Team:Macquarie Australia/Notebook

NOTEBOOK




Dry Lab
  • First team meeting was led by Ari Edmonds (2017 and 2018 Macquarie University iGEM team member), introducing synthetic biology and how the competition works. We met our advisors from the Department of Molecular Sciences and met Abigail Sison from iGEM HQ
  • Professor Robert Willows discussed theoretical aspects of H2 biosensors and NiFe hydrogenases in E. coli which guided us in researching and reading literature
  • We set up team accounts for Gmail, Facebook, Instagram and Twitter. We also brainstormed possible team names
  • Eli and Sam brainstormed ways we can address human practices through a prototype
Wet Lab
  • Anika, Daniel, Jerresa, Tasneem and Zoe prepared LB CAM and LB AMP plates. We transformed DH5ɑ with Plac, cyclic di-GMP phosphodiesterase, double terminator, RBS (parts from the 2019 iGEM distribution kit) in an attempt to build a component of our biosensor while we waited for synthesised parts to arrive
Dry Lab
  • As a team, we discussed logo ideas and potential contacts who can help to design and digitise our ideas.
  • Alice, Carly and Eli drafted emails for human practices stakeholders, industry experts and sponsorship
  • Anika, Carly, Daniel, Tasneem and Zoe brainstormed activity ideas for Macquarie University Open Day and developed an online survey based on which was shared to our social media pages
  • Jerresa, Josip, Sam, Tasneem and Zoe researched methods for detecting cyclic-di-GMP.
  • Each team member drafted their bios for the wiki.
Wet Lab
  • Transformations of Plac, cyclic di-GMP phosphodiesterase, double terminator, RBS we performed last week was unsuccessful. Anika, Daniel, Jerresa, Tasneem and Zoe reattempted these transformations.
  • We attempted to make our own competent E. coli cells.
Dry Lab
  • Everyone researched factors which may affect the functioning of cyclic-di-GMP phosphodiesterase.
  • Eli and Sam received feedback from A/Prof Louise Brown regarding their prototype design,
  • Alice and Tasneem received feedback from A/Prof Louise Brown regarding emails for human practices and sponsorship document
  • Carly, Jerresa, Josip and Sam summarised the literature we read and highlighted important points and protocols we could use for our parts
  • Ari showed us how to plan part construction using standard assembly and 3A assembly.
Wet Lab
  • We learned how to prepare an agarose gel, use the NanoDrop and Gel Visualiser
  • Anika, Daniel, Jerresa, Tasneem and Zoe miniprepped the plasmids containing Plac, cyclic di-GMP phosphodiesterase, double terminator, RBS then determined DNA concentration using the NanoDrop. We digested samples with EcoRI, PstI and XbaI then ran digests on a 1 % agarose gel.
  • Anika, Daniel, Jerresa, Tasneem and Zoe completed standard assembly for Plac and RBS
  • Anika, Daniel, Jerresa, Tasneem and Zoe completed 3A assembly for cyclic di-GMP phosphodiesterase, double terminator and TET backbone
  • After digestion, we ran samples in a 1 % agarose gel and annotated bands
Dry Lab
  • Alice finalised our sponsorship document and emails for human practices and contacted 35+ companies and organisations
  • We attended the Department of Molecular Sciences Masters of Research poster session and interacted with postgraduate research students and their supervisors. We noted what layouts and designs made posters stand out, ideas for our Jamboree poster
  • Jerresa and Tasneem contacted Team Edinburgh UG and Team NU_Kazakhstan for a possible collaboration as we are all working with hydrogen producing microbes
  • Received our hydrogenase gblock from IDT so we started planning for digestion and ligation reactions
  • Alice, Carly, Eli, Jerresa, Tasneem and Zoe attended the CSIRO ON Prime Finals at the Sydney Startup Hub to support the Macquarie Biohydrogen group
  • We started preparing a presentation for our advisors summarising the work completed during our intensive month long iGEM bootcamp
  • We received replies from Bondi Bioworks and Bioplatforms Australia, organised meetings for next week
  • Alice and Eli attended a meeting with Andrew Gilbert from Bioplatforms Australia to discuss our project
  • James Brown from Bondi Bioworks visited the team on campus to discuss entrepreneurship in synthetic biology and his expertise in the field
Wet Lab
  • No colonies from our standard assembly and 3A assembly plates
  • Digested hydrogenase gblock and KAN backbone with EcoRI and PstI, ligated then transformed. Colonies on transformed plates were inoculated to prepare liquid cultures
  • Anika, Daniel, Eli, Jerresa, Tasneem, Sam and Zoe carried out miniprep of the phosphodiesterase, followed by digestion and ran samples on an agarose gel and SDS-PAGE
Dry Lab
  • First week of semester!
  • Sam consulted our advisor, Russel, for planning our biofilm formation assay with the phosphodiesterase. Daniel, Jerresa and Sam also worked with our advisor, Dr Edward Moh to compare protocols for biofilm formation assays found in literature. We considered
  • parameters such as culture duration, dilution, media, incubation periods, wash steps and stains
  • We received a reply from Fire + Rescue NSW. Alice arranged a meeting with Paul Beylerian (Fire + Rescue NSW Scientific Officer) who will visit us on campus for an interview. We started preparing questions to ask and researched background information on Fire + Rescue NSW
  • We had our first weekly meeting with our advisors. Alice, Anika, Carly, Daniel, Eli, Josip, Sam and Tasneem presented a slideshow with our accomplishments throughout the iGEM bootcamp
  • We received our riboswitch with and without the terminator stem loop (RON and ROFF)
  • Alice and Eli met with Andrew Gilbert from Bioplatforms Australia to discuss our project
Wet Lab
  • Jerresa and Sam ran an SDS-PAGE of YHJH in Lac and YHJH in KAN
  • Sam prepared 5 liquid cultures of yhjh for the preliminary biofilm assay
  • Started assembly of our riboswitch
Dry Lab
  • Our advisor, Dr Paul Jaschke, ordered the hydrogenase component of our biosensor from Twist
  • Alice contacted various high schools who may be interested in hearing about our project
  • Alice, Zoe, A/Prof Louise Brown and Sam King attended an interview with Paul Beylerian from Fire + Rescue NSW. We developed a better understanding of the limitations of current gas detectors used by firefighters
  • We received our first silver sponsorship from Labcyte!
  • Jerresa presented the weekly update presentation to our advisors
  • Hannah joined our iGEM team! She is completing a Masters of Research and plans to help us with laboratory work and writing literature reviews for the wiki.
Wet Lab
  • Sam checked the preliminary biofilm assay for the yhjh part and performed the wash step with acetic acid and OD readings.
  • Anika transformed the ligated RON and ROFF plasmids in DH5α though plates had no colonies.
  • Carly, Daniel, Eli, Josip, Sam, Tasneem and Zoe reattempted the transformation of RON and ROFF
  • Two biofilm assays were prepared. One plate will be incubated at 28 °C, the other incubated at room temperature (kept in the drawer).
  • Jerresa prepared overnight cultures of RON and ROFF transformations. Anika, Jerresa, Daniel, Tasneem and Zoe miniprepped these transformations which was followed by digestion. Jerresa and Tasneem ran an agarose gel of the digested RON and ROFF samples.
Dry Lab
  • Alice, Carly, Daniel, Jerresa, Tasneem and Zoe have volunteered to represent our team at the Macquarie University Open Day. We started discussing ideas about engaging ways to interact with visitors and prospective students relating to our project.
  • With the help of our advisor, Dr Roy Walker, we contacted Team Edinburgh for a possible collaboration as they are using the 2017 Macquarie iGEM team’s construct.
  • Carly, Josip and Tasneem participated in a Skype call with Team NTU Singapore to discuss a possible collaboration. For the past 3 years, Macquarie Australia and NTU Singapore have collaborated by performing qPCR and SDS-PAGE for parts. We decided it would be great to continue this collaborative relationship.
  • Alice started preparing presentations for high school visits
  • Carly finalised our iGEM poster for the Macquarie University Open Day
  • The team presented the weekly update presentation to our advisors
  • Alice and Jerresa attended an interview with Macquarie University PhD recipient, Dr Vanessa Pirotta. Dr Vanessa is experienced in science communication and has a strong passion for sharing science with non-scientific audiences. She discussed how we can engage with our social media followers, gave advice on creating presentations and taught us ways to communicate complex information in inspiring and relatable terms
  • Alice drafted a second round of emails for human practices and sponsorships based on the feedback given by Dr Vanessa Pirotta
  • Alice, Ari, Carly, Daniel, Jerresa, Tasneem and Zoe represented the team at the Macquarie University Open Day. Our stall was located next to the teaching laboratories where the tours ended. This gave us the opportunity to educate tour groups about iGEM and how we plan to utilise synthetic biology to address issues. Ari demonstrated a hydrogen fuel cell toy car and we encouraged visitors to complete our short survey
Wet Lab
  • Daniel, Tasneem and Zoe repeated the agarose gel due to diffusion of the samples and distorted bands
  • Josip and Sam prepared digestion of the enzymes, PstI, SpeI, XbaI and EcoRI to check if they were still usable. Jerresa and Zoe ran a 1 % agarose gel of the digests
  • Sam prepared the next biofilm formation assay.
Dry Lab
  • We took headshots for team introductions on our social media pages
  • The team presented the weekly update presentation to our advisors
  • We were getting inconsistent results from our agarose gels. After consultation with Professor Robert Willows, we concluded that it may be due to the activity of our enzymes. Thi decided to order new enzymes (EcoRI, PstI, XbaI, SpeI, NdeI)
Wet Lab
  • Carly, Eli and Hannah digested the riboswitch with EcoRI (single) and PstI (double). Eli and Zoe ran an agarose gel of the digests. The gel did not appear to run properly (agarose didn’t melt, samples were not loaded properly) so it was repeated
  • Sam took the OD readings of last week’s biofilm formation assay and followed suggestions from our advisor, Dominic, regarding the wash step and measurement
Dry Lab
  • We received replies from Hazer Group and Maximator, organised phone call interviews for next week
  • Carly continued planning a collaboration with Team NTU Singapore
  • Carly, Jerresa and Ari participated in a Skype call with Team NU_Kazahkstan to discuss our projects and possible ways we could collaborate
  • We received a platinum sponsorship from Bioplatforms Australia and a bronze sponsorship the Macquarie University Student Representative Committee!
  • Our advisor, Samuel King, organised a human practices meeting with Alice, Anika, Carly and Daniel to discuss ways to address the medal criteria
  • Alice, Daniel, Jerresa and Zoe presented our project to high school students at Brigidine College, St Ives
  • Alice and Jerresa volunteered to represent the team at the Synthetic Biology Australasia Conference in Brisbane in October. They started planning flights, accommodation and registration with the guidance of A/Prof Louise Brown, Anwar Sunna and Louise Burton (CSIRO)
  • Josip ordered riboswitches with inducible promoters (lac and tac)
  • The team presented the weekly update presentation to our advisors
Wet Lab
  • We made observations for RON and ROFF plates
  • According to a paper found by Sam, E. coli Nissle 1917 is more efficient in producing biofilms compared to DH5α. We obtained these cells from our advisors, Dr Paul Jaschke and Dominic Logel, which were used to make competent cells
  • We decided to rebuild the cyclic-di-GMP phosphodiesterase plasmid
Dry Lab
  • Selected photos from the Brigidine school presentation to upload to our social media pages
  • Jerresa, Sam and Zoe presented the weekly update presentation to our advisors
  • Daniel started designing our team shirts and received feedback from our advisors
  • Ari, Daniel, Jerresa, Sam and Zoe participated in a photoshoot for the Faculty of Science and Engineering
  • We were approached by Team UAAAN to collaborate and planned a video call for next week
  • Alice started drafting the abstract for the wiki and Synthetic Biology Australasia Conference
Wet Lab
  • Hannah retransformed the PDE in Nissle 1917 with the following changes to our protocol
  • We sent sent RON and ROFF samples for sequencing
  • Hannah performed miniprep of PDE and miniprep of yhjh KAN and CAM
  • Hannah also helped prepare overnight liquid culture for cyclic-di-GMP and RON CAM and ROFF CAM
  • With the guidance of Professor Robert Willows, we prepared a preliminary GFP fluorescence assay using our riboswitch. Unfortunately, when we tested variables (growth media, temperature), the plate dried out and results were highly variable.
    Aleq
Dry Lab
  • Received our hydrogenase (Hyd 1A and 1B) and riboswitches with promoters. Josip started planning assembly of these parts
  • Carly, Tasneem and Sam presented the weekly update presentation to our advisors. During our meeting, we finalised the SBA and wiki abstract with feedback from advisors
  • Alice, Carly, Sam, Eli, Tasneem, Zoe, Hannah, Jerresa and Josip discussed gel image from 09/09 with Thi and Ed. When overnight cultures were made, two colonies may have been selected or incomplete digestion. Alternatively, colonies were not transformants and possible contamination from the miniprep kit.
  • Ed, Thi, Carly and Jerresa participated in a video call with NTU Singapore. We hope to send our riboswitch after sequencing confirmation so they can perform qPCR. They will send us a cell lysate and nuclear pellets for protein expression and SDS-PAGE
  • Carly and Jerresa participated in a Skype call with Team UAAAN to discuss possible ways we could collaborate
  • Carly, Jerresa, A/Prof Louise Brown, Prof Robert Willows and Macquarie biohydrogen group participated in a phone call interview with Mark Edwards (Hazer Group)
  • Jerresa and A/Prof Louise Brown submitted the team roster, abstract and title, safety form and track selection
  • Received a reply from USYD for collaboration, arranging a Skype call for next Monday
  • Alice prepared questions for the BOC interview which Carly sent and confirmed our meeting next Monday
Wet Lab
  • Josip and Tasneem used the NanoDrop to find DNA concentration of yhjh, RON CAM, ROFF CAM, cyclic-di-GMP minipreps from 06/09
  • Daniel performed an agarose gel to check the hydrogenase and cyclic-di-GMP
  • Hannah, Josip and Zoe prepared an assay for our riboswitch with different salt concentrations
  • Josip prepared liquid culture from transformant colonies from TACON, TACOFF, LACON, LAC OFF plates
Dry Lab
  • Alice, Carly, Jerresa, Sam, Zoe, Professor Robert Willows, Samuel King, Dr Tony Jerkovic attended a meeting with Chris Dolman from BOC, Australia's leading gas & safety equipment specialist
  • Eli, Josip, Jerresa, Thi and Dr Paul Jaschke planned for Gibson Assembly of hydrogenase (Twist)
  • Alice, Carly, Eli and Jerresa worked on prototype design and feedback from interviews
  • Carly and Jerresa participated in video call with Team Sydney Australia. We decided the best way to collaborate would be performing an SDS-PAGE for one of their proteins, and in return, replicate one of our GFP assays. We organised a time to exchange samples
  • Carly and Jerresa also arranged a video call with Team UAAAN for next week
  • Anika worked on the UNSW Symposium slideshow
  • Alice, Carly and Jerresa worked on content for the wiki
  • Carly and Tasneem presented the weekly update presentation to our advisors. We also did a team selection process for Boston where members pitched their contributions, team achievements and what they hope to achieve from attending the Jamboree. Carly, Eli, Jerresa, Sam, Tasneem and Zoe applied.
Wet Lab
  • Josip and Hannah performed digestion of Hyd 1A and 1B, cyclic-di-GMP using enzyme E for single digestion and a combination of E and N for the double digestion
  • Hannah prepared competent cells of Nissle and DH5α
  • Daniel and Anika performed digestion of Hyd and cyclic-di-GMP PDE
  • Eli conducted a Gibson Assembly with the hydrogenase fragments and KAN backbone, which was transformed by Tasneem in DH5α
  • Anika, Daniel, Sam and Tasneem restreaked colonies from Hydrogenase and CDG plates plus positive and negative controls
  • Anika, Daniel, Hannah and Zoe made 2 agarose gels for Tac ON/OFF; Lac ON/OFF, Hydrogenase digests and CDG PCR, W and W/O digests and annotated both gels
  • Jerresa prepared samples prepared for sequencing (CDG W KAN 3, CDG W/O CAM 2, Lac ROFF 1, Lac ROFF 2, Lac RON 1, Lac RON 2, Lac RON 4
  • Jerresa and Sam transformed PDE with DH5α (commercial and homemade)
  • Sam performed a Polymerase Chain Reaction (PCR) for the CAM backbone, KAN backbone and colony.
  • Carly, Jerresa and Zoe made LB media and plates (LB CAM and LB KAN)
  • Eli and Tasneem performed retransformation of Hyd 1A and Hyd 1B
  • Sam made Congo Red plates for the spot assay
  • Sam prepared a Congo red spot plate
  • Hannah prepared glycerol stocks of Lac ROFF red colonies CAM, Lac ROFF green colonies CAM, RON CAM 1 DH5α, ROFF CAM 1 DH5α
  • Hannah, Jerresa, Sam GFP Assay of Constitutive and Inducible Riboswitches with LB
Dry Lab
  • The team presented the weekly update presentation to our advisors
  • Carly and Jerresa visited Team Sydney Australia at their campus to exchange samples for our collaborations
  • Daniel worked on our shirt design
  • Jerresa drafted content for the wiki
Wet Lab
  • We started using our new Qiaprep spin miniprep kit (250). Miniprep and digestion of PDE + RON/OFF
  • Anika performed a digestion of the hydrogenase and Tasneem ran an agarose gel of the digests SDS-PAGE of hydrogenase
  • Hannah and Zoe prepared glycerol stocks for LacON KAN 2, LacOFF KAN 2, RON CAM, TacOFF KAN 3, TacON KAN 1, LacOFF KAN 1, LacON KAN 1, TacOFF KAN 2
  • Josip prepared sequencing reactions for CDG W, CDG W/O, Hyd 1A, PDE, RON, ROFF, LACON, PDE + RON CAM 2, PDE + RON CAM 4, PDE + ROFF CAM 1
  • Hannah prepared overnight cultures for competent cells
  • Jerresa, Hannah, Russel prepared a GFP assay for the inducible and constitutive riboswitches with salt concentrations
Dry Lab
  • Alice and Jerresa presented the weekly update presentation to our advisors and practiced their presentation for the Synthetic Biology Australasia Conference
  • Alice and Jerresa continued working on the SBA presentation with feedback from Associate Professor Louise Brown
  • Alice worked on content for the wiki
  • Eli, Josip, Sam, Tasneem, Thi, Dr Edward Moh and Professor Robert Willows discussed the spot assay results
Wet Lab
  • Josip transformed Hyd A + B colonies 4 and 7, CDG W and CDG W/O, Nissle CDG W KAN, Nissle CDG W/O CAM, DH5α A+B CAM 4, DH5α A+B CAM 7. BL21 (DE3) cells were used for transformation of Team Sydney Australia’s PsiD gene.
  • Anika, Daniel, Eli, Tasneem and Zoe performed an SDS-PAGE of the transformed PsiD (whole cell lysate, soluble fraction and pellet). Gel was repeated by Hannah
  • Sam prepared a biofilm formation assay to test the yhjh (with and without the promoter and terminator) in DH5α and Nissle 1917, yhjh + RON/ROFF in DH5α and Nissle 1917
  • Anika and Zoe performed agarose gel of digests and 3xGibson assembly
  • Jerresa prepared a GFP assay for the yhjh + RON/ROFF samples
  • Hannah and Josip miniprepped the CDG and yhjh
  • Jerresa transformed PDE + RON/ROFF CAM with Nissle 1917
Dry Lab
  • We assigned team members to each wiki page and descriptions for the special prizes on the judging form
  • Everyone worked on content for the wiki and descriptions for the judging form
  • The team presented the weekly update presentation to our advisors
Wet Lab
  • Jerresa prepared a GFP assay for yhjh + RON/ROFF with sucrose
Dry Lab
  • Alice and Jerresa travelled to Brisbane to attend the Synthetic Biology Australasia Conference and present our project to the synthetic biology community
  • Eli, Sam, Tasneem and Zoe worked on the wiki and judging form
  • Hannah wrote a report for the hydrogenase part
  • Jerresa presented the weekly advisors meeting. We received feedback on how to write our results and content for the wiki
  • All team members worked hard to finalise and upload content to the wiki and registry pages
Wet Lab
  • Sam and Tasneem redid the Congo red plates and Biofilm assay
  • Daniel performed SDS-PAGE for the Team NTU Singapore collaboration
  • We assembled the small subunit, large subunit, protease and phosphodiesterase via 3A assembly.
Dry Lab
  • All team members continued to work hard to finalise and upload content to the wiki and registry pages. Go Team HyDRA!
Wet Lab
  • Hannah and Jerresa performed an agarose gel for sequence confirmed parts. The last gel for iGEM 2019!