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IGEM:Caltech/2007/Project/Riboregulator: Difference between revisions
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==Riboregulator Design== | ==Riboregulator Design== | ||
'''Design Background''' | |||
Our riboregulators consist of two interacting parts: cis and trans. The cis sequence is located downstream from the promoter to a gene of interest and upstream of the ribosome binding site (RBS). This cis sequence is complementary to the RBS and forms a stem-loop at the 5’ end of the mRNA after transcription, thus blocking ribosome binding and translation. Gene expression is turned on with a trans noncoding RNA. This trans sequence is produced from another promoter in a different plasmid and targets the cis-repressed RNA with high specificity. When the trans transcript is available, it binds to the cis region and thus opens the RBS for ribosome docking and subsequent translation. | Our riboregulators consist of two interacting parts: cis and trans. The cis sequence is located downstream from the promoter to a gene of interest and upstream of the ribosome binding site (RBS). This cis sequence is complementary to the RBS and forms a stem-loop at the 5’ end of the mRNA after transcription, thus blocking ribosome binding and translation. Gene expression is turned on with a trans noncoding RNA. This trans sequence is produced from another promoter in a different plasmid and targets the cis-repressed RNA with high specificity. When the trans transcript is available, it binds to the cis region and thus opens the RBS for ribosome docking and subsequent translation. | ||
The cis repressive sequences are approximately 50 base pairs and do not alter the reading frame of the native gene. The trans RNA is approximately 90 base pairs and contains a nucleotide sequence that is complementary to the cis RNA. RNAstructure3 software was used to predict the secondary structure as well as the free energy of the cis and trans elements and their interactions. | The cis repressive sequences are approximately 50 base pairs and do not alter the reading frame of the native gene. The trans RNA is approximately 90 base pairs and contains a nucleotide sequence that is complementary to the cis RNA. RNAstructure3 software was used to predict the secondary structure as well as the free energy of the cis and trans elements and their interactions. | ||
'''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 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. | |||
'''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. | |||
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==Status and Future Plans== | ==Status and Future Plans== | ||
'''Deborah and Kat - please fill in here''' | '''Deborah and Kat - please fill in here''' | ||
'''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. | |||
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