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

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<h2>Pneumosensor Results</h2>
<h2>Pneumosensor Results</h2>
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<p font-size:"17px"> Detection Module </p>
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<p style= "font-size: 30px; text-align:center">Detection Module</p>
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<p>The activity of combox promoter is turned on by a specific sigma factor that is produced by a regulatory gene <i>comX</i>. The sigma factor X will bind to the combox promoter region and activate gene expression. Sigma factor X will serve as an inducer with high specificity as it binds to an area of several specific base pairs on the combox promoter. This ComX and combox system could be used as a highly specific reporting system in our Streptococcus pneumonia detection platform.
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<p>The two-component regulatory system in <i>S. pneumoniae</i>, 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 <i>S. pneumoniae</i> populations correspondingly. </p>
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However in nature, ComX protein will be degraded by clpXP enzyme which exist in E.Coli and some other bacteria. Hence, to ensure the induction of combox promoter by comX, comW protein is needed as it functions to protect ComX protein from being degraded by clpXP. ComW protein will be degraded instead, increasing the amount of ComX protein produced.
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<p><b><u>Contruct</u></b><br><br>
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The three main components of the construct are <i>comX</i> gene, <i>comW</i> gene, and combox promoter. We assemble <i>comX</i> and combox promoter in one vector plasmid, while <i>comW</i> in a different plasmid. The system will be fused with a tagging protein and a reporting protein. Tagging protein is essential for detecting the ComX and ComW protein expression by means of western blot. Reporting protein which is fluorescence protein is needed for reporting purpose, hence <i>comX</i> and combox system could serve as a specific reporting system that will be useful for many synthetic constructs. <i>comX</i> and combox promoter construct will be assembled separately in different plasmid before being combined into one plasmid. </p>
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<div class='content_1'><h3>ComX Generator construct (BBa_K1379006) and comW construct </h3>
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<div class='content_1'><h3>Construct</h3>
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<p>Backbone pSB1C3 was used for ComX generator construct and comW construct. <i>comX</i> gene / <i>comW</i> gene was fused with BBa_K880005 which contains a constitutive promoter (J23100) and strong RBS (B0032). The purpose of this strong constitutive promoter and strong RBS is to unsure the large production of ComX and ComW protein throughout time. Then, a double terminator (B0015) is fused with the promoter, RBS, and ComX. BBa_K880005 and B0015 was obtained from 2014 iGEM distribution kit. <br><br>
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<p>
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Construct using pSB1C3 backbone with only <i>comX</i> gene (BBa_K1379004), with <i>comX</i> gene and BBa_B0015 double terminator (BBa_K1379045) was also built.
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<b>Tag protein</b>
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<br>
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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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<b><u>Bacterial Strain</u></b><br>
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The bacterial strain of E.coli that were used is DH10B. Since this strain of E.Coli have clpXP degradation enzyme which target ComX for degradation, an excess amount of ComX protein are required to maintain enough amount of ComX for combox promoter induction.
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Forward primer to extract <i>comD</i> to clone into pSB1C3:
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TCTGGAGAATTCGCGGCCGCTTCTAGATGGATTTATTTGGATTTGGGACGG
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<b><u><i>comX</i> and <i>comW</i> gene</u></b><br>
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<br>
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<i>comX</i> gene and <i>comW</i> gene was cloned from NCTC 7465 E.Coli strain genomic DNA by PCR using Vent Polymerase.
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<i>[6’cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comD]</i>
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3’ primer to extract <i>comD</i> with engineered FLAG tag gene sequence:
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<b><u><i>comX</i> Tag Protein</u></b><br>
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<br><br>
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We engineered a C-myc protein tag in the 3’ ends of <i>comX</i> by including the sequence in <i>comX</i> extraction primer. <br><br>
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GCCGGACTGCAGCGGCCGCTACTAGTATTATTACTTGTCGTCATCGTCTTTGTAGTCTCATTCAAATTCCCTCTTAAATCTAATGAT
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3’ primer to extract <i>comX</i> with engineered C-myc tag gene sequence:<br><br>
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<br>
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GCCGGA
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<i>[6’ cap][21’ RFC10 suffix][6’ reverse complement stop codon][25’ reverse complement FLAG protein ][30 reverse complement Streptoccocus pneumoniae/NCTC7465/comD]</i>
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CTGCAGCGGCCGCTACTAGTA
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TTATTA
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CAGATCCTCTTCTGAGATGAGTTTTTGTTC GTGGGTACGGATAGTAAACTCCTTAAACAC
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[6’ Cap]
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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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[21’ SpeI and PstI restriction site]
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<br>
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[6’ terminator sequence]
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<br><br>
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[30’ C-myc protein]
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Forward primer to extract <i>comE</i> to clone into pSB1C3:  
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TCTGGAGAATTCGCGGCCGCTTCTAGATGAAAGTTTTAATTTTAGAAGATG
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<i>[6’ cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comE]</i>
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<b><u><i>ComW</i> Tag Protein</u></b><br>
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Reverse primer to extract <i>comE</i> to clone into pSB1C3:
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We engineered a FLAG protein tag in the 3’ ends of <i>comW</i> by including the sequence in <i>comW</i> extraction primer. <br><br>
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<br><br>
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3’ primer to extract <i>comW</i> with engineered FLAG tag gene sequence:<br><br>
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GCCGGACTGCAGCGGCCGCTACTAGTATCACTTTTGAGATTTTTTCTCTAA
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GCCGGA
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<br>
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CTGCAGCGGCCGCTACTAGTA
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<i>[6’ cap][21’ RFC10 suffix][24’reverse complement Streptoccocus pneumoniae/NCTC7465/comE]</i>
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TTATTA
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<br>
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CTTGTCGTCATCGTCTTTGTAGTC
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ACAAGAAATAAAACCCCGATTCATTACCAATT
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<i>comE</i> contained an illegal SpeI site, so we designed primers for site-directed mutagenesis:
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<br>
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[6’ Cap]
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<br><br>
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[21’ SpeI and PstI restriction site]
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Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC
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[6’ terminator sequence]
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<br>
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[24’ FLAG protein]
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[32' reverse complementary of 3’ <i>comW</i>]
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Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG
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</i>
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However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) <i>comE</i> forward primer & mutagenesis
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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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<div class='content_1'><h3>PCelA (BBa_ K1379002) and Phelicase (BBa_ K1379003) construct </h3>
 
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Backbone pSB1C3 was used for PCelA and Phelicase construct. PCelA / Phelicase gene was fused with BBa_E0240, which contains a medium RBS (BBa_B0032), GFP (BBa_E0040) and double terminator (BBa_B0015). The purpose of this GFP generator is to indicate the functionality of PCelA and Phelicase in the presence and absence of ComX protein. BBa_E0240 was obtained from 2014 iGEM distribution kit. The bacterial strain of E.coli used is DH10B.
 
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<Br><br>
 
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<b><u>PCelA / Phelicase gene</u></b><br>
 
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PCelA and Phelicase gene was cloned from the genomic DNA of E.Coli strain NCTC7465 by PCR using Vent Polymerase. The difference between these two promoters is the whole sequence of Phelicase was obtained from Wellcome Trust Sanger Institute, a British genomics and genetics research institute. (https://www.sanger.ac.uk/)
 
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<br><br>
 
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PcelA Forward primer: TTTCTGTCTAGAGTTGACCAAGGAAGACTATTTTGC<br><br>
 
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PcelA Reverse primer: GCCGGACTGCAGCGGCCGCTACTAGTAATTTTCTCCTCTCTTAGATTATTCGTAAGAGG<br><br>
 
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Phelicase Forward primer: TTTCTGTCTAGAGTGGACTTGGCCGTCCTCT<br><br>
 
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Phelicase Reverse primer: GCCGGACTGCAGCGGCCGCTACTAGTAGACGTTCTTCTTCTGTTAATTCATTCTCAG<br><br>
 
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<div class='content_1'><h3>Assembly and Characterization </h3>
 
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<p><b><u>Assembly</b></u>
 
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<i>comX</i> and <i>comW</i> construct contain 3 parts that needs to be assembled: K880005 which contains constitutive promoter and RBS, <i>comX</i> engineered with C-myc tag / comW engineered with FLAG tag, and a double terminator in pSB1C3 backbone. Promoter, RBS, <i>comX</i> engineered with C-myc tag, and double terminator were combined using traditional digestion and ligation method. The ligation product was confirmed by digestion check and sequencing. <br><br>
 
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Combox construct also contains 3 parts that need to be assembled: PcelA/Phelicase promoter, BBa_E0240 which contains RBS, GFP and double terminator, and pSB1C3 backbone. All three parts were combined using traditional digestion and ligation method. The final ligation product was confirmed by digestion check and sequencing.
 
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<Br><br>
 
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<b><u>Characterization</u></b><br>
 
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RPU (Relative promoter unit) and leakage will be measured as a characterization of 100 base pairs combox promoter (PcelA), and 160 base pairs combox promoter (Phelicase). For combox promoter characterization, ComX generator construct which contain K880005, <i>comX</i> gene, and B0015, is ligated with PcelA / Phelicase construct which contain combox promoter and GFP generator. In order to characterize the two combox promoters, ComX generator-combox construct was migrated from pSB1C3 to pSB3K3. RPU are measured with J23100 Andersen family promoter as a reference promoter.
 
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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/human_practice">Human Practice</a></li>
 
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<li class='site_link'><a href="">Team</a></li>
 
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor">Pneumosensor</a></h4>
 
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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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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_two">Expressing</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/parts">Parts</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/data">Data</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization">Characterization</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/results">Results</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/future_work">Future Work</a></li>
 
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</ul>
 
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</td>
 
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator">Riboregulator</a></h4>
 
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<ul class= 'site_list'>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/CR_TA_Feature_Page">Feature Page</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/RNA_devices_catalog">Catalog Page</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/parts">Parts</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/data">Data</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">Characterization</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/results">Results</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/future_work">Future Work</a></li>
 
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</ul>
 
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</td>
 
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice">Human Practice</a></h4>
 
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<ul class= 'site_list'>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit">Start-up Kit</a>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/handbook" >--Handbook</a></li>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/report" >--Report</a></li>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/database" >--Database</a></li>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/interview" >--Interview</a></li>
 
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</ul>
 
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</li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach">Outreach</a>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/Workshop" >--Workshop</a></li>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/talks" >--Talk</a></li>
 
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/isf_academy" >--ISF Academy</a></li>
 
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</ul>
 
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</li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/safety_and_ethics">Safety and Ethics</a></li>
 
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</ul>
 
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</td>
 
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team">Team</a></h4>
 
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<ul class= 'site_list'>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/members">Members</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/advisers">Advisers</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/instructors">Instructors</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/attribution">Attributions</a></li>
 
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<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/acknowledgement">Acknowledgement</a></li>
 
-
<li class='site_link'><a href="https://igem.org/Team.cgi?year=2014">Official Team Profile</a></li>
 
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</ul>
 
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</td>
 
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab">WetLab</a></h4>
 
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<ul class= 'site_list'>
 
-
<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/notebook">Notebook</a></li>
 
-
<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a></li>
 
-
<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/safety">Safety</a></li>
 
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</ul>
 
-
</td>
 
-
<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/Achievements">Achievement</a></h4>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/medal_requirement">Medal Requirements</a></li>
 
-
<li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/deliverable">Deliverable</a></li>
 
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</ul>
 
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</td>
 
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</tr>
 
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</table>
 
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</div>
 
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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.

Home

Pneumosensor

Riboregulator

Human Practice

Team

WetLab

Achievement

Retrieved from "http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/results/module_one"