IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation

Cell by Date: Implementation

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

First stage: Preparation of vectors and E.coli cultures

This involves a series of cloning and transformation experiments (e.g. digestion, ligation, transformation). Agarose gel electrophoresis will then be used to test for products formed. Miniprep will be performed to extract the plasmid (containing our construct) from the cells, to be used in the other stages.

This is done in conjunction with In-veso group (using same gene constructs and plasmids).

Second stage: Calibration and Measurement of Fluorescence + Absorbance

Flurorescence

This involved the use of the fluorometer to measure the fluorescence of a reporter gene (GFP), under the control of a range of promoters (pTet, pBad, pT7), at various temperatures (4^oC, 25^oC, 37^oC, etc.), over 1h time intervals for 24 hours (or however it is deemed fit).

Calibration: -Different [GFP] vs. fluorescence -intracellular [GFP] vs. lysate [GFP] -Degradation [GFP] - half-life of 24 hours

Absorbance

In addition, the culture is tested for absorbance use spectrometry to determine cell density at 600nm.

Calibration: Dilution plating (or other methods of cell counting) vs. optical density (at 600nm)

Third stage: Calibration and Measurement of Fluorescence + Absorbance with DsRed-Express

Similar to second stage, but with the reporter gene as DsRed-Express. Preparation of cultures to be done in conjunction with Hrp Characterization System.

Calibration to be done with fluorescence for DsRed-Express as well (Note response time).

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Preliminary Preparation

• Endy:Making a long term stock of bacteria
• Endy:Retrieving a Registry glycerol

Phase 1

Chassis	Phase 1	Phase 2	Phase 3
E.coli	X
In-Vitro	X	X	X
In-Veso	X	X	X

Things to consider:

• Different types of promoters in different chassis
• Different threshold
• Different degradation time
• Different reporters

Preliminery Data

Protocols Required:

• Knight:Electroporation
• Endy:High-efficiency electroporation
• Incubation

1. Degradation time

• Endy:RNA Half-life
• λ-Reporters

2. Rate of expression at constant temperature

• Fluorometer Reading
• Noting down colour change (photographs)

3. Range of Reporters

• Flurometer Reading
  • RFP
  • DsRed

• Beta-Galactosidase_Assay_(A_better_Miller)
  • β-galactoside - requires subtrates X-GAL

• Luciferase - requires substrate ATP, O₂, and luciferin

4. Range of promoters

Refer to List of Required Parts.

Data Analysis

Refer to Modelling Tab.

Validation of Model

1. Validate [Reporter]_max and V_max value

• Fluorometer Reading
• Noting down colour change (photographs)

2. Validate transient response using best promoters/reporters

• Fluorometer Reading
• Noting down colour change (photographs)