Difference between revisions of "Part:BBa K2904040"

Latest revision as of 22:05, 20 October 2019

Modular cobalamin riboswitch

Design

Background of 2019 OUC-China's project——RiboLego

Due to context-dependent performance and limited dynamic range, the widespread application of riboswitches is currently restricted. By replacing its original ORF with a new one, the structure of an aptamer domain can be subtly disrupted, resulting in a loss of ligand response. So riboswitch is still not be considered as a ‘plug and play' device. To tackle these problems, our project focuses on a standardized design principle to be used for modular and tunable riboswitch. The modular riboswitch we defined consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function.

The construction of this part

Our team’s vision is a standardized and easy adaptable design principle to be used for riboswitch of different purposes. By referencing the previous iGEM projects, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12. The cobalamin biosensor is based on a riboswitch taken from a transcribed fragment upstream of a cobalamin biosynthesis gene, cbiB, which is found in Propionibacterium shermanii and has been demonstrated to be sensitive to B12. At first, they used EGFP as their reporter gene, however, even in the absence of cobalamin, they had no GFP expression at all. Then they substituted EGFP with mRFP1 and inserted the first 24 bases of cbiB between them, the result was bad, too.

With our principle, the cobalamin biosensor can be optimized. The modular cobalamin biosensor consisting of the repressing riboswitch, the first 144bp of the original target gene and Tuner A. To test the functionality of the improved construct, the modular riboswitch was under the lac promoter and controlled the expression of mRFP1.

Result

The result by confocal microscopy

By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the original cobalamin biosensor designed by Paris_Bettencourt had mRFP1 introduced directly. It means that Stabilizer they chose was too short that couldn’t protect the structure of riboswitch. The structure offered by RNAfold also verified our prediction. On the contrary, the modular cobalamin riboswitch can express mRFP1 successfuly since Stabilizer can maintain the structure of riboswitch and Tuner has the ability to improve the function of one.

Figure1: The results by confocal microscopy, which indicates that our principle can improve cobalamin biosensor successfully. It's obvious that the modular cobalamin riboswitch can express mRFP1.

The result by microplate reader

To test the functionality of the improved construct, we measured the fluorescence level emitted by our strain in the presence of increasing levels of adocobalamin by microplate reader. We can observe an obvious decrease in mRFP1 expression with the adocobalamin concentration increased, which demonstrates that the riboswitch was improved successfully!

Figure2: The response curve of our improved cobalamin biosensor to Vb12. As we expected, an obvious decrease of mRFP1 expression was observed with the increased concentration of vb12. It demonstrates that the modular riboswitch functions properly.

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]

@@ Line 11: / Line 11: @@
 ===The construction of this part===
-Our team’s vision is a standardized and easy adaptable design principle to be used for riboswitch  of different purposes. By referencing the previous iGEM project, By referencing the previous iGEM project, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12. The cobalamin biosensor is based on a riboswitch taken from a transcribed fragment upstream of a cobalamin biosynthesis gene, cbiB, which is found in <i>Propionibacterium shermanii</i> and has been demonstrated to be sensitive to B12. At first, they used EGFP as their reporter gene, however, even in the absence of cobalamin, they had no GFP expression at all. Then they substituted EGFP with mRFP1 and inserted the first 24 bases of cbiB between them, the result was bad, too.
+Our team’s vision is a standardized and easy adaptable design principle to be used for riboswitch of different purposes. By referencing the previous iGEM projects, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12. The cobalamin biosensor is based on a riboswitch taken from a transcribed fragment upstream of a cobalamin biosynthesis gene, cbiB, which is found in <i>Propionibacterium shermanii</i> and has been demonstrated to be sensitive to B12. At first, they used EGFP as their reporter gene, however, even in the absence of cobalamin, they had no GFP expression at all. Then they substituted EGFP with mRFP1 and inserted the first 24 bases of cbiB between them, the result was bad, too.
 <br>
 <br>
@@ Line 21: / Line 21: @@
 <p>
-By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the original cobalamin biosensor designed by Paris_Bettencourt had mRFP1 introduced directly. However, the modular Btub riboswitch can express mRFP1 successfuly since Stabilizer can maintain the structure of riboswitch and Tuner has the ability to improve the function of one.
+By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the original cobalamin biosensor designed by Paris_Bettencourt had mRFP1 introduced directly. It means that Stabilizer they chose was too short that couldn’t protect the structure of riboswitch. The structure offered by RNAfold also verified our prediction. On the contrary, the modular cobalamin riboswitch can express mRFP1 successfuly since Stabilizer can maintain the structure of riboswitch and Tuner has the ability to improve the function of one.
-[[Image:T--OUC-China--parisxianwei.jpg|center|thumb|400px|'''Figure1: The results of modular Btub riboswitch containing Tuner A by microplate reader.'''  ]]
+[[Image:T--OUC-China--abali.png|center|thumb|400px|'''Figure1: The results by confocal microscopy, which indicates that our principle can improve cobalamin biosensor successfully. It's obvious that the modular cobalamin riboswitch can express mRFP1. '''  ]]
-<br>
-</p>
 ===The result by microplate reader===
+To test the functionality of the improved construct, we measured the fluorescence level emitted by our strain in the presence of increasing levels of adocobalamin by microplate reader. We can observe an obvious decrease in mRFP1 expression with the adocobalamin concentration increased, which demonstrates that the riboswitch was improved successfully!
-<p>
-The qualitative experiment is not enough to analyze the improved riboswitch. So we tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time.
-改The following chart shows the dynamic curve measured every two hours. It can prove that the modular Btub riboswitch can restore the structure of riboswitch and control the downstream gene expression during the whole cultivation period.
 <br>
 </p>
-[[Image:T--OUC-China--080microplateg.jpg|center|thumb|400px|'''Figure1: The results of modular Btub riboswitch containing Tuner A by microplate reader.'''  ]]
+[[Image:T--OUC-China--bagaipoint.png|center|thumb|400px|'''Figure2:  The response curve of our improved cobalamin biosensor to Vb12. As we expected, an obvious decrease of mRFP1 expression was observed with the increased concentration of vb12. It demonstrates that the modular riboswitch functions properly.'''  ]]
 <!-- Add more about the biology of this part here