Difference between revisions of "Team:Macquarie Australia/Notebook"

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<button class="accordion">Making Competent Cells</button>
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<li> Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22&#176;C, 200-250rpm. Alternatively, set up a starter culture (2ml) overnight and inoculate the large scale in the morning. Grow at 37&#176;C. </li>
  
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<li> A600 should be 0.2-0.8 when harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5 </li>
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<li> Remove the flask from the incubator and place on ice for 10 minutes. FROM THIS STEP, KEEP THE CELLS ON ICE AS MUCH AS POSSIBLE!</li>
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<li> Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4&#176;C</li>
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<li> Pour off and discard the supernatant, and immediately place the tube on ice.</li>
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<li> Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.</li>
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<li> Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.</li>
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<li> Incubate the tube on ice for 10 minutes.</li>
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<li> Centrifuge at 2,500 x g for 7 minutes at 4&#176;C, discard the supernatant.</li>
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<li> Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. E.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.</li>
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<li> Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.</li>
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<li> Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice. </li>
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<li> Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen or dry ice. Store cells at -80&#176;C. </li>
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Revision as of 09:23, 14 October 2017

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Dry Lab

  • The first week involved learning all about synthetic biology and what lies ahead in terms of the competition
  • Dr Louise Brown and explained all things iGEM including the biobrick system and 3A assembly
  • An overview of the chlorophyll biosynthesis pathway, photosystem II and hydrogenase activity presented by Professor Robert Willows since our project would focus on one component of this bigger picture
    • Our project being to use biosynthetic techniques to create the hydrogen producing hydrogenase
  • We started planning for creation of complete hydrogenase plasmid by combining the Ferredoxin/Ferredoxin reductase biobrick with the Hyd1 biobrick
    • From this we expect some preliminary production of hydrogen however, with the addition of maturation enzymes this production would be maximised.
  • Discussion on construction of the maturation plasmid in which HydE/HydF biobrick would be combined with HydG biobrick then the two larger constructs would be ligated together to create the complete plasmid coding for the total Hydrogenase molecular machine, fondly named Omega Ω
  • We created a “3-day plan” from 3A assembly through transformation and plating up cells to liquid media overnight cultures, miniprep kit extraction of plasmid and nanodrop concentration measurement, and finally electrophoresis gel screening of our newly made construct
  • Discussion on human outreach was started.
    • Began talking to the SDU iGEM team on collaboration
    • Discussed the creation of an iGEM Macquarie game
    • Started planning for attending synthetic biology conference and ACUR (Adelaide).

Dry Lab

  • India suggested the idea of a creating a game as her brother could help us out with that.
  • We got in contact with Joanne Jamie, an academic at Macquarie University, to gain more information about National Science Week and the university’s Orientation Day for our outreach possibilities.
    • She invited us to help out in the Chifley School Program (19th September) which we agreed would be better than the other two. We have a 1 hour time slot in which we can conduct wet and dry lab activities with gifted and talented high school science students.
  • The team was approached by Nebraska to fill out their survey
flasklogo

Wet Lab

  • Checked lab supplies to ensure we had what we needed.
  • Became familiar with protocols we will be using.
  • Poured LB plates made up with each of the three antibiotics we will be using- Amp, Kan and CAM.
  • Conducted three transformations of DHα5 cells with DNA plasmids of Fer, Hyd1 and HydEF.
    • These were grown successfully on plates.
  • Dived into our first ligation attempt for Fer/Hyd following our “3-day plan”
    • Results = no growth on the plates.
    • This lead us to testing all our restriction enzymes on familiar plasmids, which gave us a chance to pour our own electrophoresis gel, load and run it.
    • It was great to have Mike Gibbs, Dominic Logel, Thi Huynh and Ed Moh supervising and sharing their expertise.

  1. Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22°C, 200-250rpm. Alternatively, set up a starter culture (2ml) overnight and inoculate the large scale in the morning. Grow at 37°C.
  2. A600 should be 0.2-0.8 when harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5
  3. Remove the flask from the incubator and place on ice for 10 minutes. FROM THIS STEP, KEEP THE CELLS ON ICE AS MUCH AS POSSIBLE!
  4. Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4°C
  5. Pour off and discard the supernatant, and immediately place the tube on ice.
  6. Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.
  7. Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.
  8. Incubate the tube on ice for 10 minutes.
  9. Centrifuge at 2,500 x g for 7 minutes at 4°C, discard the supernatant.
  10. Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. E.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.
  11. Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.
  12. Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
  13. Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen or dry ice. Store cells at -80°C.