Team:Hong Kong HKUST/pneumosensor/results/module one

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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_one">Sensing</a></li>
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<h2>Pneumosensor Results</h2>
<h2>Pneumosensor Results</h2>
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<p align="center" style= "font-size: 30px"> Detection Module </p>
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<p style= "font-size: 30px; text-align:center">Detection Module</p>
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<p>
<b>Tag protein</b>
<b>Tag protein</b>
<br>
<br>
We engineered in a FLAG protein tag in the 3’ end of ComD by including the sequence in <i>comD</i> extraction primer.
We engineered in a FLAG protein tag in the 3’ end of ComD by including the sequence in <i>comD</i> extraction primer.
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<br><br>
Forward primer to extract <i>comD</i> to clone into pSB1C3:  
Forward primer to extract <i>comD</i> to clone into pSB1C3:  
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TCTGGAGAATTCGCGGCCGCTTCTAGATGGATTTATTTGGATTTGGGACGG
TCTGGAGAATTCGCGGCCGCTTCTAGATGGATTTATTTGGATTTGGGACGG
<br>
<br>
<i>[6’cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comD]</i>
<i>[6’cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comD]</i>
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<br><br>
3’ primer to extract <i>comD</i> with engineered FLAG tag gene sequence:
3’ primer to extract <i>comD</i> with engineered FLAG tag gene sequence:
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GCCGGACTGCAGCGGCCGCTACTAGTATTATTACTTGTCGTCATCGTCTTTGTAGTCTCATTCAAATTCCCTCTTAAATCTAATGAT  
GCCGGACTGCAGCGGCCGCTACTAGTATTATTACTTGTCGTCATCGTCTTTGTAGTCTCATTCAAATTCCCTCTTAAATCTAATGAT  
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<i>comE</i> was extracted from pKHS-<i>come</i> kindly sent to us by ??. Extraction was done using the following primers:
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<i>comE</i> was extracted from pKHS-<i>come</i> kindly sent to us by Dr. Don Morrison (Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires). Extraction was done using the following primers:
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<br>
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<br><br>
Forward primer to extract <i>comE</i> to clone into pSB1C3:  
Forward primer to extract <i>comE</i> to clone into pSB1C3:  
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TCTGGAGAATTCGCGGCCGCTTCTAGATGAAAGTTTTAATTTTAGAAGATG
TCTGGAGAATTCGCGGCCGCTTCTAGATGAAAGTTTTAATTTTAGAAGATG
<br>
<br>
<i>[6’ cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comE]</i>
<i>[6’ cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comE]</i>
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<br><br>
Reverse primer to extract <i>comE</i> to clone into pSB1C3:
Reverse primer to extract <i>comE</i> to clone into pSB1C3:
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GCCGGACTGCAGCGGCCGCTACTAGTATCACTTTTGAGATTTTTTCTCTAA
GCCGGACTGCAGCGGCCGCTACTAGTATCACTTTTGAGATTTTTTCTCTAA
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<i>come</i> contained an illegal SpeI site, so we designed primers for site-directed mutagenesis:
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<i>comE</i> contained an illegal SpeI site, so we designed primers for site-directed mutagenesis:
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Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC
Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC
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Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG
Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG
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<br><br>
However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) <i>comE</i> forward primer & mutagenesis  
However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) <i>comE</i> forward primer & mutagenesis  
reverse primer; (ii) <i>comE</i> reverse primer & mutagenesis forward primer. The two fragments were then combined through Gibson Assembly.</p>
reverse primer; (ii) <i>comE</i> reverse primer & mutagenesis forward primer. The two fragments were then combined through Gibson Assembly.</p>
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST">Home</a></h4>
 
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<li class='site_link'><a href="">Pneumosensor</a></li>
 
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<li class='site_link'><a href="">Riboregulator</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_one">Sensing</a></li>
 
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Latest revision as of 09:29, 12 October 2014



Pneumosensor Results

Detection Module

Overview

The two-component regulatory system in S. pneumoniae, consisting of the receptor ComD and its response regulator ComE was to be used in detecting the autoinducer molecule, competence-stimulating peptide (CSP) and so detect S. pneumoniae populations correspondingly.

Construct

Tag protein
We engineered in a FLAG protein tag in the 3’ end of ComD by including the sequence in comD extraction primer.


Forward primer to extract comD to clone into pSB1C3:

TCTGGAGAATTCGCGGCCGCTTCTAGATGGATTTATTTGGATTTGGGACGG
[6’cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comD]


3’ primer to extract comD with engineered FLAG tag gene sequence:

GCCGGACTGCAGCGGCCGCTACTAGTATTATTACTTGTCGTCATCGTCTTTGTAGTCTCATTCAAATTCCCTCTTAAATCTAATGAT
[6’ cap][21’ RFC10 suffix][6’ reverse complement stop codon][25’ reverse complement FLAG protein ][30 reverse complement Streptoccocus pneumoniae/NCTC7465/comD]



comE was extracted from pKHS-come kindly sent to us by Dr. Don Morrison (Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires). Extraction was done using the following primers:


Forward primer to extract comE to clone into pSB1C3:

TCTGGAGAATTCGCGGCCGCTTCTAGATGAAAGTTTTAATTTTAGAAGATG
[6’ cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comE]


Reverse primer to extract comE to clone into pSB1C3:

GCCGGACTGCAGCGGCCGCTACTAGTATCACTTTTGAGATTTTTTCTCTAA
[6’ cap][21’ RFC10 suffix][24’reverse complement Streptoccocus pneumoniae/NCTC7465/comE]



comE contained an illegal SpeI site, so we designed primers for site-directed mutagenesis:


Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC

Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG


However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) comE forward primer & mutagenesis reverse primer; (ii) comE reverse primer & mutagenesis forward primer. The two fragments were then combined through Gibson Assembly.

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