Team:SECA NZ/Results
Our Results
What we were able to achieve
Key Results
- We produced a transgenic plant which we showed contained our insert, DaIRIP4
- We tested this plant and the insert was protective at freezing and below temperatures
- An unexpected result occurred with the insert also providing a suspected change in membrane fluidity at room temperature
Creating a Transgenic Plant
The first step in our process was to ligate our synthesised fragment of DaIRIP4 into the donor gateway vector DONOR221.
The restriction digest experiment displayed in Fig1 was designed to cut once in a gene specific cut site, and once in the vector (if the multiple cloning site had been removed). The resultant number of bands would reveal the presence (or lack) of the insert.
Fortunately, as is shown, our insert was found to be present and of the correct length following digestion.
Once we had purified our recombinant donor plasmid, this was in turn ligated into the destination plasmid, pBW2G7, which carries a BASTA resistant cassette. Correct insertion was again checked by colony PCR and enzymatic degradation.
Our finalised plasmid was transformed into agrobacterium, which were then in turn used to transform Arabidopsis thaliana flowerheads.
The resultant seeds were screened using BASTA spray, and as can be seen in Fig2. we have successfully produced transgenic plants.
This process was fraught with difficulty. It is testament to utter dedication and tireless work of the lab team that even this point in the project was reached.
Fig1. PCR showing the restriction digest check of our purified DONOR221+DaIRIP4 stocks. As shown by the red arrows, there are only two bands of the expected sizes for our chosen cut sites, with the far lane showing native DONOR221 treated with the same enzymes.
Fig2. shows the few transgenic plants growing after BASTA screening, indicating the presence of the transgenic insert.
Showing the Transgenic Plants operate better than controls
An electrolyte leakage assay was performed to assess the protective qualities of the DaIRIP4 insertion. The percentage of electrolytes lost was used as a proxy for overall cellular damage.
For this assay the five healthiest of our DaIRIP4 transgenics, and three wild type controls were tested in 1 of 3 conditions; 25oC, 0oC and -20oC.
As each sample represented a separate transgenic plant, with differing insertion sites, it was expected that each plant would perform differently due to different expression and localisation of the DaIRIP4 protein in vivo.
For the -20oC trials, a clear divide between our transgenic plants developed. Transgenics A and B displayed an electrolyte leakage almost half that of the control plants, while plants C to E experienced losses similar to that of the controls.
From these results, it can be speculated that plants A and B have a more optimal level of DaIRIP4 expression compared to C-E, and so these will be the strains which we continue to characterise. The plants in questions have had their flowers staked and bagged ready for seeding, and we look forward to the harvesting of our first fully transgenic crop and starting a DaIRIP4 line.
Fig3. The percent electrolyte loss from the DaIRIP4 transgenic plants vs control at 25oC. DaIRIP4 protein offers clear protection to this condition compared to the wild type Arabidopsis.
Fig4. he percent electrolyte loss from the DaIRIP4 transgenic plants vs control at 0oC. The transgenic plants experienced similar electrolyte losses to 25oC, while the controls plants sufferred smaller losses. DaIRIP4 plants still out performed wild type.
Fig5. The percent electrolyte loss from the DaIRIP4 transgenic plants vs control at -20oC. Transgenics A and B showing greatly decreased damage compared to controls.
Of interest was that the the control plants experienced the greatest electrolyte loss (used as a proxy for cellular damage) when immersed in the room temperature water. Our transgenic plants did not experience this loss, showing DaIRIP4 offers some protection from warm temperature damage or membrane permeability changes.
We were able to make a transgenic plant that worked as intended.
Our Contribution
A review of our contribution to the BioBrick Registry
Parts Characterised:
BBa_K567018 - submitted by iGEM11_SJTU-BioX-Shanghai in 2011.
BBa_K880002 - submitted byiGEM12_Michigan in 2012
Fig1. shows no growth :( One of the many lessons we have learned this year is that science doesn’t always go as intended.
Information on our experiments
Each plasmid was provided to our team in distribution kit plate 2 (wells 1O and 17D), as an insert in pSB1C3.
The first round of testing we transformed DH5𝛼 by heat shock using 2 µL of resuspended plasmid (200-300 pg/µL) as recommended on the iGEM website.
Bacteria were plated on chloramphenicol plates (30 µg/mL) that had been previously prepared.
These were incubated overnight at 37°C.
When no growth was observed, a second transformation was attempted.
This was done using the remaining 8µL of DNA and plated on freshly made 25 µg/mL CAM plates on the suspicion that the previous plates contained a concentration of antibiotic that was too high for the expression of resistance conferred by the pSB1C3 backbone.
Once again, no growth was observed.
Hypotheses as to why these experiments did not produce the results required
- The CAM resistance provided by the pSB1C3 plasmid was either not expressed, or was too weak for the concentration of antibiotic that we used.
- Our kit plate samples may have been left at room temperature or dry for too long and thus the DNA did not suspend/transform appropriately.
- Particularly for sample BBa_K880002, other factors may have been needed and the particular method that we used to test the activity was inappropriate as we did not understand the functionality of the part.
We hope to learn from the experiments conducted this year so that future New Zealand teams make a positive impact on the registry.
Our Attributions
We would like to thank all of these people and organisations for their support
People
Associate Professor Shaun Lott
Shaun has been involved with the team for the last two years. Without his administrative and laboratory support, this project would have never gotten off the ground.
Professor Jo Putterill
Without Jo’s scientific guidance and lab space this project would have never occurred. Her vast knowledge of plant biology were vital to the lab team’s success.
Other Academics
We have recieved a tremendous amount of support from other academics around SBS and the Faculty of Science. We’d like to acknowledge Dr. Davide, Prof. Allan, Dr. Rattenbury, Dr. Lear, Dr. Domigan, Prof. Gerrard and all the other academics who have influenced our project in a positive way.
2016 UoA iGEM Students
Last year’s iGEM undergrad (now postgrad) and PhD students like Kyle, Frankie, Jess, and David, among many others, have given an enormous amount to the team in administrative, moral, and technical support.
Members of Jo’s and Shaun’s Labs
Jason, Lulu, Mau, Geoffrey, Davide, Andrew, and Wendy, have been invaluable to our lab team sparing their time and experience to guide us through the research process. A special thanks must be extended to them.
Everyone Else
Over this year there have been far too many people to thank. To those who helped us in major ways and those who have helped in ways more subtle. You know who you are. Thank you.
Organisations
The University of Auckland Faculty of Science and School of Biological Sciences
With their generous financial support we were able to register to the iGEM competition.
Biomatters - Creators of Genious
With their continued financial support and their kind offer of Genious Software licenses, we were able to make an early start on the lab work.
Chiasma
With their financial oversight and guidance we were able to find connections and set up systems that will be invaluable to future teams.
UniServices’ Return on Science committee and AgriTech committee
With their generous financial support and business advice we were able to complete our lab work and plan for the future of our project.
Staff of the New Zealand Association of Consulting Laboratories
With their generous financial support we were able to fund the final portion of our project and make it to Boston.
Synthetic Biology Australasia
With their generous support, two of our students were able to attend the SBA conference in Australia and the Australasia iGEM meetup.
Other
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