Development of the Theophylline Riboswitch 1 on 2 Meeting 09/03/13.
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Transcript of Development of the Theophylline Riboswitch 1 on 2 Meeting 09/03/13.
![Page 1: Development of the Theophylline Riboswitch 1 on 2 Meeting 09/03/13.](https://reader035.fdocuments.us/reader035/viewer/2022062504/5a4d1b6e7f8b9ab0599b4425/html5/thumbnails/1.jpg)
Development of the Theophylline Riboswitch
1 on 2 Meeting 09/03/13
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Original Riboswitch Design (Desaia and Gallivan, 2004)
5’-GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC-3’
ATG TGA
B-galactosidase: Translated Region
RBS
5 bp upstream
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RBSFwd Primer
Kpn1 Aptamer 5 bp 25 bp of RBS
B-galactosidase: Translated Region
Untranslated region8bp spacer
Original Riboswitch Design (Desaia and Gallivan, 2004)
Untranslated region
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Results
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Results
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Paper published on mechanistic Insights into Riboswithces(Lynch et al., 2007)
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Selection
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Results
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Results
8.1
8.2
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• 5 Riboswitches• Experiment with Mutation in the RBS to
increase base pairing with Aptamer• Experiment with changing positions of the
random sequencing of spacers
Final Theophyline Riboswitch(Topp et al., 2010)
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A B C
C2 D E
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RBS’ of RiboswitchesRiboswitch A B C C2 D E
Sequence AAGGGG AGGGGU AAGA AAGGGA AAGGUAA AAGGAGGU
BP with Ribosome
6 7 0 5 4 8
RBS---Shine-Dalgarno sequence: AGGAGGU
Generated a library of randomized RBS sequences to see which one based paired to Aptamer
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Results
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Plan of Action
• Write a python code that will generate randomized sequences for the 8bp spacer – The script will run the 65,536 combinations
through M-fold • Have a certain number of candidates
synthesized• Test candidates in the lab
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Aptamer
Complete Riboswitch
65,536 Riboswitches
Riboswtiches with Calculated Free
Energy
Putative Riboswitches
RBS
Mutate for 6,7,8 or base paring with
RNA polymerase
Library of RBS’s
Riboswitch w/o Aptamer
Add Riboswitch w/o apatamer
Randomize 8 bp between RBS and
aptamer
Predict free energy of aptamer
Filter out riboswitches too
large of free energy
M-fold
Add two 8 bp spacers and ATG to RBS
Riboswitches with greater free energy than aptamer