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

Cell by Date: Implementation

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

Design Focus

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
• Saving time by doing experiments in conjunction with other projects

First stage: Preparation of vectors and E.coli cultures

Introduction

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).

Materials & Methods

• List of Required Parts to be ligated into plasmid.
• General Protocols for digestion, ligation, gel electrophoresis, gene cloning, electroporation, competent cells etc. and the materials required for each.

Results

A summary of the stage results. More details in lab notebook.

Second stage: Calibration and Measurement of Fluorescence + Absorbance

Introduction

1. Fluorescence

This involves the use of the fluorometer to measure 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:

• fluorescence vs. intracellular [GFP]
• intracellular [GFP] vs. lysate [GFP]
• Degradation [GFP] - half-life of 24 hours

2. 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)

Materials & Methods

• List of Required Parts to be tested under the conditions stated above.
• Fluorometer Reading
  • aka. 'Twinkle' - plate reader fluorometer that measures 96 well plates
  • It is hoped that absorbance@600nm and fluorescence emisssion of GFP & DsRed-Express can be measured at set time intervals
    • We can thus derive GFP molecules synthesized cfu^-1 s^-1
  • To be installed on 3^rd Aug @10:30am by engineer.
  • General protocols for fluorometer usage to be finalised.

• Mass Spectrometry (can the 'Twinkle' replace this?)
• Digital Camera
  • To take digital pictures of our results. Might not be useful to GFP, but colour scheme can be done for DsRed-Express.

• Calibration Curves
  • Aim is to create a more generic unit to allow for modular design and easier repetition of experiments - GFP molecules synthesized cfu^-1 s^-1.
  • Required for absorbance and fluorescence measurements.

1. Absorbance@600nm vs. Colony Forming Units (CFU)

Example of Absorbance Calibration Curve

Absorbance of the cultures in the wells are measured to allow to convert our absorbance raw data into a cell count, so that cell we can relate GFP synthesis per cell. As cultures of E. coli are grown to various cell densities, a sample of these cultures are taken and OD₆₀₀ measured. Dilution plating is then carried out to work out approximate CFU ml^-1 of culture. This data set allows the conversion of absorbance levels at OD₆₀₀ to cell density measured by CFU ml^-1.

Limitations of calibration:

• Sources of error in plate counting (The Great Plate Count Anomaly)
• Different strains of bacteria will require different calibration curves.

2. Fluorescence vs. intracellular [GFP]

Example of Fluorescence Calibration Curve

Calibrating intracellular [GFP] to that of fluorescence is tricky. This will however allow us to relate [GFP] to fluorescence readings, and would provide us with valuable information for the modelling studies. Three curves are required to describe this data set.

• Fluorescence vs extracellular [GFP] molecules. Known concentrations of GFP are diluted into a range of dilutions and are then mixed with cell lysate of a particular chassis.

• Fluorescence vs Cultures of varying unknown intracellular [GFP]. Measuring the fluorescence of cultures of cells at set time intervals would measure a set of unknown intracellular [GFP]. For our objectives, we would need to compare this unknown concentration in terms of its extracellular content. This problem can be circumvented with the following experiment.

• Fluorescence vs Cultures of varying unknown extracellular [GFP]. Lysing samples of Experiment 2 will allow the relation of an unknwon intracellular [GFP] to that of extracellular [GFP]. The results of comparing Experiments 2 & 3 can then be inferred with respect to the calibrations made in experiment 1 where we used known concentrations of GFP. The result is the correlation of intracellular [GFP] to that of fluorescence observed. This is important to the creation of our generic unit of measure.

Results

A summary of the stage results. More details in lab notebook.

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

Introduction

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).

Materials & Methods

• List of Required Parts to be ligated into plasmid.
• General Protocols for digestion, ligation, gel electrophoresis, gene cloning, electroporation, competent cells etc. and the materials required for each.
• Fluorometer Reading
• Mass Spectrometry

Results

A summary of the stage results. More details in lab notebook.