Introduction

The group proposed to discuss and investigate the working range of the parts and devices of our system

• The proposed variables to investigate are:
  

i) Concentration of activator

ii) Time

iii) Temperature

Characterization of Parts

We are interested in characterizing the following parts:

i) Lactose/IPTG sensitive Promoter pLac [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]

ii) Iron Sensitive Promoter pFe [http://partsregistry.org/wiki/index.php?title=Part:BBa_I716014 BBa_I716014]

iii) AI2 senstive Promoter pLsrA
The characterization is carried out by ligating the desired promoter with the Green Fluorescence protein: [http://partsregistry.org/Part:BBa_E0240 BBa_E02040]

• For the pLac BBa_R0010, it is already available in the form that we desired in the registry as [http://partsregistry.org/wiki/index.php?title=Part:BBa_J04430 BBa_J04430]
• For the pLsrA promoter, the Yellow Fluorescence protein was used instead to differentiate between our two different systems.

The FLx800™ Fluorescence Microplate Reader

The FLx800™ Fluorescence Microplate Reader was available for our use courtesy of Division of Bio-engineering lab. It was purchased from BioTek and for more information about the reader, you can refer to [http://www.biotek.com/products/product_detail.php?pid=117 The FLx800™ Fluorescence Microplate Reader]

The FLx800™ Fluorescence Microplate Reader has a computer software KCJunior that was installed in a computer adjacent to the reader for collection and processing of data from our samples.

Protocols

Characterization of standard Biobrick via GFP Activity

The objectives of the characterization experiment are to investigate the effects of concentration of inducer, temperature and time on the production of Green Fluorescence protein (GFP) by the Biobrick promoter part being experimented. A stronger fluorescence, as measured using NTU FLx800™ Fluorescence Microplate Reader will indicate more GFP proteins being synthesised. This also corresponds to higher promoter activity.

Top10 competent cells are transformed with plasmid carrying the following gene sequence:

The cells are cultured and upon addition of the inducer, the reaction mixtures (with both cells and inducer) are fed into the Fluorescence Microplate Reader. This machine has inbuilt software, KCJunior which will translate the results into excellent data suitable for analysis. The samples’ fluorescence was measured for a period of 12 hours, at the temperatures of
i) 25°C
ii) 37°C
and iii) 42°C.

The readings were than plotted to produce a 3-dimentional graph for detail analysis.

For more information please refer to Characterization of pLacI-GFP

Characterization of pLsrA - YFP

1) The successfully ligated plasmid with pLsrA-YFP gene was first transformed into chemically competent LuxS(-) cells.

2) The next day, one colony of cell with pLsrA-YFP plasmid was inoculated in 5ml LBA for 16 hours at 37oC and shaked at 225 rpm.

3) Overnight cell culture was then centrifuged at 4000 rpm and 4oC for 10 minutes.

4) The supernatant was discarded and cell pellets were re-suspended in 5ml of Ampicilin-containing M9 medium. The amount of M9 medium was adjusted until cell suspension had an OD600 of 1.

5) The cell suspensions were then pipetted into 96-well microplate wells. This was followed by the adding of 50µl AI-2-containing supernatant into corresponding wells.

6) There were 6 different supernatant solutions used, which correspond to the time points when they were obtained: 2, 3, 4, 5, 6, 8 hours.

7) There were 3 different negative control samples being used for this study.

  * The First control sample contained cell suspension only. 

  * The Second control sample was cell suspension with 50µl pure water added.

  * And as the supernatants also contain large amount of LB, cell suspension with 50µl 

    LB added was also used as another control sample. 

8) YFP measurement was carried out by VICTOR 3 multilabel reader at excitation wavelength of 490 nm and emission wavelength of 535 nm.

9) Data were automatically gathered every 10 minutes and temperature was set at 37oC.