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Team:Macquarie Australia/trial
From 2012.igem.org
Contents |
Tuesday 07/08/12
(Click on headings to visit methods)
Making liquid media, plates & buffers
- Liquid LB Media
- SOC Solution
- SOB Solution
- LB Agar Plates
Ampicillin LB Agar Plates: 31 plates
Chloramphenicol LB Agar Plates: 33 Plates
Kanamyacin LB Agar Plates: 32 Plates
- TB buffer.
- TAE buffer.
- EDTA buffer.
Designing Gibson Assembly Fragments
- Haemoxygenase Gene
- Deinococcus radiodurans Bacteriophytochrome Gene
- Agrobacterium tumefaciens Bacteriophytochrome Gene
Tuesday 14/8/12
(Click on headings to visit methods)
Making Competent Cells
- Choosing Biobricks
- Making competent cells
- Transformation Protocol
Outreach Planning Labwork
- Open Day
- Secondary Education Seminars
Wednesday 15/8/12
Our open day preparation did not go as planned. Most of our transformations were unsuccessful, we believe this was because SOC was added after the heat shock. As disappointing as this was, there was one little piece of good news.
A single colony of Top 10 E.coli containing GFP insert was identified on a plate of LB agar media.
LB agar and nutrient agar plates were struck using this colony. The colony was also used to inoculate 5mL of nutrient broth. Plates and stock were then incubated at 37°C overnight. Both of the streaked plates and the broth showed significant growth of E.coli colonies which could fluoresce when exposed to UV light.
Unfortunately, Qantas declined to sponsor the effort of getting our team to Hong Kong. This was soon forgotten when the Macquarie University School of Medicine, Agilent and the Defence Science Institute (thanks Yagiz) both agreed to sponsor our team. Hong Kong here we come.
Tuesday 21/8/12
Outreach Planning Labwork
- Secondary education seminars
- Reattempting glowing bacteria
Making Plates
Tuesday 28/8/12
Outreach Planning
- High School Presentations: Preparation for 4th September
- Open Day: Preparation for 8th September at Macquarie University
- Writing the Abstract for the project due 7th September
Outreach Lab Work
- Fluorescent Bacteria
Competent Cell Testing
Friday 31/8/12
gBlock fragments
- gBlock fragments from IDT
Tuesday 04/09/12
Gibson Assembly
- Gibson Assembly Protocol
Outreach
- Visit to High School
- Outreach administration
- Media preparation for more Fluorescent bacteria
Competent Cell Protocol
- Generation of Competent Cells
Wednesday 05/09/12
Matt looked at the transformed cells we cultured the previous day (04/09/12). Two small colonies of the transformed Deinococcus bacteriophytochome without the T7 promoter were grown on kanamycin. The positive control was successful, so we could infer that the reactions were proceeding as expected. The positive control was also cultured on XGal and incubated overnight.
The Deinococcus was cultured in 2 tubes of LB broth (2 mL) containing kanamycin. The transformations performed on the previous day were done again with a slightly different method. We choose to redo the transformations as the efficiency was relatively low. The transformations were re-innoculated onto fresh plates and incubated over night.
Thursday 06/09/12
We continued on from the Gibson assembly and transformations we performed on the 04/06/12. Daniel did a plasmid prep using a QIAprep Spin Miniprep Kit by Qiagen, for two colonies from Deinococcus.
QIAprep Spin Miniprep Kit by Qiagen
Friday 07/09/12
Plasmid Preparation was continued from Thursday, by Daniel and Harry, using the QIAprep Spin Miniprep Kit by Qiagen. A total of 7 colony cultures were used as the remaining did not grow. Successful cultures included 6 colonies from 1C and 1 colony from 3K. The DNA concentration was then determined for each sample by NanoDrop, with EB buffer from the Miniprep Kit used as a blank.
NanoDrop Results
Sequencing Results
Purified Plasmid Samples then underwent sequencing at the Macquarie University Sequencing Facility. Each sample contained 10 uL Plasmid (>50ng/ul) and 1 uL of BioBrick forward and reverse sequencing primers.
Tuesday 11/09/12
The team received excellent news from the sequences performed on Friday. We had produced our first gene that is suitable for a biobrick! We had successfully produced the T7 promoter containing Heme Oxygenase. We then took this plasmid and inserted into a BL21 strain of E. coli to overproduce the protein.
We also performed another round of Gibson assembly. As the T7 Heme Oxygenase was successfully produced we decided to focus on the Deinococcus and Agrobacterium bacteriophytochromes.
Results of Gibson Assembly
- Further Gibson Assembly lab work
- Transformation
- More plate preparation - Kanamycin & Chloramphenicol
Wednesday 12/09/12
Colony Count Results
Monday 17/09/12
Tuesday 18/09/12
Wednesday 19/09/12
Plates from the transformations of Top 10 cells with the ligation products were inspected. No colonies from the 2K-4K ligations grew. 9 colonies from the 1C-3C and single colony from 1C-5C colonies had grown. These colonies were used to inoculate into LB Kanamycin and left overnight for growth.
Rob and Matt spun down the BL21 colonies that had been transformed with plasmids containing the T7-HO genes (1C) and had been growing overnight after inducing with IPTG in the presence of ALA. 2 of the cultures were not induced but still had ALA added. These cultures had varying levels of green pigment visible in the pellet after being spun down. One culture was subjected to an additional 10ul of 100mM ALA and left to grow in the refrigerator. This culture will be left to grow for a further 48-72 hours to assess if there is any increase in pigmentation. Pellets from the remaining treatments are in the -18 freezer and will be used to run SDS-PAGE.
The liquid cultures used for the earlier minipreps were used to streak agar plates and these are now in the incubator. The cultures were then transferred into glycerol stocks and frozen.
