Team:Bologna

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= Project Description =
 
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'''Which is our idea?'''
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= Project Summary =
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Our project aims the realization of a device for gene expression regulation at translation level, regardless of the target gene.
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'''Our idea'''
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The aim of our project is the design of a standard device to control the synthesis of any protein of interest. This "general-purpose" device, implemented in <i>E. coli</i>, acts at the translational level to allow silencing of protein expression faster than using regulated promoters. We named this device <b>T-REX</b> (<b>T</b>rans <b>R</b>epressor of <b>Ex</b>pression).
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'''How T-REX works'''
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'''How can we achieve this?'''
 
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We designed a 50bp non coding sequence (CIS - repressing) to be assembled upstream of the target gene RBS, while its complementary sequence (TRANS - repressor) is to be placed under the control of another promoter. The TRANS - repressing contains also a cover, that is a short sequence  complementary to the RBS.
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When the TRANS-repressor element is present, it binds to the CIS-repressing, forming a RNA duplex and producing an obstruction that prevents the ribosome binding to the RBS.
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The device consists of two new BioBricks:
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<li><font color="#000080"><b>CIS-repressing</b></font>, to be assembled upstream of the target coding sequence.
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<li><font color="#000080"><b>TRANS-repressor</b></font>, complementary to the CIS-repressing and placed under the control of a different promoter.  
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CIS-repressing and TRANS-repressor sequences were designed by [[Team:Bologna/Software#1|BASER]] software.
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Transcription of the target gene yields a mRNA strand - containing the CIS-repressing sequence at its 5' end - available for translation into protein by ribosomes (<i>see Fig. 1, left panel</i>). When the promoter controlling the TRANS coding sequence is active, it drives the transcription of an oligoribonucleotide complementary to the CIS mRNA sequence. The TRANS/CIS <b>RNA duplex</b> prevents ribosomes from binding to RBS on target mRNA, thus <b>silencing protein synthesis</b>. The amount of the TRANS-repressor regulates the rate of translation of the target mRNA (<i>see Fig. 1, right panel</i>)
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[[Image:project3b.png|center|950px|thumb|<center>Figure 1 - T-REX device</center>]]
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'''How we can test the device'''
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'''What can we use it for?'''  
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After doing some tests to prove that the presence of CIS – repressing doesn't alter the cell normal activities, we are trying to realize some simple circuits in order to characterize our device.
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In order to test and characterize our T-REX device, we developed the following genetic circuit (Fig 2):
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Several are the possible future applications, but at the moment we are aiming to oscillators and flip-flop memories.
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More details about our work in the [[Team:Bologna/Project|Project]] section.  
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[[Image:circuit2OK.jpg|center|900px|thumb|<center>Figure 2 - Genetic Circuit to test CIS and TRANS' mRNA affinity</center>]]
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== Project Abstract ==
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More details about our work are reported in the [[Team:Bologna/Project|Project]] section.
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The project aims to realize a device with standard biological parts for the post-transcriptional control of gene expression, regardless of the gene sequence to be silenced.
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To achieve this, we designed the T-REX device, composed of two standard parts: the TRANS-repressor and the CIS-repressing elements. The TRANS-repressor is a non-coding DNA sequence, the transcript of which acts as a silencer of the CIS-repressing RNA target. This target includes a region complementary to the sequence of the TRANS-repressor antisense, ends with a ribosome binding site (RBS), and is assembled upstream of the coding sequence of the gene to be silenced. Upon binding of TRANS-repressor and CIS-repressing RNAs, the access to RBS by ribosomes is hampered, silencing translation from the downstream transcript. Accordingly, The amount of TRANS-repressor controls the translation rate of the regulated gene.
 
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The TRANS-repressor sequence was determined by a computational analysis performed to minimize the interference with the genomic mRNAs and to maximize the base-pairing interaction to the CIS-repressing RNA.
 
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The T-REX device is proposed as a universal and fast switch in synthetic gene circuits.
 
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= Acknowledgements =
= Acknowledgements =
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* ''' [http://www.unibo.it/Portale/default.htm University of Bologna] '''
* ''' [http://www.unibo.it/Portale/default.htm University of Bologna] '''
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* <font color=#0000cd>'''Cultural Association San Sebastiano'''</font>
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Latest revision as of 03:12, 22 October 2009

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Project Summary


Our idea

The aim of our project is the design of a standard device to control the synthesis of any protein of interest. This "general-purpose" device, implemented in E. coli, acts at the translational level to allow silencing of protein expression faster than using regulated promoters. We named this device T-REX (Trans Repressor of Expression).


How T-REX works


The device consists of two new BioBricks:

  • CIS-repressing, to be assembled upstream of the target coding sequence.
  • TRANS-repressor, complementary to the CIS-repressing and placed under the control of a different promoter.

CIS-repressing and TRANS-repressor sequences were designed by BASER software.

Transcription of the target gene yields a mRNA strand - containing the CIS-repressing sequence at its 5' end - available for translation into protein by ribosomes (see Fig. 1, left panel). When the promoter controlling the TRANS coding sequence is active, it drives the transcription of an oligoribonucleotide complementary to the CIS mRNA sequence. The TRANS/CIS RNA duplex prevents ribosomes from binding to RBS on target mRNA, thus silencing protein synthesis. The amount of the TRANS-repressor regulates the rate of translation of the target mRNA (see Fig. 1, right panel)

Figure 1 - T-REX device



How we can test the device


In order to test and characterize our T-REX device, we developed the following genetic circuit (Fig 2):

Figure 2 - Genetic Circuit to test CIS and TRANS' mRNA affinity




More details about our work are reported in the Project section.


Acknowledgements


  • [http://www.unibo.it/Portale/default.htm University of Bologna]


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  • [http://serinar.criad.unibo.it Ser.In.Ar. Cesena]


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  • Cultural Association San Sebastiano
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