Hi, the name is Brourd. I am currently creating this page to test my wiki editing skills. If all else fails, we may just need to bring in an wiki crash cart. More info coming soon...
I wish I had watched that EteRNA University Program about how to use the wiki!
Outline for "A Method for Experimental Testing and Validation of Multistate RNA Systems" (tentative title)
- Assemble a team of players, to brainstorm for ideas, criticism, and development of riboswitch constructs.
- Creation of multiple FMN mimic sequences, for use in the competition structure.
- Determine if any other options for aptamer competition constructs work, and implement if they do.
- Determine the timeframe for experiments, and options we have for experimental validation.
- Assign basic reading material to team members, to make them familiar with previous work on riboswitches, and the problems currently associated with multistate RNA systems.
- Determine if a new scoring function is needed for riboswitches, as well as the development of a custom scoring function, if necessary.
- Begin the summary of riboswitch work from 2012 and 2013.
- Initial Testing of FMN Mimic Competition Construct
- Hopefully, the team can come up with various examples of FMN constructs.
- 10-30 slots for each construct.
- If any slots remain, they will be used for existing FMN switch constructs.
- Design of FMN riboswitch constructs, based on knowledge gained from the results of previous labs, as well as anything that may be gleaned from the published literature.
- Hand pick FMN riboswitches from previous labs, for testing.
- Hand pick FMN riboswitch sequences from previous labs, for testing.
*NOTE* First riboswitch constructs must be simple. No pseudoknots, ribozyme sequences, or triple stranded, water mediated, RNA structures, allowed.
- Continued synthesis of competition constructs
- First FMN Riboswitch constructs will be synthesized.
- Each sequence will take up 3-4 synthesis slots; 1 synthesis slot for the WT sequence, and 2-3 for mimics.
- Sequences may be a mix of player created and team created, details will be worked out in the first interim period.
- Begin testing of sequences from previously synthesized FMN riboswitches.
- Round 2 will be solely focused on sequences from work done with FMN riboswitches in 2012.
- May use this round to test the unsynthesized hand and finger lab from late 2012.
- Analyze results of Round 1
- Some select FMN Mimic Competition constructs will have be synthesis in round 3 with a limited mutate and map(M2), protocol developed by the Das lab, and ran through REEFIT, developed by Pablo Cordero.
- Continue further development of FMN riboswitch constructs.
- The complexity of the riboswitches will be increased, from simple structures, to somewhat less simple strutures. (additional stems, loops, etc.)
- Select 2013 FMN Riboswitch designs for synthesis with mimics.
- Request additional synthesis slots, for the increased thorughput of the designs being analyzed using M2.
- Synthesis of more complex riboswitch structures.
- M2 Synthesis of competition constructs.
- Synthesis of 2013 FMN Riboswitches.
After this point, the cycle of analysis, synthesis, analysis, and synthesis, will continue on until the middle/end of the summer of 2014. The FMN riboswitch constructs will continue to become more and more complex, with the addition of structural features such as pseudoknots, multibranch loops, and ribozyme sequences. After some future point in time, a small number of select FMN competition constructs will have FMN titration tests run for them. Using this data, the hope is for the creation of a FMN mimic that can adequately model the free energy contribution gained from FMN at a specific concentration, or at least be pretty close.
- Synthesis of the best candidate sequences and structures gained from the previous rounds of synthesis, using the "ideal" FMN mimic
- Request that the Das Lab run the next round of synthesis with FMN in the solution, in order to test the ideal structure and sequence candidates.
- Begin compiling data, hopefully with promising results!
Round X+1 +FMN
- Synthesis of the most promising FMN riboswitch sequences and structures, with FMN in the solution, to determine if the FMN mimic construct provides an accurate prediction of sequences that will change conformation when in the presence of a ligand. (way to verify or falsify this?)
At this point, further rounds of synthesis may need to be made. Results will continue to be compiled, and the first draft of the paper may be done by the end of the summer, with luck. Input from the Das lab for any of this would be greatly appreciated.
Join the group
If you would like to join the player research group that will be taking part in this "Paper Lab Pilot" please feel free to send a private message to Brourd, in EteRNA, or leave a comment on the discussion page in the wiki.