Rstclair2012 (Talk | contribs) |
Rstclair2012 (Talk | contribs) |
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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> | ||
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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> | ||
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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 | ||
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<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 |
Revision as of 18:42, 31 October 2017
Florida_Atlantic
Experiments
Wet Lab Protocols
LB Broth 25g LB Broth, Miller 1L Water -Mix LB powder into water, autoclave for 15 minutes. Supplement with antibiotics (Chloramphenicol, 35μL/mL) as needed. LB Agar 25g LB Broth, Miller 15g Granulated Agar 1L Water -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.
Transformation of Competent Cells
50μL Competent Cells (DH5α E. coli) 10ng Plasmid DNA 450μL LB LB/Chloramphenicol Plate -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.
Dry Lab Protocols
Protein Reverse Translation -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.
Machine Learning Protocols
LSTM model was coded using Tensorflow library and imported to Jupyter notebooks for 3 main experiments.
Artemisinin Binding
- Import LSTM model. - Collected and import 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. - Train model to learn binding vs. not binding. - Establish consistent parameters for learning process (number of proteins per dataset, learning rate, network size, and sub-sequence length viewed). - Run test and repeat to establish consistency.
Artemisinin Consensus Sequence
- Import LSTM. - Import dataset (from previous model^). - Train model to look at specified sequence length for binding vs. not binding. - Establish consistent parameters for learning process (number of proteins per dataset, learning rate, network size, and sub-sequence length viewed). - Set variables consistent (batch size, epochs, and sequence length to run in range loop). - Run test and repeat to establish consistency.
Homeobox Consensus Sequence
- Import LSTM. - Import dataset from Uniprot data website for proteins containing homeo-domain sequence and proteins and proteins not containing homeodomain sequence. - Train model to look at specified sequence length for binding vs. not binding. - Establish consistent parameters for learning process (number of proteins per dataset, learning rate, network size, and sub-sequence length viewed). - Set variables consistent (batch size, epochs, and sequence length to run in range loop). - Run test and repeat to establish consistency.