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Team:Groningen/Project/Motility
From 2013.igem.org
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<h1>Heat Motility</h1> | <h1>Heat Motility</h1> | ||
<p> | <p> | ||
| - | In case of a low yield we want a targeted secretion only near our (that we want to coat with silk). | + | <st>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. | + | 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.</st> |
| + | |||
</p> | </p> | ||
| - | <h2> | + | <h2>A cold inducable promoter</h2> |
<p> | <p> | ||
| - | + | Rigid membrane | |
| - | cascade that has to maintain the membrane liquidity. It does this by phosphorylating DesR, | + | |
| - | phosphorylated | + | DesK is an membrane localized protein that senses temperature of its enviroment. DesK is part of a |
| - | + | cascade that has to maintain the membrane liquidity. It does this by phosphorylating DesR, when DesR is | |
| + | phosphorylated it activates the promoter of <i>des</>. The gene <i>des</i> is translated in a fatty acid desaturase, that changes the fluidity of the membrane. | ||
| + | <br> | ||
<h3>The promoter activity of <i>des</i></h3> | <h3>The promoter activity of <i>des</i></h3> | ||
<img src="https://static.igem.org/mediawiki/2013/d/d7/Promoter-des-activity.jpg" width="50%"> | <img src="https://static.igem.org/mediawiki/2013/d/d7/Promoter-des-activity.jpg" width="50%"> | ||
<br>(a)<b>Pattern of P<i>des</i>-<i>lacZ</i> expression on a temperature downshift.</b> <i>B. subtilis</i> 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 P<i>des</i>-<i>lacZ</i> expression in a <i>des</i>‾ background. <i>B. subtilis</i> 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 P<i>des</i>-<i>lacZ</i> expression pattern. <i>B. subtilis</i> AKP4 cells were grown at 37 °C to an optical | <br>(a)<b>Pattern of P<i>des</i>-<i>lacZ</i> expression on a temperature downshift.</b> <i>B. subtilis</i> 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 P<i>des</i>-<i>lacZ</i> expression in a <i>des</i>‾ background. <i>B. subtilis</i> 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 P<i>des</i>-<i>lacZ</i> expression pattern. <i>B. subtilis</i> 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 <i>desKR</i> disruption on P<i>des</i>-<i>lacZ</i> expression. <i>B. subtilis</i> 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 | + | 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 <i>desKR</i> disruption on P<i>des</i>-<i>lacZ</i> expression. <i>B. subtilis</i> 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). |
| - | at the indicated times (a, b, c, or d). | + | |
</img> | </img> | ||
Revision as of 14:50, 8 September 2013
Heat Motility
A cold inducable promoter
Rigid membrane
DesK is an membrane localized protein that senses temperature of its enviroment. DesK is part of a
cascade that has to maintain the membrane liquidity. It does this by phosphorylating DesR, when DesR is
phosphorylated it activates the promoter of des. The gene des is translated in a fatty acid desaturase, that changes the fluidity of the membrane.
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
Bacterial movement is based on flagella (tail like structures) and utilizes a counter-clockwise (CWW) 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 principle
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.
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).
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.
