iGEM 2015 — Team Reading

# 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)

![](https://static.igem.org/mediawiki/2015/1/17/ReadingWindowSill.jpeg)
  **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!

![](https://static.igem.org/mediawiki/2015/1/1a/ReadingFlasks.jpeg)
  **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

![](https://static.igem.org/mediawiki/2015/9/95/ReadingTom-Lab.jpeg)
  **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.

![](https://static.igem.org/mediawiki/2015/2/20/ReadingMatt-Lab.jpeg)
  **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

![](https://static.igem.org/mediawiki/2015/9/90/Reading-cuvets.jpeg)![](https://static.igem.org/mediawiki/2015/a/ac/Reading-cuvetscomments.jpeg)
  **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.