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Team:Groningen/Project/Motility
From 2013.igem.org
Contents |
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 article mentions that DesK is most active onces the temperature drops below 30 C, however the autokinase activity is still present at 42 C though drastically less. DesK is a real cold sensor, which is natively present in bacillus. We want to use the DesK in our heat sensing construct (how we do this is under motility), by PCR'ing the pdes promotor out of B. subtilis and Hereby activating the motility gene that we fuse after it.
References
Regulation of Bacillus subtilis DesK thermosensor by lipids , Mariana MART ́N1 and Diego DE MENDOZA2 , Biochem. J. (2013) 451, 269–275
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
The three adaptation systems of Bacillus subtilis chemotaxis , Christopher V. Rao1, George D. Glekas2 and George W. Ordal2 , 2008 Elsevier Ltd. All rights reserved. CHEMOTAXIS IN BACILLUS SUBTILIS: HOW BACTERIA MONITOR ENVIRONMENTAL SIGNALS, LIAM F. GARRITY and GEORGE W. ORDAL* , Pharmac. Ther. Vol. 68 No.1 pp. 87-104, 1995
