Difference between revisions of "Team:Florida Atlantic/Experiments"
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| − | <h1>Experiments</h1> | + | <center><h1 style="font-size: 26px">Experiments</h1></center> |
| − | < | + | <h2>Wet Lab Protocols</h2> |
<p style="font-size: 18px"> | <p style="font-size: 18px"> | ||
LB Broth</br> | LB Broth</br> | ||
25g LB Broth, Miller</br> | 25g LB Broth, Miller</br> | ||
1L Water</br> | 1L Water</br> | ||
| − | Mix LB powder into water, autoclave for 15 minutes. Supplement with antibiotics | + | -Mix LB powder into water, autoclave for 15 minutes. Supplement with antibiotics |
(Chloramphenicol, 35μL/mL) as needed.</br> | (Chloramphenicol, 35μL/mL) as needed.</br> | ||
</br> | </br> | ||
| Line 33: | Line 33: | ||
15g Granulated Agar</br> | 15g Granulated Agar</br> | ||
1L Water</br> | 1L Water</br> | ||
| − | Mix LB powder and Agar into water. Heat until agar is dissolved and solution is clear, avoid | + | -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, | boiling over. Autoclave for 15 minutes. Supplement with antibiotics (Chloramphenicol, | ||
35μL/mL) as needed. Pour 20mL into sterile culture plates and let cool.</br> | 35μL/mL) as needed. Pour 20mL into sterile culture plates and let cool.</br> | ||
| Line 44: | Line 44: | ||
450μL LB</br> | 450μL LB</br> | ||
LB/Chloramphenicol Plate</br> | LB/Chloramphenicol Plate</br> | ||
| − | Thaw Competent cells on ice and add plasmid DNA. Let sit for 20 minutes on ice. Heat shock | + | -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 | 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 | for 2-3 hours. Plate 100μL of the cells on a LB/Chloramphenicol plate and incubate at 37 o C | ||
| Line 53: | Line 53: | ||
<p style="font-size: 18px"> | <p style="font-size: 18px"> | ||
Protein Reverse Translation</br> | Protein Reverse Translation</br> | ||
| − | Isolate the protein sequence of interest and reverse translate using the E. coli preferred codon | + | -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 | 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 | 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> | 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
