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!

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.
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.
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

26/08/15

Our lab becomes engulfed in the tides of cuvettes arriving

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 as