"
Team:Groningen/Project/Motility
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.
-----------------------------------------------------
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 strait swimming. On the other hand if the flagella are spinning clockwise (CW) then the flagella will disperse over the membrane and cause tumbling.
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
|
| Figure 1: Pattern of Pdes-lacZ expression on a temperature downshift.(a) B. subtilis AKP3 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. The first was transferred to 25 °C (●) and the second was kept at 37 °C (○). (b) Pattern of Pdes-lacZ expression in a des‾ background. B. subtilis AKP4 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. One fraction was transferred to 25 °C (●) while the other was kept at 37 °C (○). (c). Effect of exogenous fatty acids on Pdes-lacZ expression pattern. B. subtilis AKP4 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. Each fraction was supplemented with palmitic (●) or oleic acid (■) and growth was continued at 25 °C. (d) Effect of desKR disruption on Pdes-lacZ expression. B. subtilis AKP21 cells were grown at 37 °C to an optical density of 0.4 at 525 nm and then divided into two fractions. One of the fractions was transferred to 25 °C (●) and the other one was kept at 37 °C (○). Optical density at 525 nm (inserts) and β-galactosidase specific activity were determined at the indicated times (a, b, c, or d). |
| 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).
|
| Figure 2: |
|
| Figure 3: |
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).
|
| Figure 1: Colony PCR of the des knock out |
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. 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).
|
| Figure 2: Motility assay results after 16 hours of growth |
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 knockout 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.
