Team:Victoria Australia/Notebook

- Team meeting in which we discussed ideas of how we are going to get the biological lighting system to work

- Discussed potential sponsors and other fundraising ideas

- Team meeting discussed fundraising ideas – a silent auction cocktail was an idea

- Came up with two systems to light the biological lighting system:

- Cell free systems

- Blue tongue orbivirus system

- Team meeting brainstormed ideas for the silent auction, made a list of people to invite and things to auction of.

- Discussed the advantages and disadvantages of the two systems

- Found protocols to put the fluorescent protein into the E.coli system

- Began working on the Sponsorship template

- Team meeting came to a vote to use the cell free systems for our biological lighting system and discussed using E.coli strain from cell free chassis submitted to iGEM parts registry

- Found GFP parts available through iGEM toolkit

o BBa_E0030 – Yellow Fluorescent protein (A. Victoria)

o BBa_E0020 – Cyan Fluorescent protein (A. Victoria)

o BBa_E0010 – Red Fluorescent protein (D. Striata)

o BBa_E0040 – Green Fluorescent protein (A. Victoria)

- Contacted Airline companies in regards to sponsorship

- Contacted New England Biolabs in regards to E.coli strains

- Received an email from New England Biolabs in regards of the E.coli strains- as we decided to use the protein expression strains i.e. PR1031 or ER2508

- Two students along side our supervisor went to Japan for an iGEM workshop

- Team meeting – discussed logo, began thinking of t-shirt designs and team names

- Found protocols regarding E.coli and wheat germ cell free systems - Continued working and finalizing our sponsorship template

- A list of reagents and products was made from the protocols was items were found from the www.sigmaldrish.com catalogue

- Had a team meeting Caroline Northwood to discuss our approach to sponsorship and our sponsorship template

- Reagent list to be ordered was given to Wade to be ordered

- Sent out sponsorship template to all potential airline sponsors

- Team meeting – voted on team name, logo and mascot

- Discussed our progress in individual research on the project

- Began working on the Funding proposal to the RMIT Vice Chancellor

- Meeting with the Vice Chancellor

- Began working in the Lab

o LB- Media was made and autoclaved which went to make the LB-Agar plates

o Ampicillin was made

o SOC media was made and autoclaved

o Magnesium Chloride solution and Sodium Chloride solution were also made and autoclaved

-We did the transformation of the GFP and the BFP; this was completed by Pre-incubating the LB-agar plates while thawing the component cells. Plasmid DNA was added to the tube followed by a heat shock. 800μl of SOC medium is added and incubated for 45 mins. The transformed cells were then spread thoroughly on the LB agar plates and were left to incubate overnight.

- Went in to the lab to see the colonies that grew on the LB- Agar plate

-We began the protein expression by using previously autoclaved LB media and placing 100ml into two conical flasks we then transferred a single colony from the transformed bacteria of the GFP and BFP into the flasks. We left these to incubate overnight.

-We continued with the protein expression by dividing the incubated GFP and BFP into two falcon tubes and centrifuging them. The supernatant was emptied and the pellet was resuspended. We then read the absorbance of the solution at 600nm.

o GFP=0.93A

o BFP=0.95A

-Two conical flasks were set up with SOC solution and Ampicillin with GFP and BFP in them. They were incubated for an hour then the absorbance of the solution was obtained:

o GFP= 0.7A

o BFP = 0.64 A

- The proteins were then induced and centrifuged then stored at -80ºC (Phil Poronnik) .

- Went into the lab to make reagents

o Lysis buffer

o Phosphate buffer

o Chaps

o 1M sucrose solution

o 1M imidazole solution

o 6M Guanidine hydrochloride solution

o Tris Buffer solution (pH 7.8)

-We began the purification of the GFP and BFP by taking the pallets from the -80ºC (Phil Poronnik) freezing and resuspending them with Lysis buffer. These cells were then sonicated for 3 cycles and then centrifuged. The supernatant was then transferred to eppendorf tubes and resin was added then tubes placed on suspension mixer. Phosphate buffer was then added to the pellet and centrifuged. The resulting pellet was then resuspended with the elution buffer and centrifuged again and the supernatant formed contained the GFP and BFP.

-Team meeting – discussed what part we were going to add to the iGEM Biobrick and decided to modify an existing fluorescing protein to allow it to fluoresce blue.

-We did the transformation of the Vic GFP, YFP AZ, blueberry cherry and the BFP; this was completed by Pre-incubating the LB-agar plates while thawing the component cells. Plasmid DNA was added to the tube followed by a heat shock. 800μl of SOC medium is added and incubated for 45 mins. The transformed cells were then spread thoroughly on the LB agar plates and were left to incubate overnight.

-Went into the lab to see which LB agar plates we had colony growth on.

- Team meeting- discussed what had been done on the wiki, and what else needed to be added

- Discussed with team members what was being done in the lab

- Discussed which fluorescing protein would be best to modify to made our BFP for the Biobrick

- Celebrated our team members 21st Birthday

- Began the preparation of the yellow plasmid this was done by placing LB medium in a falcon tube with the E.Coli bacteria allowing it to incubate overnight.