IGEM:Caltech/2007/Project/Riboregulator: Difference between revisions

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'''Cis Repression'''  
'''Cis Repression'''  


In order to find a riboregulator with the necessary dynamic range, a total of eight cis repressive elements were designed (cr1-cr8) (Fig. 2). These consisted of the same overall stem-loop structure, but had varying strengths of complementarity within the stem, as seen by their free energies (Table 1). The designs of cr1-cr4 were based on previous work done by Collins et. al. and those for cr5-cr8 were based on work done by the University of California Berkeley 2006 International Genetically Engineered Machines team.  
In order to find a riboregulator with the necessary dynamic range, a total of eight cis repressive elements were designed (cr1-cr8). These consisted of the same overall stem-loop structure, but had varying strengths of complementarity within the stem, as seen by their free energies. The designs of cr1-cr4 were based on previous work done by Collins et. al. and those for cr5-cr8 were based on work done by the University of California Berkeley 2006 International Genetically Engineered Machines team.  
In order to determine the optimal level of repression, various aspects of RNA secondary structure were considered, including inner loops, single base pair bulges, and varying loop sizes. Higher free energies (i.e. less complementarity due to base pair mismatches) favor activation by taRNA because they destabilize the stem and facilitate the open RBS form upon addition of taRNA. The cis elements were inserted downstream of the Ptet promoter. The stem consisted of approximately 20-nt and the loop ranged from 6 to 10-nt. The YFP gene was inserted directly downstream of the cis sequence. Flow cytometry measurements were taken to quantify the expression of the cis riboregulated YFP gene.  
In order to determine the optimal level of repression, various aspects of RNA secondary structure were considered, including inner loops, single base pair bulges, and varying loop sizes. Higher free energies (i.e. less complementarity due to base pair mismatches) favor activation by taRNA because they destabilize the stem and facilitate the open RBS form upon addition of taRNA. The cis elements were inserted downstream of the Ptet promoter. The stem consisted of approximately 20-nt and the loop ranged from 6 to 10-nt. The YFP gene was inserted directly downstream of the cis sequence. Flow cytometry measurements were taken to quantify the expression of the cis riboregulated YFP gene.  


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'''Trans Activation'''  
'''Trans Activation'''  


To initiate translation, five trans-activating elements (ta1-ta5) were designed. Each sequence binds to the cis-repressive elements and opens up the RBS(Fig. 5). For ta1-ta4, part of the sequence was complementary to the hairpin loop of the cr element and extending in the 5’ direction. Element ta5 was designed to bind to the 5’ end of the cis regulator and open up the hairpin in the 3’ direction. ta1 and ta2 were complimentary to cr1-4; ta3-5 were complimentary to cr5-8. The energies of the trans and trans and cis combinations are given in Table 2. The activation with these ta elements has not yet been determined.  
To initiate translation, five trans-activating elements (ta1-ta5) were designed. Each sequence binds to the cis-repressive elements and opens up the RBS. For ta1-ta4, part of the sequence was complementary to the hairpin loop of the cr element and extending in the 5’ direction. Element ta5 was designed to bind to the 5’ end of the cis regulator and open up the hairpin in the 3’ direction. ta1 and ta2 were complimentary to cr1-4; ta3-5 were complimentary to cr5-8. The energies of the trans and trans and cis combinations are given in Table 2. The activation with these ta elements has not yet been determined.  




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'''Initial Results with Cis Repressors'''
'''Initial Results with Cis Repressors'''


Control plasmids with no cis repressive elements showed increased YFP fluorescence with no aTc (2.5%) and with 1ug/mL aTc (71.7%), using the YFP gene alone under TetR control as the 100% marker. Plasmids with cis-repressive elements were grown in cells under the same conditions. The cr7 cultures showed repression of YFP fluorescence at 0.1ug/mL (1.1%) and at 1ug/mL (3.7%), using the same plasmid without insert as a 100% marker (Fig. 3 & 4). The cr3 repressor showed 2.9% fluorescence and cr4 showed 0.84% fluorescence at 1ug/mL aTc, using the YFP gene alone under TetR control as the 100% marker. Table 1 lists fluorescence for all cr inserts measured to-date. Repression was observed for all variants measured.  
Control plasmids with no cis repressive elements showed increased YFP fluorescence with no aTc (2.5%) and with 1ug/mL aTc (71.7%), using the YFP gene alone under TetR control as the 100% marker. Plasmids with cis-repressive elements were grown in cells under the same conditions. The cr7 cultures showed repression of YFP fluorescence at 0.1ug/mL (1.1%) and at 1ug/mL (3.7%), using the same plasmid without insert as a 100% marker. The cr3 repressor showed 2.9% fluorescence and cr4 showed 0.84% fluorescence at 1ug/mL aTc, using the YFP gene alone under TetR control as the 100% marker.Repression was observed for all variants measured.  
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