanted to continue with the theme portrayed in a previous 'teaser' we produced. The hydroponics switching circuit was finalised by the engineers, this controlled the day and night cycle to simulate specific environments for the seeds. The Hydroponics system aesthetics were also worked on, we applied spray paint and stencils to make it more visually appealing. At the end of the week MMatt Burridge (One of our masters students) conducted a workshop to teach people how to write and run protocols on the OOpentrons OT-2 robot.
In the lab we: amplified the backbone of pSBS1C3 through thermocycling using primers for the 2kb sequence required for assembly and this allowed the plasmid to be linearized and prepare for later insertion of the gblocks for the naringenin operon,
ran this PCR product using agarose gel electrophoresis to check for separation of the backbone required from the rest of the plasmid, purified the PCR product and quantified the DNA concentration thus making the backbone ready for Gibson Assembly.
Towards the end of the week amplified the positive control for Gibson assembly practice using the NEBuilder HiFi DNA Assembly Cloning Kit.
Week commencing 13/08/2018
We were in the final stages of building the hydroponics system this week, we have determined optimal wavelengths to run the system at. Chris 3D printed a series of trays for seeds to fit in. We were also lucky enough to have a meeting with Chris Tapsell From KWS (the 4th largest seed compay in the world). We outlined our project and discussed the issues surrounding GM crops and how the industry and wider public respond to GM use. Chris seemed to be passionate about the project and we intend to stay in contact with each other.
In the lab wade competent DH5α E. coli cells for Gibson transformation through the use of repeated centrifugations and the reagents MgCl2, CaCl2 and Glycerol.
We also transformed these chemically competent cells with the positive control from Gibson assembly and found that the Gibson Assembly method was successful, as the positive control grew on ampicillin plates due to the DH5α cells taking up the positive control overlapping dsDNA fragments containing ampicillin resistance. No growth was observed on the negative control plates with un- transformed DH5α cells, as they are not naturally resistant to ampicillin.
Acknowledgements
1. Credit: Dr. Alice Banks
2. Credit: Joshua Loh