Week Commencing - 16/07/2018

This week we started our trials for the qualitative chemotaxis assay (v1) on 0.75% LB agar. This was done with Echerichia coli in order to practice technique and identify flaws in the protocol. We did not observe any evidence of chemotactic behaviour, which was as we expected. However, we did also note that the growth rate of the E. coli was substantially slower on the plates containing naringenin than the control. As such, we decreased the naringenin concentration from 100µM to 50µM

We also spent this week identifying other potential avenues through which we could demonstrate chemotaxis.

Week Commencing - 23/07/2018

With help from Dr. Maria Del Carmen Montero-Calasanz, we isolated our Pseudomonas fluorescens and Azospirillum brasilense from freeze-dried pellets and successfully produced multiple agar and liquid cultures. After allowing the colonies on these plates to grow to sufficient size, we produced 30% glycerol stocks for E. coli,P. fluorescens, and A. brasilense.

Week Commencing - 30/07/2018

After obtaining experience in the previous weeks, we performed the qualitative chemotaxis assay again but with A. brasilense. However, the results did not indicate chemotactic behaviour which was not expected. We hypothesised that this was either due to the naringenin concentration originally being too low after the previous reduction or that it was unable to diffuse through the agar with ease and as such the concentration was even further reduced.

We also isolated the other two nitogen fixing bacteria (Azorhizobium caulinodans and Herbaspirillum seropedicae) from freeze-dried pellets and prepared to repeat chemotaxis assays with these species

Week Commencing - 06/08/2018

This week we decided to characterise the behaviour of our bacteria on LB agar. We recorded the colony size after 24 and 48 hours of incubation at their recommended optimal temperatures as well as recording the colony morphology. This included the margin, border, elevation and pigmentation of the colony. Results of this characterisation can be found here: link to be added.

Week Commencing - 13/08/18

We produced dose-response kill curves for E. coli and A. brasilense for naringenin concentrations between 0 and 150μM, increasing by 10μM intervals. These kill curves were produced by observing changes in absorbance at 600nm over 24 hours by using a plate reader.

During this week we also performed the semi-qualitative chemotaxis assay (link) on 0.5% LB agar with our 3 nitrogen fixing bacteria and E. coli. However, results once again did not indicate positive chemotaxis. After this, we attempted the quantitative capillary-based chemotaxis assay for all bacteria species. This method is based on the premise that if a capillary contains a chemoattractant and the end of said capillary is submerged in bacterial solution then the bacteria will move into the capillary at a higher rate than a species that is not attracted.

The results of this experiment were largely unexpected as no agar colonies grew from the capillary contents for 4 species, suggesting no movement at all (even in the controls). Herbaspirillum seropedicae was the only species to show movement into the capillary, however, the difference between the control and naringenin capillaries was not significant (p>0.05).

Week Commencing - 20/08/2018

After multiple issues with many assays not showing any evidence of chemotaxis, we decided that future repeats should include malate as a positive control due to the literature also indicating that this is a chemoattractant for our nitrogen-fixing bacteria [1-3].

While doing said repeats, we also noticed that on lower percentage agars, the 'halo' around the H. seropedicae colony was more constricted on plates containing higher concentrations of naringenin (>100μM). After researching this, we found that naringenin can repress chemotaxis genes at higher concentrations which may provide a reason as to why we have not seen positive chemotaxis in this species.

Week Commencing - 27/08/2018

Thanks to our sponsors ibidi, Cells in Focus, we were able to obtain specialised chemotaxis micrscopy slides. The μ-slideIII 3-in-1 chemotaxis slide allows for real time visualisation of bacterial movement in response to controllable chemical gradients. This equipment will be highly valuable to the Alternative Roots project as we use it to verify results as well as provide visuals to help improve our understanding. We spent this week familiarising ourselves with this product so that we could start using it in a timely manner.

During this week we also began to grow our nitrogen-fixing bacteria and E. coli on Minimal A Salt agar in preparation for the semi-quantitative chemotaxis assay as we were not sure if the agar could support growth of all our bacteria.

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 and 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 we made 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.

Week commencing 20/08/2018

The week started with a visit to Scotland to meet the Edinburgh iGEM team. The visit was extremely useful as it allowed us to share our progress with Edinburgh and vice versa and we also gave each other feedback on our respective projects. Additionally, we discussed the potential for collaborations, ate lots of pizza and even got to see some of the shows in the Edinburgh Fringe festival. We thoroughly enjoyed our time in Edinburgh and meeting the Edinburgh team, who were very hospitable and we hope to maintain a close relationship with Edinburgh iGEM moving forward.
Two members of the team, Will and Umar, also travelled to London to meet with Richard Ballard, co-founder of Growing Underground. We were shown around their tunnels of hydroponics which grow approximately 33 metres below the busy streets of Clapham. Growing Underground sustainably grow fresh micro greens and salad leaves, which are unaffected by weather and seasonal changes, before providing these to wholesalers and local restaurants such as M&S and Ocado. The meeting was very productive, we discussed how our project may be beneficial to the company and, also, how the company can help our project develop. We discussed the project at length and considered how the project might be refined to better meet the needs and concerns of a company such as Growing Underground. We hope to continue working with Richard and the Growing Underground team as we continue to develop the project.
This week in the lab, we resuspended the gblocks and primers from IDT. We also calculated the volumes of pSB1C3 and gblock inserts required for specific concentrations in the Gibson assembly and conducted a Gibson Assembly using the NEBuilder HiFi DNA Assembly Reaction Protocol. We then transformed the Gibson Assembly using the NEBuilder HiFi DNA Assembly Protocol, and then spread the reaction mixture onto chloramphenicol selection plates for overnight incubation. There was no colony growth on any of the plates therefore transformation was unsuccessful.

Week commencing 27/08/2018

On the chemotaxis side of things, we spent the week producing kill curves for each of our bacteria as to determine whether our concentration of naringenin was having an adverse effect on the growth of our bacteria. This was as we knew naringenin possessed antimicrobial properties thus it was important to work out if this was the reason that we found chemorepulsion for E. coli instead of no effect. During this week we also secured a sponsorship from ibidi who we are happy to say provided the team with their specialised μ-slideIII 3-in-1 chemotaxis slide which allowed us to visualise cell movement in response to naringenin via timelapse videos. It was also during this week that we reinvented our chemotaxis assay to be more in line with the work of Reyes-Darias et al. (2014) by reducing our agar percentage even further and utilising large, square plates in order to run multiple replicates and test distances on a single plate.
We also conducted a second attempt of Gibson with re – calculated values for the inserts. This was transformed as before and there was no growth on the plates. As this was thought to be due to the backbone concentration being far too low after purification, another PCR to linearize the backbone was conducted. This was ran on a gel and quantified to have a much higher concentration.

Week commencing 03/09/2018

This week we carried out our reinvented chemotaxis assays that involved using minimal agar on square plates, and setting up a concentration gradient of naringenin, and obtained results which showed positive chemotaxis of Azospirillum brasilens towards 100uM narinegenin at distances between 0.5cm and 2.5cm! The most successful result was when the bacteria was at a distance of 1.5cm, so we will carry out repeats of this distance.
A third Gibson attempt with the new backbone and the gblocks was done. Following transformation this reaction yielded 2 colonies showing that the plasmid with chloramphenicol resistance had been taken up. In order to see whether the rest of the operon had been integrated into the plasmid, colony PCR and plasmid miniprep was conducted on both colonies. However only the plasmid had been taken up without the operon.

Week commencing 10/09/2018

This week we were able to successfully transform Pseudomonas fluorescens with a gentamicin resistance gene by electroporation.
We also tested methods of sterilising plant tissue, such as using different concentrations of ethanol and bleach. Seeds and seedlings were inoculated with wild type P. fluorescens.
Performed square plate assays with Herbaspirillum seropedicae and Azorhizobium caulinodans, but results were inconclusive as colonies grew too large and overlapped. Repeats will be performed with bacteria inoculated at greater distances from each other.
As wiki freeze draws closer, we are trying to develop more pages of our wiki and keep on top of it to hopefully lighten the load later when the deadline becomes imminent.

Acknowledgements

1. Credit: Dr. Alice Banks
2. Credit: Joshua Loh