Team:SECA NZ/Notebook
Notebook
Beginning Weeks (November 2016 to Week 18, 2017)
Prior to the 19th week of 2017 our team has spent a significant amount of energy outside of the wet lab work that makes up the majority of the iGEM competition. This ranged from important decisions such as our name to the selection of team members and project ideas. that being said there are some important points to note.
Being a new team to, and university, to the iGEM competition the finding of lab space in which we could work was a massive effort carried out mostly by our team leader Peter. Ultimately we were able to secure space in Professor Joanna Putterill's lab, where we can make mess, progress and hopefully a model organism.
Building upon the contacts created by the UoA iGEM 2016 team we were able to present the basic idea of our project to the Biomatters Ltd group. They provided four of us with invaluable feedback and funding which will allow us to start the lab work ASAP. This was our first expedition into the 'real-world' and the photo below captures our excitement!
Week 19 (8-14th May)
This Friday saw the beginning of our official lab work. With the aid of Geoffrey Thomson, a PhD student in Prof Joanna Putteril's lab, we constructed and ordered the DAIRIP4 gene for gateway plant expression. This involved the addition of attB1 and attB2 sequences to each end of the gene for insertion into the pDONAR221 vector. Furthermore we optimized 6 codons for synthesis within plants.
We have been given a place in the Papakura High School which we can use to run our lab plan. From here we need to provide an official, and fleshed out, plan to the school and set up a time which doesn't clash with our lectures.
Registration is still in Progress, there is some paper work required from our PI Shaun Lott. Our Finance head Alastair will follow this up. Otherwise the drive for funding continues with applications to many different organisations, including Crown Research Institutes where we have been passed between many people with no definite responses.
Finally, the Chiasma MOU with regards to our account, funds and changing over to next years team has been finalized and needs to be sent to the Chiasma team to review.
Week 20 (15-21st May)
Initial development complete!. Finished generating the reverse transcript genes with the added his tag and pelB fragment overlaps for the gibson reaction. From these generated the PCR primers for gene amplification. As all genes have the same insertion location and overlap, the same primers can be used for all of them. Also generated PCR primers for the pET20 vector which would eliminate the MCS.
Talked to Shaun. Lab does not have a stock of pET20b+ vectors, but does have pET22b+. Functionally different only in that pET22b+ has a lacI region. His tag and pelB localisation sequence is identical, so constructs are compatible.
Finished adjusting the genes for sequencing. Had to change a few of the codons fro reduce GC content, and change the codons in the his tag due to inability to synthesis 6 repeat sequences in a row. Added on an additional overlap to the his tag end to act as the homologous region for the Gibson’s reaction now that the his tag is not identical to the one included in the vector. However, we cannot claim free DNA from IDT until registration is finalised.
Our Finance head Alastair has been officially recognized by Chiasma and can now authorize payments directly!!
Otherwise the funding is progressing very slowly with organisations such as the Tertiary Education Fund operating outside our scope. Furthermore, Plant and Food Research can't give us assistance without the go ahead from higher powers so looks like we are back to the drawing board.
Week 21 (22-28th May)
With our plant genes ordered we are treading water before the lab work proper can begin. In other teams nothing huge has happened as we keep moving forwards with various funding and community outreach attempts.
Week 22 (29th May- 4th June)
Jess, Alastair and Judith completed the Plant Containment course required for entry into the PC2 Plant Hall under instruction from Davide Zazzaro. This enables us to start working with transgenic plants for all of our future experiments
Our gene DAIRIP4 has arrived, this was re-suspended and placed in the freezer for us by Geoffrey.
Using our new access we have been able to Plant Arabidopsis!!! The procedure for this was shown to us by Andrew (one of Jo's students), and we managed to plant two trays of wild-type Arabidopsis. This involved the sterilization of seeds using bleach and cold treatment for 3 days to promote germination.
The seeds (Arabidopsis seeds are super tiny!) were planted in Rockwool - the preferred planting media for Arabidosis. Our sowing number: Q523, Wt Columbia, 01/06/17, JC and JG. To successfully plant we removed seeds from the tube by swirling with a toothpick. Then using a second tooth pick, collect one seed at a time and place onto the surface of the rockwool. Place two seeds for each hole, in-case one does not germinate. At a later date seedlings will be thinned. Finally, we made a germination tent which acts to increase the humidity to promote fast germination. Construct with clear plastic and kebab sticks.
With the study break beginning and exams looming not much has been happening outside the burgeoning lab work. Each team is slowly working towards their end goals.
Week 23 (5-11th June)
While the rest of the iGEM team is on break Judith, and the lab team, have been hard at work taking our lab work to the next stage.
At this point that progress basically involves watering our plants and waiting for them to grow!
Outside lab work the iGEM team is on mostly hold as we take a well deserved rest.
Week 24 (12-18th June)
This week we had the first major growth of our seedlings, in fact they had grown so much we had to remove some. This is called thinning and allows the successful growth which should happen very soon.
Outside lab work the iGEM team is on mostly hold as we take a well deserved rest.
Week 25 (19-25th June)
Began seed sterilization at 10am with Andrew (A student in Jo's Lab). Together we sterilized enough seeds for three blocks, in case we have issues during transformation and require more plants. However only a couple of healthy plants are required for transformation, so the spares will be used as experimental controls.
Outside lab work the iGEM team is on mostly hold as we take a well deserved rest.
Week 26 (26th June- 2nd July)
Sowed a new batch of seeds from those sterilized last week. Overall we planted out three trays of Arabidopsis. These were registered under the following details: 'Sowing 2: Q567, Columbia, 260617, JG/JC'
At the end of this week we were able to collect our primers. These can be suspended in TE buffer (10 mM Tris, 0.1mM EDTA, pH 8.0) or Nuclease free water. In the end we choose to go with Nuclease free water. Spun down the tubes to ensure no DNA had been dislodged from the pellet and could be lost.
Outside lab work the iGEM team is on mostly hold as we take a well deserved rest.
Week 27 (3-9th July)
Removed the germination tent from the seedlings, watered plants and trimmer the flower heads.
Began the Gateway Cloning Protocol, combining our pDONOR221 vector with the DaIRIP4 fragment. With the help of Lulu, we successfully completed the Gateway Cloning Protocol, combining our pDONOR221 vector with the DaIRIP4 fragment. Success was verified by growth under selection and colony PCR.
On this gel we were to ran three samples:
- DaIRIP4 fragment, 744 bp long.
- pDONOR221, 4762 bp
- pB2GW7, 10882
The high mass ladder, loaded in the second well has bands corrisponding to 10 kb, 6kb, 4kb, 3kb, 2kb and 1kb in decending order. The pDONOR221, loaded in the second well sits just above the 4kb band. This corresponds well to its exected length, and as there is not evidence of small bands, we can be safe in the assumption that the vector has not degraded.
pB2GW7 can be seen to sit just above the the 10kb marker, which also corresponds well to its effected length. There is a second band, located at 10kb which is unexpected, but compared to the main band it is at an extremely low concentration.
The gene band, highlighted by the blue arrow, is very faint, which is expected condisering the loss of material during loading. However, is does fall just below the 800bp line on the low mass dna ladder, which proves the the fragment is likley the size we ordered.
Week 28 (10-16th July)
This week we grew up, purified and checked our successful DONOR+DaIRIP4 plasmids. Purification was carried out with a Miniprep kit, and we ensured the insert was present and of the correct size using a restriction digest. We also began our plant experiments – individual leaves were placed in water for the electrolyte leakage assay, and whole plants placed in darkened, sealed containers for the whole plant survival. The appropriate samples were then exposed to room temperature of 4 degrees Celsius for 24 hours. The plants were monitored for survival following treatment. Preformed the second gateway reaction with pB2GW7 to produce our final plasmid construct.
Outside lab work the iGEM team is on mostly hold as we take a well deserved rest.
Week 29 (17-23rd July)
Moved on to protein prep work. Talked to Jason (from Shaun Lott's Lab) about next steps, and found we need to produce a new batch of pET22b + plasmid for recombination.
As there was no pet22b+ plasmid purified, we need to grow some more up from a frozen glycerol stock of bacteria. Bacteria do not like to be freeze/thawed, so we took a pipette tip and scraped some of the top layer of the frozen tube and plated those. Plate was a standard ampicillin plate.
In the plant area we have suffered a set-back following an email from IDT stating that our gene specific primers were missing in transport, and would likely be further delayed. As a result we were forced to use Geoffrey's non-gene specific primers for our plasmid so we can proceed with transformations next week and be relatively sure our insert is present.
Outside lab work the iGEM team is on mostly hold as we take a well deserved rest.
Week 30 (24th-30th July)
This week saw us transform our Agrobacterium tumefaciens in preparation for the Arabidopsis transformation. Using two plasmids we carried out four separate electroporation transformations (Two for each). With one plasmid we experienced significant amounts of arcing, suggesting the plasmid could contain too much salt for successful transformation.
After incubating our Agrobacterium colonies for four days we preformed a DNA extraction to check the plasmid levels. The DNA concentrations were as follows:
- Colony 1: 26.7ng/uL
- Colony 2: 87.45 ng/uL
- Colony 3: 99.800 ng/uL
- Colony 4: 40.550 ng/uL
To check our Agrobacterium strains carried the correct insert we used electroporation to introduce the extracted DNA from Colonies 2 & 3 back into E.coli which were incubated for future PCR experiments. Note: Due to the low DNA conc Colonies 1&4 were discarded.
This marks the last week of our inter-semester break so we should be back at work next week
Week 31 (31st July- 6th August)
We have filled our final Lab position with Sneha joining the team, welcome and good luck! Otherwise the lab work is continuing in all areas.
In preparation for transformation on Thursday our Arabidopsis plants were trimmed back, removing extra flowers
To check our plasmids carried the correct insert we preformed a standard 35-cycle PCR upon the transformed E.coli strains. The resulting DNA was then run on a 1% agarose gel, and reassured we moved to the transformation of Arabidopsis.
The transformation of Arabidopsis is a complicated procedure, for more info refer to our experiment page. After preparing the media required we grew a pre-inoculum of our transformed Agrobacterium overnight. The next morning we used Strain 3 to inoculate our transformation media, discarding strain 2 due to a lack of growth. On Thursday (Day 3 of the procedure) we carried out the actual transformation, using the vacuum assisted floral dip method we transformed 10 separate plants.
The Lab guide for our trip to Papakura High School has been finalized by the Community engagement team and sent away to be approved by the High School. A big Thank you to Saajidah and Miquela for getting this done.
Not a huge amount to report this week. We have identified a potential source in the Vice Chancellor Student Support Fund which Alastair applied for this week. Otherwise we just keep sending out emails hoping for a response.
Week 32 (7-13th August)
It's getting closer and closer to the end of iGEM 2017!! This week our meeting produced a rough calendar of important dates coming up, and its getting busy. Otherwise not a huge amount happened this week as we all get ready for the big push to come.
Still trucking along. This week our plants have been left to grow so we are focusing on the expression of proteins in E.coli, with a large amount of planning occurring between our lab heads and the PIs.
Week 33 (14-20th August)
Plants is rolling forward nicely, the little guys are growing up and we will soon be ready to begin. Sadly most of the E. coli work is still on hold as we sort our the lab spaces. However we are able to check our vectors in preparation for future transformations.
Using the pET vector prepared on Thursday 27-07-2017 we set up some PCR reactions to check the inserts. The amplified pET22b + linear PCR fragments were run on a 1% agarose gel for 35min at 90V. Found there to be multiple bands with a background smear, after talking to the experts (Ass. Prof. Shaun Lott) we decided the annealing temperature too low and hence non-specific.
To find the correct annealing temperature we used a gradient PCR machine to check annealing between 55-62 degrees celsius. The results are shown below with a different annealing temperature- ranging from tube 1 at 55C, to tube 12 at 62C. The wells containing PCR temperatures of 60.7C, 61.4C, and 61.8C (10, 11 and 12 respectively) showed single bands, indicating successful reactions had occurred.
Recruitment drive is still going strong, messages to the course coordinators is a successful method of gathering interest. As a result we have up to 10 people to interview over the coming week.
The synapse event was a broad success leading to contacts and new people being introduced. A new fund for entrepreneurial students has been released which would be perfect for our group! A new member, Hadley, has significant connections to funding and is extremely keen to take the team forward over the next few weeks and even into next year.
Week 34 (21-27th August)
Finally sorted out our importation documents so the DNA kit is on its way! Sadly we will need to resynthesis the gene for submission but that shouldn't be a long term issue. In the lab we have experienced the Dark Days of bad Gels!! None of our reaction temperatures seem to work, but a fix should soon be in sight.
First contact with Onehunga High School where we will hopefully present to a group of keen biology students, fingers crossed!! Otherwise the plan for our big engagement event is progressing smoothly, heres hoping everything goes well.
Excitement this week!! We have been awarded the Vice Chancellor Student Support Fund to get us over to the Giant Jamboree. Therefore a big thank you to the University of Auckland, without your support we wouldn't be able to do what we do. More Mundane matters has led to the development of a lab payment method, our supervisors will be happy!!
Week 35 (August 28 - September 3rd)
Lab work is mostly on hold this week with most of our team members focussing on Uni tests and assignments. However, even while university work calls the iGEM project must continue with the completion of our Abstract and finalised title. Welcome to project "Frozen in Thyme"
Two major pieces of news this week. The first is the announcement by SBA (see below) which allows our team member, Alastair Harris, to travel to Sydney later this month! We are extremely excited by this opportunity to attend an international iGEM Meetup and look forward to engaging with all the teams who attend this meeting; more info to follow.
Excitement this week for both the community engagement and finance teams with word coming from the Synthetic Biology Australia conference. They have announced the support of a New Zealand iGEM trip to the SBA conference to Sydney on the 21st of September. A big Thank You to the SBA Conference for their support!! In other news we have registered our third team member for the Giant Jamboree and we edge closer to the ultimate goal of flights and accommodation for Boston 2017.
Week 36 (4-10 September)
This week we have continued to trouble shoot our Gibson preparation PCR of the pet22b vector and daIRIP gene. We have confirmed bands at the expected length, but it appears some purification will be required due to a second smaller band (See Gel below). Hopefully next week we will get into the E.coli model.
This week sees our first major contact with high school students!! Its been a long time coming but we were extremely excited to visit the Papakura High School. A practical and informal lab was run at the school, in a basic microbiology lab format. The students swabbed various items such as phones, chairs and tables and transferred them to Petri dishes for incubation. We also discussed the various reasons why microbiology was important relating them back to things like Saccharomyces cerevisiae used in brewery, winery and bakery. After the lab work the students were encouraged to stay and discuss the different pathways into science and general university life.
While it's not really engagement with the Community this weekend marks the first BBQ of the 'Summer' (although some may argue it is actually Spring!). Some of the iGEM team got together to share some stories and enjoy the sun.
With university exams and assignments finally at an end (mostly!) this week turned into a bit of a rush for the Finance Team. The final pitch document for presentation to Return on Science was due so all of our team has been hard at working putting the facts together. Late on Wednesday afternoon however, the document was complete so bring on the presentation next week!! In other news one of our Lab Leaders, Jess Chase, has been awarded another travel to attend the SBA conference in a couple of weeks, so thank-you once again to the team at SBA!
Week 37 (11-17th September )
This week we began by harvesting both out transformed and wild type seeds. This involved releasing the seeds from their siliquas, and filtering out the chaff. On Tuesday we sterilised approximate 10000 seeds of the transformed line, which were sown out to undergo selection on Friday. The seeds were sown onto two blocks in rows using pipettes.
This week shows a massive burst of Community work going on across the board. On Monday two of our team, Judith and Alastair, made a trip to Onehunga High School to talk about the ethics of GMOs compared to Selective Breeding. The presentation went fantastically with the students asking some insightful questions about the future impacts of GMOs in the environment. In fact it went so well the H.O.D of Biology has invited us back to present to another class. Friday marked the second lab with Papakura High School students. We showed them the gross yet cool things they had grown on their plates.(insert the pictures I forwarded to you). We ran through some of the common bacteria and fungi and identified some of the major groups the students had grown. (maybe insert a picture of a plate with the yellow bacterial fungi as they were Streptococcus aureus). After we had finished with our lab work we were able to facilitate a fantastic discussion about the practical importance of microbiology and the work we had done. The students were very engaging and developed some interesting ideas around the future of microbiology, and the science industry in New Zealand.
EXCITEMENT!! The finance team has been hard at work this week and as a result we have finally raised enough money to make it to Boston. A big thank you to the team at NZ Association of Consulting Laboratories who donated a substantial amount to cover the final hurdle towards flights. Now all we need is enough money for accommodation and to pay back our lab supplies. However, no need to worry with the Pitch to Return on Science on Thursday (Lead by or Captain Peter) going extremely well we will hopefully have reached our goals for this year. Fingers crossed.
Week 38 (18-24th September)
Our seeds for selection had germinated over the weekend, and we continued to water them and slowly remove the germination tent to reduce the humidity, and prepare them for their first round of selection at 7 days old. The tDNA construct with which we transformed the DaIRIP gene into the plants also contains the gene for BASTA resistance, so it is this herbicide which we will be using to select for the transformants. We mixed up a 120mg/L solution of BASTA spray, and went over the rows of seedlings twice to ensure good coverage. This will be repeated on 10 and 13 days. Also this week we again attempted to PCR amplify our pET22b+ vector fragment and DaIRIP for protein expression, to no avail.
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The final financial hurdle has been achieved with funding coming in from the Return on Sciences committee to the tune of $3,750. With this final burst we have reached our goal for the year and are able to relax on the finance side and enjoy the fireworks. We would like to take this chance to say a big thank you to all our sponsors and most especially to the finance team who have put in some many hours making our iGEM dream a reality! Next stop is Boston, Finance out.
Week 39 (25th September - 1st October)
Observed die off of large quantities of seedlings, as expected. Some promising candidates observed, but still some areas which appear more like they did not receive sufficient spray for selection on Monday. Second and Third sprayings were completed, and some definitive survivors in the fields of death have emerged. Following another PCR attempt with further adjusted annealing temperatures and times, we have decided to move forward with the vector product which contained a minor band, with the assumption any recombination with this fragment would not confer antibiotic resistance when transformed into E.coli. Completed Gibson reactions for both the DaIRIP/pET22b+ and DaIRIP/(Insert the back vector name here.) Sterilised wild type Columbia seeds for sowing next week.
Wrapping Up (October)
The last weeks in our lab have been a very hectic time, pushing to get the biobricks completed in time for the deadline! Essentially we've been doing rounds of digestion and then ligations. Following that we've been transforming, one round of electroshock into dh5alpha, and three of heat shock into b221 because the electroshock arced and our Biobrick lead Jess Chase was worried that the high concentration would make it an ongoing problem in later rounds. The bacteria have then been plated onto CAM plates. Each time they were grown overnight before checking if anything grew (answer was no). Each time we used our digestion/ligation product and (separately) the biobricks provided in kit plate 2, wells 1o and 17d
After weeks of desperate struggle however we finally managed to send our brick to the iGEM headquarters!!! Lab work done.
To round out the year we went back to where it all began and gave a presentation to Biomatters to practice for the Boston event. It was great to show the company all we had managed to achieve throughout the year, which couldn't have been done without their initial support. The presentation also gave us valuable feedback and allowed Biomatters to meet the beginnings of next years iGEM team!
