Difference between revisions of "Team:Cardiff Wales/Team PlantP"

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<p align="left" style="background-color:#ffffff;"> <br><br><br>Team Plant Promoter consisted of Ryan and Niall. Their objective was to isolate four plant promoters that are found in <i> Arabidopsis thaliana </i> and create gene constructs with either luciferase or our TSH antagonist as the coding sequence. Another objective was to create a new variant of luciferase (LucX5) and express the P19 gene to get higher transgene expression levels. We could not get these to grow in <i> E. coli </i>, however. The plant promoters were isolated by grinding up <i> Arabidopsis thaliana </i> tissue, extracting the DNA, and amplifying the promoter sequences using PCR, with primers that would incorporate the BsmB1 type IIS restriction endonuclease recognition site, and only amplify the 3'-most 500bp of promoter DNA. These were then excised from a gel and purified. The DNA was put into the level 0 plasmid and can be seen on the <a href="https://2017.igem.org/Team:Cardiff_Wales/basicparts">basic parts</a> page. These promoters are PDF1.2 (jasmonic acid inducible), PR2 and GST6 (salicylic acid inducible), and WRKY30 (DAMP response inducible). These were then used to create level 1 constructs containing Luc+ or our TSH antagonist with 'His'-tags. Unfortunately, the promoter:luc+:NosT constructs grew in <i> E. coli </i>, but not in <i> Agrobacterium </i> when we ran out of lab time, so these constructs could not be tested. WRKY30 constructs with our TSH antagonist did not grow either. However, the TSH-antagonist <i> Agrobacterium</i>-constructs with PDF1.2, PR2, and GST6 were produced, and can be seen on our <a href="https://2017.igem.org/Team:Cardiff_Wales/compositeparts"> composite parts</a> page. These were used to transform our <i> Nicotiana benthamiana </i> plants, and had protein extractions performed on them following induction with the associated acid for ~6 hours. Sadly, these gels were not conclusive, with no bands present definitively in the samples with nickel beads to isolate the protein exclusively. Regardless, these gels can be seen on our <a href="https://2017.igem.org/Team:Cardiff_Wales/results"> results </a> page.
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<p align="left" style="background-color:#ffffff;"> <br><br><br>Team Plant Promoter consisted of Ryan and Niall. Their objective was to isolate four plant promoters that are found in <i> Arabidopsis thaliana </i> and create gene constructs with either luciferase or our TSH antagonist as the coding sequence. Another objective was to create a new variant of luciferase (LucX5) and express the P19 gene to get higher transgene expression levels; We could not get these to grow in <i> E. coli </i>, however. The plant promoters were isolated by grinding up <i> Arabidopsis thaliana </i> tissue, extracting the DNA, and amplifying the promoter sequences using PCR, with primers that would incorporate the BsmB1 type IIS restriction endonuclease recognition site, and only amplify the 3'-most 500bp of promoter DNA. These were then excised from a gel and purified. The DNA was put into the level 0 plasmid and can be seen on the <a href="https://2017.igem.org/Team:Cardiff_Wales/basicparts">basic parts</a> page. These promoters are PDF1.2 (jasmonic acid inducible), PR2 and GST6 (salicylic acid inducible), and WRKY30 (DAMP response inducible). These were then used to create level 1 constructs containing Luc+ or our TSH antagonist with 'His'-tags. Unfortunately, the promoter:luc+:NosT constructs grew in <i> E. coli </i>, but not in <i> Agrobacterium </i> when we ran out of lab time, so these constructs could not be tested. WRKY30 constructs with our TSH antagonist did not grow either. However, the TSH-antagonist <i> Agrobacterium</i>-constructs with PDF1.2, PR2, and GST6 were produced, and can be seen on our <a href="https://2017.igem.org/Team:Cardiff_Wales/compositeparts"> composite parts</a> page. These were used to transform our <i> Nicotiana benthamiana </i> plants, and had protein extractions performed on them following induction with the associated acid for ~6 hours. Sadly, these gels were not conclusive, with no bands present definitively in the samples with nickel beads to isolate the protein exclusively. Regardless, these gels can be seen on our <a href="https://2017.igem.org/Team:Cardiff_Wales/results"> results </a> page.
 
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Revision as of 11:44, 22 October 2017




Team Plant Promoter









Team Plant Promoter consisted of Ryan and Niall. Their objective was to isolate four plant promoters that are found in Arabidopsis thaliana and create gene constructs with either luciferase or our TSH antagonist as the coding sequence. Another objective was to create a new variant of luciferase (LucX5) and express the P19 gene to get higher transgene expression levels; We could not get these to grow in E. coli , however. The plant promoters were isolated by grinding up Arabidopsis thaliana tissue, extracting the DNA, and amplifying the promoter sequences using PCR, with primers that would incorporate the BsmB1 type IIS restriction endonuclease recognition site, and only amplify the 3'-most 500bp of promoter DNA. These were then excised from a gel and purified. The DNA was put into the level 0 plasmid and can be seen on the basic parts page. These promoters are PDF1.2 (jasmonic acid inducible), PR2 and GST6 (salicylic acid inducible), and WRKY30 (DAMP response inducible). These were then used to create level 1 constructs containing Luc+ or our TSH antagonist with 'His'-tags. Unfortunately, the promoter:luc+:NosT constructs grew in E. coli , but not in Agrobacterium when we ran out of lab time, so these constructs could not be tested. WRKY30 constructs with our TSH antagonist did not grow either. However, the TSH-antagonist Agrobacterium-constructs with PDF1.2, PR2, and GST6 were produced, and can be seen on our composite parts page. These were used to transform our Nicotiana benthamiana plants, and had protein extractions performed on them following induction with the associated acid for ~6 hours. Sadly, these gels were not conclusive, with no bands present definitively in the samples with nickel beads to isolate the protein exclusively. Regardless, these gels can be seen on our results page.