Difference between revisions of "Team:Sorbonne U Paris/Parts"

 
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<h2 class="title-h2">pSAD</h2>
 
<h2 class="title-h2">pSAD</h2>
 
<br>
 
<br>
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<p>pSAD is constitutive promoter pSAD from Chlamydomonas reinhardtii. It is a high constitutive expression promoter that encodes for a ferrodoxin-binding protein of photosystem I. This pSAD promoter was submitted in 2014 by the Concordia team (BBa_K1547005). To be compatible with the PhytoBrick standard (RFC1000), an illegal BpiI restriction site was removed (A289T). We cloned P pSAD by PCR amplification in A2-A3 fusion site to use it for our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM9121. We sequenced again the sequence to be sure there is no unwanted mutations. <br><br>
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<p>pSAD is a constitutive promoter in Chlamydomonas reinhardtii. It is is a high expression constitutive promoter that controls the expression of the gene encoding for a ferrodoxin-binding protein of photosystem I. This pSAD promoter was submitted in 2014 by the Concordia team (BBa_K1547005). To be compatible with the PhytoBrick standard (RFC1000), an illegal BpiI restriction site was removed (A289T). We cloned pSAD by PCR amplification in A2-A3 fusion site to use it for our retrotransposon design. The acceptor plasmid is the MoClo Universal Level 0 plasmid (pL0): pAGM9121. We sequenced again the sequence to be sure there are no unwanted mutations. <br><br>
−
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intented to be use as constitutive promoter for the transcription unit of paromomycine in the CARGO of our synthetic retrotransposons for directed evolution on the Chlamydomoas. It is part of our reporter system to characterize the activity and transposition rate.</p>
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<h6>Role in our project </h6>
 +
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intented to be used as a constitutive promoter for the transcription unit of paromomycine in the CARGO of our synthetic retrotransposons for directed evolution in the <i>Chlamydomonas</i>. It is part of our reporter system to characterize the activity and transposition rates.</p>
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<h2 class="title-h2">paromycin resistance gene</h2>
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<br>
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<p> The sequence of the paromomycine-resistance marker is an aminoglycoside antibiotic. The aphVIII gene from Streptomyces rimosus encodes an aminoglycoside 3’-phosphotransferase gene (aph). From the aphVIII gene sequence of the BioBrick BBa_K1884013 (iGEM16_SZU-China) BpiI estriction sites were removed ( G551C) by PCR point mutation. We cloned aphVIII by PCR amplification in B4-B5 position of the MoClo standard2 to use it in our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM91212. We sequenced again the sequence to be sure there is no unwanted mutations.
 +
<br><br>
 +
<h6>Role in our project </h6>
 +
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intended to be used as a reporter gene in the CARGO to test the functionality of our synthetic retrotransposons for directed evolution in C. reinhardtii.</p>
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<h2 class="title-h2">RBCS2i1</h2>
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<br>
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<p>This is the second intron of the rubisco protein from Chlamydomonas reinhardtii. </P>
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<h2 class="title-h2">3'LTR_Cr</h2>
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<br>
 +
<p>Long-Terminal-Repeat in the 3'end of a retrotransposon (or retrovirus). It contains the information necessary to start the reverse transcription. <p>
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<h2 class="title-h2">5'LTR</h2>
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<br>
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<p>Long-Terminal-repeat in the 5'end of a retrotransposon (or retrovirus). It contains the regulatory elements to start the transcription of the retrotransposon. </p>
  
 
<h2>Parts used in our project but not submitted to the registry</h2>
 
<h2>Parts used in our project but not submitted to the registry</h2>
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Latest revision as of 01:29, 18 October 2018

Parts

New parts submitted to the registry



Parts name Type Part number
RBCS2i1 Protein domain BBa_K2703000
pSAD Regulatory BBa_K2703002
3'LTR_Cr Regulatory BBa_K2703003
5'LTR Regulatory BBa_K2703004
paromycin resistance gene Coding BBa_K2703008


pSAD


pSAD is a constitutive promoter in Chlamydomonas reinhardtii. It is is a high expression constitutive promoter that controls the expression of the gene encoding for a ferrodoxin-binding protein of photosystem I. This pSAD promoter was submitted in 2014 by the Concordia team (BBa_K1547005). To be compatible with the PhytoBrick standard (RFC1000), an illegal BpiI restriction site was removed (A289T). We cloned pSAD by PCR amplification in A2-A3 fusion site to use it for our retrotransposon design. The acceptor plasmid is the MoClo Universal Level 0 plasmid (pL0): pAGM9121. We sequenced again the sequence to be sure there are no unwanted mutations.

Role in our project
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intented to be used as a constitutive promoter for the transcription unit of paromomycine in the CARGO of our synthetic retrotransposons for directed evolution in the Chlamydomonas. It is part of our reporter system to characterize the activity and transposition rates.

paromycin resistance gene


The sequence of the paromomycine-resistance marker is an aminoglycoside antibiotic. The aphVIII gene from Streptomyces rimosus encodes an aminoglycoside 3’-phosphotransferase gene (aph). From the aphVIII gene sequence of the BioBrick BBa_K1884013 (iGEM16_SZU-China) BpiI estriction sites were removed ( G551C) by PCR point mutation. We cloned aphVIII by PCR amplification in B4-B5 position of the MoClo standard2 to use it in our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM91212. We sequenced again the sequence to be sure there is no unwanted mutations.

Role in our project
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intended to be used as a reporter gene in the CARGO to test the functionality of our synthetic retrotransposons for directed evolution in C. reinhardtii.

RBCS2i1


This is the second intron of the rubisco protein from Chlamydomonas reinhardtii.

3'LTR_Cr


Long-Terminal-Repeat in the 3'end of a retrotransposon (or retrovirus). It contains the information necessary to start the reverse transcription.

5'LTR


Long-Terminal-repeat in the 5'end of a retrotransposon (or retrovirus). It contains the regulatory elements to start the transcription of the retrotransposon.

Parts used in our project but not submitted to the registry



s
Parts name Type Part number
otsA Coding Bba_K2703001
pL0-pSAD_A2-B1 Regulatory Bba_K2703005
otsB Coding Bba_K2703006
Bba_K2703006 Regulatory Bba_K2703007
5' UTR pSAD Regulatory Bba_K2703009
Gag Pol Coding Bba_K2703010
pAGM9121 backbone Bba_K2703011

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