Revision as of 11:18, 10 August 2011

Chemotaxis Results

28th of July

-We have made tryptone broth, which will be used to grow E. coli (K-12 D10b strain) overnight before the chemotaxis
experiments, for them to develop flagella and therefore they will be capable of chemotaxis. To make tryptone broth look
at protocols.

-Cells have been transformed with backbone plasmid pSB1C3 carrying biobrick BBa_K398500, with constitutive promoter J23100,
protocol for transformation can be found here.

Chapter 1 Assembly

3rd of August

-transformation of PA2652 fragments into E coli competent cells. Fragment numbers 22 & 23. -midiprep of the 22 & 23 cells to obtain DNA. -Due to this we have transformed 5a strain with a high copy plasmid containing ampicillin and kanamycin resistance (AK3 backbone)and sfGFP. These cells have been numbered 17.

Chapter 3: Experiments

Experiments involving chemotaxis can be split to two categories, qualitative and quantitative. In the qualitative experiments, we are able to show that bacteria, which we study do or do not move towards a source, however it does not inform us at all about the cell count.

5th of August - Agar plug in assay

The simplest method for studying chemotaxis is to use agar plug in assay, which is a simple experimental method to show bacterial movement towards a localised chemical source.

Experiment is performed as follows, bacteria are added into the plate on one side, and the attractant to the other. Result is then the shape of the formed colony which grows into distorted elipse shape within the agar if the attractant is present, however colony shape remains circular if no attractant was added.

In our experiment we have used 5-α Escherichia coli cells. Cells used as positive control were transformed with pSB1C3 plasmid backbone, which conferred chloramphenicol resistance. These cells have not been modified in any other way and were used to show 5-α E. coli are capable of chemotaxis, using serine as attractant, which acts as a ligand for Tsr, an endogenous chemoreceptor. 5-α Escherichia coli competent cells were also transformed with pRK415 a working plasmid with mcpS gene and tetracycline resistance. These cells express mcpS gene from P. putida KT2440, which is chemoreceptor with malate as its ligand, and therefore malate was used as attractant for these cells during agar plug in assay. As a negative control, no attractant was added into the plate.

Amount of attractant added contributes greatly towards the result, as too little attractant added will not stimulate bacteria to move towards source, however too much attractant will result in the saturation of the medium and the bacteria will not chemotax towards source. Values (concentration of attractant, distance between colony and attractant at start etc.), necessary to perform this experiment have been worked out using modelling.
These are the values of attractant concentration we have used, when 5µl sample was added into the plate:

1

2

3

4

5

Concentration

1.884x10^-3 mol/L

1.884x10^-2 mol/L

1.884x10^-1 mol/L

0.5

mol/L

0.75

mol/L

Amount

9.42x10^-9 mol

9.42x10^-8 mol

9.42x10^-7 mol

2.5x10^-7 mol

3.75x10^-6 mol

Note: Concentration values for 4 & 5 were not obtained from models. They were overestimations to show possible saturation of the medium with attractant.

Results: possible movement towards the source.

@@ Line 12: / Line 12: @@
 -transformation of PA2652 fragments into E coli competent cells. Fragment numbers 22 & 23.
 -midiprep of the 22 & 23 cells to obtain DNA.
-For experiments involving modified E. coli, we will need positive and negative control. In order to set up a positive control, E. coli expressing GFP is used for observing endogenous chemotaxis system of E. coli. After growth and observation under microscope we have found out that K-12 DH10b does not develop flagella and therefore can not move. Due to this reason we are using another strain, BL21 DE3 which is motile. These bacteria however had no plasmid and to observe them under wide field microscope a flourescent expression is necessary. Due to this we have transformed BL21-DE3 strain with a high copy plasmid containing ampicillin and kanamycin resistance (AK3 backbone)and sfGFP. These cells have been numbered 17.
+-Due to this we have transformed 5a strain with a high copy plasmid containing ampicillin and kanamycin resistance (AK3 backbone)and sfGFP. These cells have been numbered 17.
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Team:Imperial College London/Project/Chemotaxis/Results

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