Rstclair2012 (Talk | contribs) |
Rstclair2012 (Talk | contribs) |
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− | <p> | + | <center><h1 style="font-size: 26px">Experiments</h1></center> |
− | + | ||
+ | <h2>Wet Lab Protocols</h2> | ||
+ | <p style="font-size: 18px"> | ||
+ | LB Broth</br> | ||
+ | 25g LB Broth, Miller</br> | ||
+ | 1L Water</br> | ||
+ | -Mix LB powder into water, autoclave for 15 minutes. Supplement with antibiotics | ||
+ | (Chloramphenicol, 35μL/mL) as needed.</br> | ||
+ | </br> | ||
+ | LB Agar</br> | ||
+ | 25g LB Broth, Miller</br> | ||
+ | 15g Granulated Agar</br> | ||
+ | 1L Water</br> | ||
+ | -Mix LB powder and Agar into water. Heat until agar is dissolved and solution is clear, avoid | ||
+ | boiling over. Autoclave for 15 minutes. Supplement with antibiotics (Chloramphenicol, | ||
+ | 35μL/mL) as needed. Pour 20mL into sterile culture plates and let cool.</br> | ||
</p> | </p> | ||
− | </ | + | <h3>Transformation of Competent Cells </h3> |
− | + | <p style="font-size: 18px"> | |
− | < | + | 50μL Competent Cells (DH5α E. coli)</br> |
− | + | 10ng Plasmid DNA</br> | |
− | + | 450μL LB</br> | |
− | + | LB/Chloramphenicol Plate</br> | |
− | + | -Thaw Competent cells on ice and add plasmid DNA. Let sit for 20 minutes on ice. Heat shock | |
− | + | cells at 45 o C for 30 seconds and then return to ice for 2 minutes. Add LB and incubate at 37 o C | |
− | </ | + | for 2-3 hours. Plate 100μL of the cells on a LB/Chloramphenicol plate and incubate at 37 o C |
− | + | overnight.</br> | |
− | + | </p> | |
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− | </ | + | |
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+ | <h3>Dry Lab Protocols</h3> | ||
+ | <p style="font-size: 18px"> | ||
+ | Protein Reverse Translation</br> | ||
+ | -Isolate the protein sequence of interest and reverse translate using the E. coli preferred codon | ||
+ | library in SnapGene. After reverse translation, look for out-of- frame coding regions and alter the | ||
+ | codons so that no transcription is likely to occur. Finally, run a BLASTX protocol to ensure that | ||
+ | the nucleotide sequence still encodes the protein of interest.</p> | ||
+ | </br> | ||
+ | </br> | ||
+ | <h2>Machine Learning Protocols</h2> | ||
+ | <p style="font-size: 18px"> | ||
+ | LSTM model was coded using Tensorflow library and imported to Jupyter notebooks for 3 main experiments.</p> | ||
+ | </br> | ||
+ | <h4>Artemisinin Binding</h4> | ||
+ | <p style="font-size: 18px"> | ||
+ | - Created LSTM model. </br> | ||
+ | - Collected and imported positive dataset for proteins that bind to Artemisinin and negative dataset for proteins that do not bind to Artemisinin by hand from NCBI and literature.</br> | ||
+ | - Train model to learn binding vs. not binding. | ||
+ | - Established consistent parameters for learning process (number of proteins per dataset, learning rate, network size, and sub-sequence length viewed). </br> | ||
+ | - Ran test and repeated to establish consistency.</p></br> | ||
+ | <h4>Artemisinin Consensus Sequence</h4> | ||
+ | <p style="font-size: 18px"> | ||
+ | - Created LSTM. </br> | ||
+ | - Imported dataset (from previous model^). </br> | ||
+ | - Trained model to look at specified sequence length for binding vs. not binding. </br> | ||
+ | - Established consistent parameters for learning process (number of proteins per dataset, learning rate, network size, and sub-sequence length viewed). </br> | ||
+ | - Set variables consistent (batch size, epochs, and sequence length to run in range loop). | ||
+ | - Ran test and repeated to establish consistency.</p></br> | ||
+ | </p> | ||
+ | </br> | ||
+ | <h4>Homeobox Consensus Sequence</h4> | ||
+ | <p style="font-size: 18px"> | ||
+ | - Created LSTM. </br> | ||
+ | - Imported dataset from Uniprot data website for proteins containing homeo-domain sequence and proteins and proteins not containing homeodomain sequence. </br> | ||
+ | - Trained model to look at specified sequence length for binding vs. not binding. </br> | ||
+ | - Established consistent parameters for learning process (number of proteins per dataset, learning rate, network size, and sub-sequence length viewed). </br> | ||
+ | - Set variables consistent (batch size, epochs, and sequence length to run in range loop). </br> | ||
+ | - Ran test and repeated to establish consistency.</p></br> | ||
+ | </p> | ||
+ | </br> | ||
+ | </feildset> | ||
+ | </form> | ||
</div> | </div> | ||
</html> | </html> |
Latest revision as of 21:15, 1 November 2017
Florida_Atlantic