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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 2 bacteria uptake into roots

Wednesday, 3 August 2011

One important part of our project is uptake of our bacteria into plant roots. The observation that this occurs (albeit under controlled lab settings) is new and was only published last year. We attempted to replicate these findings.

In preparation, we met with Dr Martin Spitaler who advised us on how to prepare samples for the confocal microscopy. Confocal microscopy is much more precise than conventional light field microscopy as it eliminates background light by focusing the laser through a pinhole (Mark Scott, oral communication). The confocal microscopy will focus on imaging GFP expressing bacteria inside Arabidopsis roots to show that uptake of the bacteria takes place.

Staining of wt roots with DiD, a lipophilic dye that stains the plant membranes and does not interfere with the absorption or emission spectra of GFP and Dendra, was unsuccessful. However, natural fluorescence was measured in a root in a spectrum that does not interfere with measuring GFP. We should therefore not need to dye the roots before imaging.

INSERT MARTIN’S ROOT PICTURE

The root can be imaged at around __nm

We may also try to stain the roots with propidium iodide, which is also a strong indicator of cell wall break down.

Thursday, 4 August 2011

We prepared the GFP-expressing bacteria for plant infection. They were spun down and media was exchanged prior to incubation at 37°C to reach exponential phase. Bacteria were then spun down and resuspended in wash buffer (5mM MES) to reach OD 30. 8ml, 4ml and 2ml were added to separate flasks, containing 100ml of half-MS media each. 4ml and 6ml of wash buffer were added to the flasks containing 4ml and 2ml bacteria, respectively. 8ml of wash buffer was added to the negative control. Ten Arabidopsis seedlings were distributed into each of the flasks. Incubation was carried out for 15 hours prior to imaging.

Friday, 5 August 2011

Prior to imaging, roots were washed in PBS to wash off bacteria and facilitate imaging. We imaged the plants incubated with 8ml of bacteria and were able to find bacteria inside one of the roots. A 3D picture was taken of uninfected roots and roots containing bacteria by taking a Z stackk image using confocal microscopy.

This video shows a zoom from the top to the bottom of the root. It was put together from successive images taken at different depth levels. The GFP-expressing bacteria are clearly visible within the root.

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.

12th of August - Swarm plate assay

This experiment uses logic of attractant concentration gradient just like other chemotaxis assays, however this one relies on bacteria generating the attractant gradient by metabolism. Semi solid agar plate contains evenly distributed concentration of attractant. Bacteria are inoculated at one position on the plate, and as they start to use the attractant source, the concentration of attractant in the immediate vicinity will decrease. This results in higher concentration of attractant to be present some distance away, causing chemotactic bacteria to move towards it. At specific positions bacteria will locate optimum concentration, and at these swarm rings will form. Different number of concentric rings is formed due to a different nubmer of attractants inserted into the semi solid agar.