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Team:Alberta/Project/promotor
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We finished the first part of our biological circuit construction (promoter switching) and discovered that the red fluorescent protein (rfp) gene was only expressed from the two strongest promoters, 4 (second strongest) and 5 (strongest), out of nine different promoters tested. The blue pigment protein (bpp) gene was only expressed from promoter 2, which is the second weakest of the tested promoters..Expression of the green fluorescent protein (gfp) gene was not detected using any promoter, unexpectedly. These results indicate that the recruitment of the RNA polymerase to initiate transcription and expression of these pigment genes can be accomplished using promoter 2 or higher. However, the strength of the promoter does affect visible color development in E. coli colonies. A larger amount of RFP is required as expression was found to only be driven by the strongest promoter while BPP was produced at sufficient levels using a weaker promoter. This may also indicate that BPP is toxic at higher levels driven from stronger promoters. The lack of GFP production may be a result of cloning errors or secondary structure of mRNA blocking RBS from ribosome binding. Therefore, We have decided to construct plasmids to test the different strengths of RBS and investigate the toxicity of colour proteins. We have also begun cloning transcriptional repressors in order to modulate level of pigment gene expression. | We finished the first part of our biological circuit construction (promoter switching) and discovered that the red fluorescent protein (rfp) gene was only expressed from the two strongest promoters, 4 (second strongest) and 5 (strongest), out of nine different promoters tested. The blue pigment protein (bpp) gene was only expressed from promoter 2, which is the second weakest of the tested promoters..Expression of the green fluorescent protein (gfp) gene was not detected using any promoter, unexpectedly. These results indicate that the recruitment of the RNA polymerase to initiate transcription and expression of these pigment genes can be accomplished using promoter 2 or higher. However, the strength of the promoter does affect visible color development in E. coli colonies. A larger amount of RFP is required as expression was found to only be driven by the strongest promoter while BPP was produced at sufficient levels using a weaker promoter. This may also indicate that BPP is toxic at higher levels driven from stronger promoters. The lack of GFP production may be a result of cloning errors or secondary structure of mRNA blocking RBS from ribosome binding. Therefore, We have decided to construct plasmids to test the different strengths of RBS and investigate the toxicity of colour proteins. We have also begun cloning transcriptional repressors in order to modulate level of pigment gene expression. | ||
Revision as of 22:03, 30 August 2012
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description
Description
Stage One
We finished the first part of our biological circuit construction (promoter switching) and discovered that the red fluorescent protein (rfp) gene was only expressed from the two strongest promoters, 4 (second strongest) and 5 (strongest), out of nine different promoters tested. The blue pigment protein (bpp) gene was only expressed from promoter 2, which is the second weakest of the tested promoters..Expression of the green fluorescent protein (gfp) gene was not detected using any promoter, unexpectedly. These results indicate that the recruitment of the RNA polymerase to initiate transcription and expression of these pigment genes can be accomplished using promoter 2 or higher. However, the strength of the promoter does affect visible color development in E. coli colonies. A larger amount of RFP is required as expression was found to only be driven by the strongest promoter while BPP was produced at sufficient levels using a weaker promoter. This may also indicate that BPP is toxic at higher levels driven from stronger promoters. The lack of GFP production may be a result of cloning errors or secondary structure of mRNA blocking RBS from ribosome binding. Therefore, We have decided to construct plasmids to test the different strengths of RBS and investigate the toxicity of colour proteins. We have also begun cloning transcriptional repressors in order to modulate level of pigment gene expression.
Stage Two we replaced constitutive promotor into inducible lac promotor to control the color gene expression and introduced (consetitutive) promotor 1, 2, 3, or 4 to control Lac I repressor expression. According to one of our experiment results, we have transformed
