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Team:Alberta/Project/promotor
From 2012.igem.org
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A promoter is a genetic element that initiates transcription. To initiate transcription, the promoter must have a binding site for a certain RNA polymerase. There are often certain sigma factors in prokaryotes that aid the polymerase in finding the promoter region. Promoters can be either inducible or constitutive.
- A constitutive promoter is a promoter that is constantly driving transcription, regardless of transcription factors and environmental conditions. A constitutive promoter is useful for achieving high levels of expression of a recombinant protein, or to produce a transcription factor that is part of a regulatory transcription system (as we did in our project with lacI). These promoters have constant output levels, and these levels can only be changed by modifying the actual sequence of the promoter to weaken it or strengthen it.
- An inducible promoter is a promoter that can be activated or inactivated when a specific biotic or abiotic factor is present or absent. This type of promoter can be used to engineer regulation systems in vivo with clear and calibrated outputs. As well, an inducible promoter usually utilizes other transcriptional regulators such as repressors which can be de-repressed. In our project, we used the lac operating system. In this system, the lac operator is the inducible promoter that drives transcription of a gene of interest.
we replaced constitutive promoter with inducible lac promoter to control the color gene expression and introduced (consetitutive) promoter 3 to control Lac I repressor expression. We also replaced wild tyepe ori promoter with wild type -10 and -35 region and modification of lac operator between -10 and -35 region, in order to control plasmid replication under selective conditions; We have similar type of replacement with wild type ori promoter by switching promtor to Tet promoter to regulate the plasmid replication under selective condition.
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
Using theold Ribosome binding site (RBS) generated by a computer program and unique foreach colour gene, we achieved tiny, colorless or dull colonies. Therefore, weswitched the lacking RBS with a new known RBS, which was acquired from adifferent iGEM group.
The two differences between the old and new plasmids are that all the colour genes now share anidentical RBS, and the novel RBS has been previously confirmed as working. We attached the new RBS by performing PCR and transformed it in Top10 E.coli cells. As a result, the yellow colonies expressed a vivid colour but still grew quickly, indicating that the over expression experienced with the blue gene’s original RBS was not a problem with the yellow gene’s modified RBS. The blue gene with the new RBS, however,is not expressed. Over expression, resulting in toxicity because of high concentrations of pigments, and under expression, resulting in death from low concentration of antibiotic resistance may be possible explanations. The redgene shows the same expression compared to the old RBS.
