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Team:Victoria Australia/Notebook
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-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 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. | ||
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| + | <div id=" .2F1_September_2009"> | ||
| + | <div id=" .2F2_September_2009"> | ||
| + | ==1 September 2009== | ||
| + | </div></div> | ||
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| + | -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. | ||
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| + | o GFP=0.93A | ||
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| + | o BFP=0.95A | ||
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| + | -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: | ||
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| + | o GFP= 0.7A | ||
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| + | o BFP = 0.64 A | ||
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| + | - The proteins were then induced and centrifuged then stored at -80ºC (Phil Poronnik) . | ||
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| + | <div id=" .2F3_September_2009"> | ||
| + | <div id=" .2F4_September_2009"> | ||
| + | ==4 September 2009== | ||
| + | </div></div> | ||
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| + | - Went into the lab to make reagents | ||
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| + | o Lysis buffer | ||
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| + | o Phosphate buffer | ||
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| + | o Chaps | ||
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| + | o 1M sucrose solution | ||
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| + | o 1M imieazole solution | ||
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| + | o 6M Guanidine hydrochloride solution | ||
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| + | o Tris Buffer solution (pH 7.8) | ||
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| + | <div id=" .253_September_2009"> | ||
| + | <div id=" .2F6_September_2009" | ||
| + | <div id=" .2F7_September_2009"> | ||
| + | <div id=" .2F8_September_2009"> | ||
| + | ==7 September 2009== | ||
| + | </div></div> | ||
| + | </div></div> | ||
| + | |||
| + | -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. | ||
Revision as of 02:36, 21 October 2009
Notebook
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14 May 2009- 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
21 May 2009- 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
28 May 2009- 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
29 May 2009- Found protocols to put the fluorescent protein into the E.coli system - Began working on the Sponsorship template
4 June 2009- 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
5 June 2009- Contacted New England Biolabs in regards to E.coli strains
11 June 2009- 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
25 June 2009- Two students along side our supervisor went to Japan for an iGEM workshop
29 June 2009- Team meeting – discussed logo, began thinking of t-shirt designs and team names
30 June 2009- Found protocols regarding E.coli and wheat germ cell free systems - Continued working and finalizing our sponsorship template
1 July 2009
3 July 2009- Had a team meeting Caroline Northwood to discuss our approach to sponsorship and our sponsorship template
6 July 2009
- Sent out sponsorship template to all potential airline sponsors
24 July 2009- Team meeting – voted on team name, logo and mascot - Discussed our progress in individual research on the project
11 August 2009- Began working on the Funding proposal to the RMIT Vice Chancellor
20 August 2009- Meeting with the Vice Chancellor 21 August 2009- 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
25 August 2009-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.
26 August 2009- Went in to the lab to see the colonies that grew on the LB- Agar plate
31 August 2009-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.
1 September 2009-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) .
4 September 2009- Went into the lab to make reagents o Lysis buffer o Phosphate buffer o Chaps o 1M sucrose solution o 1M imieazole solution o 6M Guanidine hydrochloride solution o Tris Buffer solution (pH 7.8)
<div id=" .2F6_September_2009" 7 September 2009-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. |
