Difference between revisions of "Part:BBa K1017603"

Latest revision as of 15:42, 25 September 2013

37℃ RBS + LuxR

This unit includes 37℃ RBS and LuxR. 37℃ RBS, a RBS which is controlled by temperature. LuxR, a protein which can activate P_lux after binding with AHL. This unit act as a biotheometer, which can act on temperature changing. When the temperature is higher than 37℃, the downstream gene LuxR is expressed. Therefore , P_lux is activated and the downstream gene, sRNA, can be produced, then the sRNA will bind to the rRBS so the Shine-Dalgarno sequence can't be exposed. Without the ribosome binding, causing the downstream gene of the rRBS can't be translated. When lower than 37℃, P_lux is no longer activated , which means sRNA production shuts down. Thus, the downstream gene of the rRBS, mRFP or mYFP, can be expressed normally.

Mechanism

Unlike normal RBS, the 37℃ RBS has a unique rather stable hairpin structure which shape like a lock. If the temperature drops below 37℃, the base-pairing neucleotides on the RNA will form a stable hairpin structure, occluding the Shine-Dalgarno sequence thus disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded within.When the temperature is higher than 37℃, the bonds will be broken, the hairpin would be unfolded, causing the Shine-Dalgarno sequence to be exposed and permitting the binding of the ribosome, which then starts the translational machinery so the downstream gene, luxR, can be expressed.

Fig 1. When the temperature increases, the secondary structure near the SD site would change its conformation and allow the ribosome to bind to initiate translation

Data

From chart 1, we can clearly found that the time to reach same O.D. 0.8 various as the temperature changes. The expression of mGFP under 37°C and 42°C reached a higher OD value in less time than the expression under 32°C and 27°C. This demonstrates the fact that 37°C RBS functions more efficiently above or at 37°C. In our project, our goal is to get a certain product by a certain condition, but when under 37°C, the RBS also functions, which is out of our expectation. As a result, we cannot make sure that our system produces the only protein under 37°C.

Chart 1. 37°C RBS efficiency under different temperatures.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K1017603 short</partinfo>
-This unit includes 37℃ RBS and LuxR. 37℃ RBS, a RBS which on/off is controlled by temperature. LuxR, a protein which can activate lux promoter after binding with AHL. This unit act as a biotheometer, producing lux protein when the environment is higher than 37℃ and stop expressing when lower than 37℃.
+This unit includes 37℃ RBS and LuxR. 37℃ RBS, a RBS which is controlled by temperature. LuxR, a protein which can activate P<sub>lux</sub> after binding with AHL. This unit act as a biotheometer, which can act on temperature changing.
+When the temperature is higher than 37℃, the downstream gene LuxR is expressed. Therefore , P<sub>lux</sub> is activated and the downstream gene, sRNA, can be produced, then the sRNA will bind to the rRBS so the Shine-Dalgarno sequence can't be exposed. Without the ribosome  binding, causing the downstream gene of the rRBS can't be translated. When lower than 37℃, P<sub>lux</sub> is no longer activated , which means sRNA production shuts down. Thus, the downstream gene of the rRBS, mRFP or mYFP, can be expressed normally.
-When the temperature is higher than 37℃, the downstream gene LuxR is expressed. Therefore , P<sub>lux</sub> is activated and the downstream gene, sRNA, can be produced, then the sRNA will bind to the rRBS so the Shine-Dalgarno sequence can't be exposed. Without the ribosome  binding, causing the downstream gene of the rRBS can't be translated. When lower than 37℃, P<sub>lux</sub> is no longer activated , which means sRNA production shuts down. Thus, the downstream gene of the rRBS, RFP or GFP, can be expressed normally.
 == Mechanism ==
-<br>Unlike normal RBS, the 37℃ RBS has a unique rather stable hairpin structure which shape like a lock. If the temperature drops below 37℃, the base-pairing neucleotides on the RNA will form a stable hairpin structure, occluding the Shine-Dalgarno sequence thus disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded within.When the temperature is higher than 37℃, the bonds will be broken, the hairpin would be unfolded, causing the Shine-Dalgarno sequence to be exposed and permitting the binding of the ribosome, which then starts the translational machinery so the downstream gene, mGFP, can be expressed. [[File:NCTU RNAthermometer.jpg|center|Fig 1. When the temperature increases, the secondary structure near the SD site would change its conformation and allow the ribosome to bind to initiate translation]]
+<br>Unlike normal RBS, the 37℃ RBS has a unique rather stable hairpin structure which shape like a lock. If the temperature drops below 37℃, the base-pairing neucleotides on the RNA will form a stable hairpin structure, occluding the Shine-Dalgarno sequence thus disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded within.When the temperature is higher than 37℃, the bonds will be broken, the hairpin would be unfolded, causing the Shine-Dalgarno sequence to be exposed and permitting the binding of the ribosome, which then starts the translational machinery so the downstream gene, luxR, can be expressed. [[File:NCTU RNAthermometer.jpg|600 px|center|Fig 1. When the temperature increases, the secondary structure near the SD site would change its conformation and allow the ribosome to bind to initiate translation]]
 == Data ==
-<br>From chart 1, we can clearly found that the time to reach same O.D. value(0.8) various as the temperature changes. The expression of mGFP under 37°C and 42°C reached a higher OD value in less time than the expression below 37°C. This demonstrates the fact that 37°C RBS functions more efficiently above or at 37°C. On the other hand, the fact that 37°C RBS leads to moderate expression even under 37°C degree might be a problem, since then we cannot make sure that only single gene is expressed under 37°C.
+<br>From chart 1, we can clearly found that the time to reach same O.D. 0.8 various as the temperature changes. The expression of mGFP under 37°C and 42°C reached a higher OD value in less time than the expression under 32°C and 27°C. This demonstrates the fact that 37°C RBS functions more efficiently above or at 37°C. In our project, our goal is to get a certain product by a certain condition, but when under 37°C, the RBS also functions, which is out of our expectation. As a result, we cannot make sure that our system produces the only protein under 37°C.
 [[File:NCTU 37RBS result.png|center|600px|Chart 1. 37°C RBS efficiency under different temperatures.]]