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From 2013.igem.org

Page set-up

Why, what

What are we doing
Why is it important
Which genes are involved
for motility, The che genes (most important ones), what they do in b.sub
For temperature sensing, The des pathway

Construct

All the pictures of our construct.
Short explanation.

Scenario's

Moving towards heat
Not moving towards heat
Moving towards cold.

Results

Labresults, cheY is inmobile.
Moddeling results, short summary and a link to the modelling page.

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Back-up plans

Swirling mechanism collecting all the strep
(Biofilm +killswitch)

The Coating Mechanism

Our initial idea was to let bacteria produce the silk in a bath and of the implant is put into the bath the implant will be coated. Because a low yield can be expected and with the recent developments of porous implants a more elegant solution is needed, because the silk has to be produced on site. Therefore the heat motility was developed. with the use of heat motility the silk will be produced on site, this will also save energy in the form of nutritions and energy of heating the bath.

In case the silk cannot be secreted the coating will be done by a biofilm formation on the implant. (need to improve this)

Native Motility

The Motility in most bacteria is governed by the che proteins, they control if the bacterium is strait swimming or tumbling (changing directions). They do this by controlling the flagella. If the flagella spin counter-clockwise (CCW) they will group together in one pole of the bacterium, causing straight swimming. On the other hand if the flagella are spinning clockwise (CW) then the flagella will disperse over the membrane and cause tumbling.

cheY

The che proteins are present in many motile bacteria, however they can have different effects depending on the species. The cheY protein in Bacillus subtilis, for example, has the complete opposite effect as in Escherichia coli. CheY is an important protein for controlling the spinning of the flagella. When the concentration of phosphorylated cheY (cheY-p) is sufficiently high the flagella turn CCW (straight swimming), but when the concentration of cheY decreases the chance of tumbling also increases, and the bacterium will reorient themselves more often.

Attractant Receptor

The chemotaxis process is initiated at the receptor, which can sense the concentration of an attractant (or repellent). Increasing concentrations of attractant correspond to an increased chance of swimming. If the concentration decreases, the bacteria will start to tumble more frequently, and will reorient its swimming direction, hopefully to more desirable regions.

cheA & cheCD

Binding of the attractant receptor causes straight swimming via a small cascade. When the receptor is bound, the cheA protein (which is attached to it) gets phosphorylated. The cheA-p phophorylates cheY, which then causes straight swimming. The protein complex cheC-cheD causes dephophorylation of cheY-p (when receptor is bound) resulting in a negative feedfoward. Two more negative feedback systems are also activated following the binding of attractant, which also result in decreased values of cheY-p. In such a way, cheY-p adaption occurs, and B. subtilis is ready to sense new changes in its environment. For more information about how this pathway works please visit the heatmotility section LINK

Heat Motility

To get heat regulated motility, we first made a system in which the motility of Bacillus subtilis could be controlled via knocking out the motility gene cheY. We could now introduce any particular promoter in front of our cheY gene and thus controlling the motility. This system in combination with our general chemotaxis model allow for good control of the cell. The promoter from the thermosensing des pathway, that is natively present in Bascillus subtilis, was fused to a cheY gene. In the end the thermotaxis model showed that this approach for controllable motility worked.

Thermal control of fatty acid synthesis.

In order to maintain the fluidity of the cell membrane when the environmental temperature is decreasing, B. subtilis (among other bacteria) adapts the membrane by increasing the fraction of unsaturated phospholipids acyl chains.

The desaturation of the membrane starts with the membrane protein, DesK. DesK senses temperature of its environment and when the temperature is <30 °C, DesK autophosphorylates its conserved histidine. Sequentially the phosphoryl group is transferred to the aspartate residue in desR that activates the promoter of des. The gene des is translated into a fatty acid desaturase (Δ5-Des), that changes the fluidity of the membrane by introducing double bonds into pre-existing saturated fatty acyl chains.

The promoter activity of des

Strain	Description
JH642	trpC2 pheA1
AKP3	JH642 amyE::[Pdes(-2269 to +31)lacZ]
AKP4	AKP3 des::kan
AKP21	AKP3 desKR::kan

Bredeston et al. 2011

Motility

Bacterial movement is based on flagella (tail like structures) and utilizes a counter-clockwise (CCW) and clockwise (CW) motion. When the flagella turn CCW they gather in one area resulting in bacteria that move straight. When the flagella move CW they disperse all over the cell membrane, resulting in the bacteria tumbling in random directions. When bacteria sense an attractant the flagellar motion will turn CCW, when the concentration of the attractant reduces the flagella will turn CW.

The receptor is displayed in the figure on the page below. The letters are all Che proteins, with this cascade of proteins motility is coordinated. When a attractant is bound to the receptor, CheA phosphorylizes CheY into CheY-P. CheY-P causes the CCW motility in the flagella, it also causes (by forming a CheY-p/CheC complex) that CheD is pulled off the receptor. CheD and CheC forms also a complex which dephosphorylizes CheY-p into CheY thus resetting the receptor.

The principle

CheY is a mayor factor in spinning the flagella CCW. When cheY is absent, cells are significant less motile[ref]. Because the promoter of des is active at low temperatures (25 °C) we placed cheY under control of the promoter of des (figure 2).

Biofilm

(..)

The coating

The silk proteins are secreted with a Strep tag. The implant will be covered with biotin (vitamine B8). The Strep tag will automatically form a Van der Waals binding with the biotin and thus, if the silk is secreted closely the implant will be coated automatically. B.subtilis has a tendency to form a biofilm on a structure, or in our case an implant (insert proof that biofilm can grow in plastic/titanium) . To coat the implant we have thought of to option 1 via secretion and 2 via a biofilm formation. The secretion process is the most elegant and best option, but secretion of large proteins is proven to be difficult and has never been done before with silk. So a ‘backup’ plan was needed.

Animation

cheY and des knockout

For our heat motility model we need at least a double knockout strain of B.subtilis. A knockout of both cheY and des are necessary. To obtain the double knockout strain, first a knockout of cheY is made after which the des knockout is inserted.

Correct insertion of the des knockout

A knockout of gene des is inserted into the genomic DNA of B.subtilis strain 168 with a tetracyclin resistance marker. Colony PCR showed that des is indeed transformed into the genomic DNA (Figure 1).

Motility of the knockout strains

To observe whether or not the mutant strains are less motile than the wild type strain. Two different tests are done.

Motility assay

To compare the motility of the wildtype strain with the two knockout strains, ΔcheY and ΔcheYΔdes, a motility assay is made. All the results obtained from this assay is from an experiment performed in triplo. When the strains are grown on a 0.4% LB agar plate, after 16 hours of growth it is visible that the wildtype strain shows more swimming behaviour than both of the mutant strains (Figure 2). A comparison between the two mutant strains indicates that ΔcheY has better swimming behaviour than ΔcheYΔdes.
When the plates are incubated for 21 hours, it is more distinguishable that the wildtype strain shows more swimming behaviour than the mutant strains. A comparison at two temperatures, 25°C and 37°C, is made. A comparison between the strains at both temperatures shows that the wildtype strain swims better than the ΔcheY strain and the ΔcheY swims better than the ΔcheYΔdes strain. It is also visible that cells grow better when they are in a warmer environment (Figure 3).

Microscope movies

Another way of analyzing the swimming behaviour is to make microscope movies (4x real time). These movies are made for the wildtype, the ΔcheY and the ΔcheYΔdes strain. These movies show that the wildtype strain (Movie 1) is more motile than both mutant strains. The comparison between the movie of the ΔcheY (Movie 2) and ΔcheYΔdes (Movie 3) strain shows just as seen in the motility assay, that the ΔcheYΔdes strain is less motile than the ΔcheY strain.

Movie 1: Motility of the wild type strain

Movie 2: Motility of ΔcheY

Movie 3: Motility of ΔcheYΔdes

References

Mariana Martin and Diego de Mendoza , Regulation of Bacillus subtilis DesK thermosensor by lipids, Biochemical Journal (2013), Vol 451 No 2, pp. 269–275 Christopher V. Rao, George D. Glekas and George W. Ordal, The three adaptation systems of Bacillus subtilis chemotaxis, Trends in Biology (2008), Vol. 16 No 10, pp. 480-487.
Liam F. Garrity and George W. Ordal, Chemotaxis in Bacillus Subtilis: How bacteria monitor environmental signals, Pharmacology and Therepeutics (1995), Vol. 68 No.1, pp. 87-104. Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis* Received for publication, January 3, 2011, and in revised form, January 31, 2011 BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 5, pp. 362–366, 2011