Team:EPF-Lausanne/Our Project/T7 promoter variants/t7prom

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(Characterization of Randomer Variants)
(Non-random T7 promoter variants)
 
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We made two families of T7 promoter variants. Each family has six designed mutants and three randomer mutants. The only difference between the two families is the addition of a lac operator sequence downstream of the T7 promoter. These promoter variants were characterized using fluorescence, revealing a wide range of promoter efficiencies.
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We made two families of T7 promoter variants. Each family has six designed mutants and three randomer mutants. The only difference between the two families is the addition of a lac operator sequence downstream of the T7 promoter.  
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[[File:t7_lac_rbs_lysis_term.png|470px|center]]
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[[File:t7_rbs_lysis_term.png|400px|center]]
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These promoter variants were characterized using fluorescence, revealing a wide range of promoter efficiencies. To find out more about how these T7 promoter variants were made, please click [[Team:EPF-Lausanne/Our Project/T7 promoter variants/t7prom/making|here]].
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Here is a version of the T7 promoter consensus sequence:
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[[File:t7_logo.png|400px|center]]
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It is taken from Schneider and Stephens, ''Sequence Logos: A New Way to Display Consensus Sequences'' Nucleic Acids Research, 18 6097-6100, 1990
===Non-random T7 promoter variants===
===Non-random T7 promoter variants===
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The T7 promoter variants in this section were designed based on considerations from literature. These sequences, and their corresponding predicted strengths, also come from literature. The  
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The T7 promoter variants in this section were designed based on considerations from the relevant literature. These sequences, and their corresponding predicted strengths, also come from literature. The exact source is Imburgio, D., Rong, M., Ma, K. & McAllister, W.T. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39, 10419–10430 (2000).
Variants without additional lac operator (constitutive):
Variants without additional lac operator (constitutive):
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For each family, we tested the randomers and the designed variants separately. To characterize the promoter strengths, we used RFP as the reporter gene and used a platereader to test for fluorescence during and after induction with IPTG.  
For each family, we tested the randomers and the designed variants separately. To characterize the promoter strengths, we used RFP as the reporter gene and used a platereader to test for fluorescence during and after induction with IPTG.  
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[[File:non_random_dose_response.png|700px]]
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[[File:non_random_response.png|700px]]
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The six designed T7 promoter variants are named as a function of their predicted promoter efficiency, relative to the wildtype. For example, T7 14 has a predicted efficiency of 14% compared to the consensus T7 promoter, whereas T7 111 is predicted to be 111% more efficient than the wildtype. In the chart above, each of the designed promoter variants for both the T7 with and without the lac operator are arranged in increasing predicted efficiency. Contrary to our expectation, some variants that were designed to have a lesser efficiency than the wildtype (e.g. T7 54) seem to have a much higher strength (as measured by fluorescence at saturation, normalized by the optical density). Already in the designed variants, we see a substantial difference in the behaviour of the promoters that have a lac operator as opposed to those that do not. The data for this graph was produced in triplicate, so the error bar represents the standard error across those three measurements.  
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The six designed T7 promoter variants are named as a function of their predicted promoter efficiency, relative to the wildtype. For example, T7 14 has a predicted efficiency of 14% compared to the consensus T7 promoter, whereas T7 111 is predicted to be 111% more efficient than the wildtype. In the chart above, each of the designed promoter variants for both the T7 with and without the lac operator are arranged in increasing predicted efficiency. Contrary to our expectation, some variants that were designed to have a lesser efficiency than the wildtype (e.g. T7 54) seem to have a much higher strength (as measured by fluorescence at saturation, normalized by the optical density). The data for this graph was produced in triplicate, so the error bar represents the standard error across those three measurements.
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[[File:induction_ratio.png|700px]]
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=== Characterization of Randomer Variants ===
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In addition to fluorescence at saturation, another way to characterize promoter strength is to look at its induction ratio, which is the ratio, at saturation, of fluorescence produced by induction with IPTG versus fluorescence produced without induction. In layman's terms, it indicates how strongly the promoter reacts to induction. Here again, our results indicate that some promoter variants (T7 80 in particular) stand out with respect to this feature. Here too the importance of the lac operator in producing high induction ratios is not to be underestimated.
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We used the same platereader protocol to test the efficiency and induction ratios of the randomers using RFP fluorescence. Twelve colonies per variant were tested. The results indicate that the T7 -10 randomers, both the non-lac and lac varieties, have the highest fluorescence saturation levels (normalized by OD). The other randomer types seem to have negligible strength. [[File:randomers.png|700px]]
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=== Characterization of Randomer Variants ===
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[[File:randomers.png]]
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On the whole, the T7-lac promoters with random mutations at position -10 have higher fluorescence expressions than their T7 non-lac counterparts.  
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{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 02:11, 22 September 2011