"

From 2013.igem.org

Heat Motility

In case of a low yield we want a targeted secretion only near our (that we want to coat with silk). In order to achieve this we want to have a bacillus that will move towards heat. If the implant is heated it will attract our silk secreting bacillus.

The cold sensor DesK

Use of DesK as an sensor for temperature. DesK is a membrane protein (a kinase), it is part of a cascade that has to maintain the membrane liquidity. It does this by phosphorylating DesR, this phosphorylated DesR activates a promotor (pdes) that expresses the des gene, changing the membrane in the process.

The promoter activity of des

(a)Pattern of Pdes-lacZ expression on a temperature downshift. 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).

Motility

The motility part of our construct is based on two articles, a very old (1995) article with general information about motility and a newer one focusing on the attractant/repellent sensor cascade.

General

Bacterial movement based on flagella (tail like structures) and utilizes CCW(counter-clockwise) and CW (clockwise) movement. When the flagella spin CCW they gather in one area resulting in bacteria that move strait. When the flagella move CW they disperse all over the cell membrane, resulting in the bacteria spinning in random directions (tumbling). When the bacteria senses an attractant it will go CCW, till the concentration gets lower after which it will go CW resulting in a change in direction. Bacteria will move towards attractants and away from repellents.

The receptor and our Idea

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. What we want is to knock out the natively CheY gene, which the article from 1995 mentions so it is possible. And then fuse a CheY gene to the pdes promoter. The result is that CheY is only present when the temperature is low (below 30 C). Causing a very particular movement, two ways of movements are theorized.

Claudio's theory

The DesK activates when cold, so the bacteria could swim forward when it is still cold. This is dependent on the fact if the attractant receptor is bound at the moment of expression of CheY. Causing a pseudo random movement. As soon as it gets warmer the bacteria will stop being able to move cause no CheY will be present, therefore staying in place. The result will be a bacteria that will stay in a warmer area’s, cause as soon it gets cold again it will be able to move again.

Inne's theory

The pseudo random movement claudio proposes is correct, however could be improved by continually stimulating a repellent receptor. This will cause the Bacillus to move away as soon it gets into a cold area. It guarantees that CheY will be phosphorylated as soon as it is transcribed/ translated, and therefore the bacteria can move. Both systems are not directly influencing movement, but cause the organism to have a bias towards warmer area's. Note: chemotaxis is kind of confusing when applied to different organisms, e.coli for example uses the same proteins but they have the effect of these proteins is the total opposite. Namely CheY causing tumbling.

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.