Team:Reading/Notebook

Lab notes

This page is an account of the daily events in our lab and project. Dates are in the UK format DD/MM/YY. 6803 refers to the cyanobacterium used - Synechocystis sp. PCC 6803

29/06/15
  • Cultured a sterile 500ml conical flask using 100 ml BG-11 and 5 loopfuls of 6803 Wild Type (WT). (Flask 1)
  • Cultured 5 streak plates of 6803 WT on BG-11 Agar

    • All placed on the windowsill in our lab under natural light and at room temperature (~25°C)


    02/07/15
  • Three new sterile 500ml conical flasks set up on the shaker at 58rpm and 30°C. Each contains 5 loopfuls of 6803 WT and 100ml of BG-11. They are exposed to the overhead lights in the lab 24/7. (Flasks 2, 3, 4)
    • 07/07/15
  • Flask 5 cultured containing 10ul 6803 WT in 100ml BG-11. This flask will be used for preliminary growth curve measurements. The flask was placed on the shaker with flasks 2,3 and 4 all at 58rpm and 30°C under lab overhead lights.
    • 10/07/15

    2 Litres of water collected from Whiteknights Lake on campus for SODIS, method outlines in protocols.

    13/07/15

    Estimating the Cells per ml for the flask from 29/06 1 a.u at 750nm = 1.6x108

  • OD750 = 0.028
    • Therefore
  • 0.028x1.6x108 = 4.48x106 cells per ml

    • OD750 measurement and cells per ml estimated for flasks cultured on 02/07

    14/07/15

    The lamp safety testing completed so flasks placed under the bright white light bulb

    21/07/15

    Flasks are getting very green, definitely a good sign!


    23/07/15

    Flask 2 discarded as it is not showing growth, and has signs of contamination. We received most of the lab equipment we have ordered.

    27/07/15 BG-11 order arrived finally!

    1. Miniprep of plasmid DNA: We carried out extraction of biobrick construct plasmids from E.coli NEB-5α glycerol stocks.

      We extracted pSB1K3 plasmid backbones combined with biobricks containing the following genes:

      • PilA1
      • PsaD
      • PetF
      • PilT

      This gave us 4 samples of plasmid DNA, which were placed in the freezer at -20°C.

    2. BG-11 cultures: We added ~200ml of BG-11 to two sterile 500ml conical flasks. We inoculated them as follows;
      • Flask 5 - 1ml of Wild type 6803 from flask 3 as this flask had the best growth.
      • Flask 6 - 10µl of stock Howe Lab triple mutant 6803.
    3. SODIS lakewater cultures: We added ~200ml of SODIS lakewater to three sterile 500ml conical flasks. We inoculated them as follows;
      • SODIS 1 and 2 - 1ml of Wild type 6803 from flask 3 as this flask had the best growth.
      • SODIS 3 - 1ml of stock Howe Lab triple mutant 6803.

    All flasks cultured today were placed in the incubator, being shaken at 58rpm, 30°C, and under bright white light

    05/08/15

    200ml of BG-11 was added to flasks 3 and 4 which already contained ~100ml of ~2 a.u density 6803 WT.

    Competition Assay

    200ml of BG-11 added to a new sterile conical flask

    added 10ul of WT 6803 and 10ul of MT3 6803

    Flask was placed in the incubator, being shaken at 58rpm, 30°C, and under bright white light


    06/08/15

    DNA concentration from miniprep

    • PilA1 = 11.16ns/ul
    • PsaD = 13.01ns/ul
    • PetF = 0.38ns/ul
    • PilT = 8.58ns/ul

    *no DNA due to miniprep from Glycerol Stock

    07/08/15

    Made LB Agar tranplates for culturing

    • PilA1
    • PsaD
    • PetF
    • PilT
    10/08/15

    No growth on plates from 07/08

    Cultured plates of LB Agar from all stocks (glycerol stocks)

    11/08/15

    Growth on many the stocks from 10/08, replated onto LB kan 50 plates

    12/08/15

    Competition Assay had begun to turn a light green colour.

    13/08/15

    Stock growth onto LB kan 50 plates unsuccessful.


    21/08/15

    Potassium ferricyanide reagent arrives, protocols start being drawn up

    22/08/15

    Nanowires concluded to be a non-fruitful venture

    24/08/15

    Reculturing flasks 2, 3, 4, and 5 into repeat flasks respectively

    26/08/15

    Our lab becomes engulfed in the tides of cuvettes arriving


    27/08/15

    Unsuccessful ferricyanide assay, protocol will be revised

    28/08/15

    Competition assay starting to reach log phase

    31/08/15

    LB BG-11 Kan plates and LB BG-11 plates made up to plate the competition assay on

    01/09/15

    Competition assay plated and growth will be monitored

    02/09/15

    Starting to prepare poster and presentation for the UK iGEM meet up hosted by Westminster

    04/09/15

    Enjoyed a fun day presenting in Westminster and talking to the other teams

    07/09/15

    Another ferricyanide ran with a mildly improved protocol but still yielded inconclusive results

    11/09/15

    Ran a ferricyanide assay not to give comparable data but data which can be interpreted to give the correct concentrations of Potassium Ferricyanide used

    14/09/15

    A successful ferricyanide assay was run using a new and improved protocol #Winning

    18/09/15

    The fuel cell was assembled and we put in the Cyanobacteria to measure the voltage given seeing a vast improvement over last years project, and we lived happily ever after without iGEM.