Difference between revisions of "Part:BBa K1017602"

Revision as of 09:31, 22 September 2013 (view source) Mike821005 (Talk | contribs) ← Older edit		Latest revision as of 15:37, 29 September 2013 (view source) Yizaochen (Talk | contribs) (→‎Data)
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	<partinfo>BBa_K1017602 short</partinfo>		<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. <br>When the temperature is higher than 37℃, 37℃ RBS functions normally and the downstream gene can be expressed. In contrast with high temperature,37℃ RBS forms a hairpin structure that block the ribosome binding when lower 37℃. Thus, the downstream gene, mGFP, is not able to translate.[[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]]	+	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<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 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. 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." ]]
−	<br>From chart 1., we can clearly found that the time to reach same O.D. value(0.8) various as the temperature changes.
−	[[File:NCTU_37RBS_result.png|center|600px|Chart 1.37°C RBS efficiency under different temperatures.]]
−
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here
	===Usage and Biology===		===Usage and Biology===
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	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>
−	<partinfo>BBa_K1017602 SequenceAndFeatures</partinfo>	+	<partinfo>BBa_K1017603 SequenceAndFeatures</partinfo>
	<!-- Uncomment this to enable Functional Parameter display		<!-- Uncomment this to enable Functional Parameter display
	===Functional Parameters===		===Functional Parameters===
−	<partinfo>BBa_K1017602 parameters</partinfo>	+	<partinfo>BBa_K1017603 parameters</partinfo>
	<!-- -->		<!-- -->

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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.

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.

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.

Sequence and Features