Team:Newcastle/Notebook/Operon
Alternative Roots
Naringenin Operon Biosynthesis Notebook
NOTEBOOK
Follow the Newcastle iGEM team on their journey
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