Difference between revisions of "Part:BBa K1017602"

Revision as of 14:54, 24 September 2013

37℃ RBS + mGFP

This biobrick is comprised of 37 degrees Celsius RBS and mGFP. 37℃ RBS, a RBS which on/off is controlled by temperature. mRFP is a red fluorescent protein. In our project, we use this part to conduct the test of temperature regulated-system. By adding a P_cons promoter at the upstream gene , mRFP is constantly expressing and produce red fluorescent protein. Then measure the O.D. value to determine the level of expression at different temperature.

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, mGFP, 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. 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.

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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-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 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]]
+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]]
 == Data ==