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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)

Heat Motility

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

Correct insertion of the des knock out

A knock out 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 knock out 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 knock out strains, ΔcheY and ΔcheYΔdes, a motility assay is made. 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).

Microscope movies

Another way of analyzing the swimming behaviour is to make real time microscope movies. These movies are made for the wildtype, the ΔCheY and the ΔCheYΔDes strain. Visible is that the wildtype is motile whereas the knock out strains are not (Movie 1-3).
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.