Difference between revisions of "Part:BBa K1017602"

Latest revision as of 15:37, 29 September 2013

37℃ RBS + mGFP

This biobrick is comprised of 37℃ RBS and mGFP. 37℃ RBS, a kind of RBS which is controlled by temperature. mGFP is a green fluorescent protein. In our project, we use this part to conduct the test of temperature regulated-system. P_cons promoter is a constitutive promoter. This promoter can be used to tune the expression level of constitutively expressed parts. By adding a P_cons promoter and controlling temperature , we can measure the O.D. value to determine the level of GFP's expression.

Mechanism

Unlike normal RBS, the 37℃ RBS has a unique 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. 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.

As shown the following figure the normalized expression, obtained by dividing the florescence expression (emmision 612 nm and excitation 584 nm) with the OD600 value, of GFP under 37 °C is much higher than the expression under 25 °C. Such result demonstrates the fact that 37 °C RBS can effectively regulate gene expression by responding to temperature. The increased kinetic energy at 37 °C is sufficient to cause the 37 °C RBS to unfold and become available for ribosome binding. At 25 °C, however, there isn't sufficient kinetic energy to unfold the hairpin structure and the structure is preserved. As a result, the translational efficiency is very low at 25 °C.

Figure 7. The normalized expression (florescence expression/ OD value) under 37 °C is higher than the expression under room temperature by about 6 folds."

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_K1017602 short</partinfo>
-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.
+This biobrick is comprised of 37℃ RBS and mGFP. 37℃ RBS, a kind of RBS which is controlled by temperature.  mGFP is a green fluorescent protein.
-In our project, we use this part to conduct the test of temperature regulated-system. By adding a P<sub>cons</sub> 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.
+In our project, we use this part to conduct the test of temperature regulated-system. P<sub>cons</sub> promoter is a constitutive promoter. This promoter can be used to tune the expression level of constitutively expressed parts. By adding a P<sub>cons</sub> promoter and controlling temperature , we can measure the O.D. value to determine the level of GFP's expression.
 == 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]]
+Unlike normal RBS, the 37℃ RBS has a unique 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|600 px|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.]]
+<p>As shown the following figure the normalized expression, obtained by dividing the florescence expression (emmision 612 nm and excitation 584 nm) with the OD600 value, of GFP under 37 °C is much higher than the expression under 25 °C. Such result demonstrates the fact that 37 °C RBS can effectively regulate gene expression by responding to temperature. The increased kinetic energy at 37 °C is sufficient to cause the 37 °C RBS to unfold and become available for ribosome binding. At 25 °C, however, there isn't sufficient kinetic energy to unfold the hairpin structure and the structure is preserved. As a result, the translational efficiency is very low at 25 °C.</p>
+[[File:600px-NCTU_Test_functional_test_of_37RBS.png|center|600px|Figure 7. The normalized expression (florescence expression/ OD value) under 37 °C is higher than the expression under room temperature by about 6 folds." ]]
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