Difference between revisions of "Team:UCSC/Logs"
Line 273: | Line 273: | ||
<div id="collapseOne" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne"> | <div id="collapseOne" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne"> | ||
<div class="panel-body"> | <div class="panel-body"> | ||
− | + | <p><strong>June 2015</strong></p> | |
− | + | <p><strong><strong> </strong></strong></p> | |
− | Wednesday, June 24 | + | <p><strong>Wednesday, June 24</strong></p> |
− | Discussed overall project goals for the research period | + | <ol> |
− | Need to test the efficiency of multiple cellulases | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Discussed overall project goals for the research period</span></li> |
− | preferably those that are already present in halophiles and is compatible with Haloferax volcanii | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to test the efficiency of multiple cellulases</span></li> |
− | identified the positions of the four different cellulases associated with Halorhabdus utahensis | + | </ol> |
− | Cellulosome construction possibility | + | <ul> |
− | + | <ul> | |
− | Thursday, June 25 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">preferably those that are already present in halophiles and is compatible with </span><em><span style="font-weight: 400;">Haloferax volcanii</span></em></li> |
− | Looked through the halophile phylogenetic tree and found that Haloquadratum walsbyi (Hwa) is closer to Haloferax volcanii, and has cellulase that occurs in the natural metabolic pathway as well | + | </ul> |
− | Did a pairwise alignment between Halorhabdus utahensis (Hut) and Haloquadratum walsbyi (Hwa) | + | </ul> |
− | Concluded that it was probably not a good idea to risk using Haloquadratum walsbyi since it only contains a | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">identified the positions of the four different cellulases associated with </span><em><span style="font-weight: 400;">Halorhabdus utahensis</span></em></li> | |
− | Friday, June 26 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Cellulosome construction possibility</span></li> |
− | Learned how to design primers for Gibson Assembly | + | </ol> |
− | Have two potential cellulase candidates | + | <p><strong><strong> </strong></strong></p> |
− | one from H.tiamaten | + | <p><strong>Thursday, June 25</strong></p> |
− | one from H.utahensis | + | <ol> |
− | Using pTA963 as an expression plasmid | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Looked through the halophile phylogenetic tree and found that</span><em><span style="font-weight: 400;"> Haloquadratum walsbyi </span></em><span style="font-weight: 400;">(Hwa) is closer to</span><em><span style="font-weight: 400;"> Haloferax volcanii</span></em><span style="font-weight: 400;">, and has cellulase that occurs in the natural metabolic pathway as well </span></li> |
− | Most of the cellulases seen need C-terminal His Tag since the N-terminus is found in the transmembrane | + | </ol> |
− | Need to use inverse PCR to linearize and amplify plasmid. This will also remove 6x His Tag and stop codon | + | <ul> |
− | His Tag and stop codon will be added using flagged primers | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Did a pairwise alignment between </span><em><span style="font-weight: 400;">Halorhabdus utahensis</span></em><span style="font-weight: 400;"> (Hut) and </span><em><span style="font-weight: 400;">Haloquadratum walsbyi </span></em><span style="font-weight: 400;">(Hwa)</span></li> |
− | Introduced to idea of codon optimization | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Concluded that it was probably not a good idea to risk using Haloquadratum walsbyi since it only contains a “probable” cellulase, and it does not contain any other beta-glucanase enzymes</span></li> |
− | Worked on the pI Finder program, which takes in a FASTA amino acid sequence and calculates the theoretical isoelectric point | + | </ul> |
− | Narrowed criteria for usable cellulases: | + | <p><strong>Friday, June 26</strong></p> |
− | Signal Peptide for excretion or transmembrane position | + | <ol> |
− | pI range between 4 and 5 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Learned how to design primers for Gibson Assembly</span></li> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Have two potential cellulase candidates </span></li> | |
− | Monday, June 29 | + | </ol> |
− | Worked with different plasmid editors and decided on Geneious | + | <ul> |
− | Narrowed down the cellulase candidates from CAZy with respect to the signal peptide sequences | + | <ul> |
− | Introduction to PredSignal. Software that uses hidden markov models to predict the presence and purpose of archaeal signal peptides | + | <li style="font-weight: 400;"><span style="font-weight: 400;">one from</span><em><span style="font-weight: 400;"> H.tiamaten</span></em></li> |
− | Selection options for testing with cellulose: | + | <li style="font-weight: 400;"><span style="font-weight: 400;">one from </span><em><span style="font-weight: 400;">H.utahensis</span></em></li> |
− | Rayon | + | </ul> |
− | Filter paper | + | </ul> |
− | Avicel microcrystalline cellulose | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Using pTA963 as an expression plasmid </span></li> | |
− | Tuesday, June 30 | + | </ol> |
− | Continued the narrowing down of the cellulase candidates with respect to the presence of signal peptide | + | <ul> |
− | Found the sequence of the cellulase (Hu-CBH1) from H. utahensis that has been proven to work in the paper | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Most of the cellulases seen need C-terminal His Tag since the N-terminus is found in the transmembrane </span></li> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to use inverse PCR to linearize and amplify plasmid. This will also remove 6x His Tag and stop codon </span></li> | |
− | July 2015 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">His Tag and stop codon will be added using flagged primers </span></li> |
− | + | </ul> | |
− | Wednesday, July 1 | + | <ol> |
− | Have enzymes categories as beta glucanase and alpha amylase, but also looking for endo glucanase and exo glucanase | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Introduced to idea of codon optimization</span></li> |
− | Discuss whether or not we can utilize the cellobiohydrolase Hu-CBH1 since it is patented | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Worked on the pI Finder program, which takes in a FASTA amino acid sequence and calculates the theoretical isoelectric point</span></li> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Narrowed criteria for usable cellulases: </span></li> | |
− | Thursday, July 2 | + | </ol> |
− | Weekly team meeting (Moved to Thursday due to 4th of July Weekend) | + | <ul> |
− | Discussed a more narrow focus on Exoglucanases, Endoglucanases and B-glucosidases | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Signal Peptide for excretion or transmembrane position</span></li> |
− | We did not want to focus on Alpha amylases since they are used for breaking down starch | + | <li style="font-weight: 400;"><span style="font-weight: 400;">pI range between 4 and 5</span></li> |
− | + | </ul> | |
− | Monday, July 6 | + | <p><strong>Monday, June 29</strong></p> |
− | Design protocols for inverse PCR and Gibson Assembly | + | <ol> |
− | Performed inverse PCR to linearize and amplify our expression plasmid (pta963) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Worked with different plasmid editors and decided on Geneious </span></li> |
− | Protocols labelled as | + | </ol> |
− | + | <ol> | |
− | Tuesday, July 7 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Narrowed down the cellulase candidates from CAZy with respect to the signal peptide sequences </span></li> |
− | Acquired Q5 2x Master mix for PCR reactions | + | </ol> |
− | H.tiamaten cellulase | + | <ul> |
− | Gene is about 2,700 nucleotides long x 3 ~ 8,100 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Introduction to PredSignal. Software that uses hidden markov models to predict the presence and purpose of archaeal signal peptides </span></li> |
− | We have about 4 gene candidates + H.tiamaten cellulase that has already been tested | + | </ul> |
− | + | <ol> | |
− | Wednesday, July 8 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Selection options for testing with cellulose: </span></li> |
− | GOALS | + | </ol> |
− | Thursday 7/9 | + | <ol> |
− | Friday 7/10 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Rayon</span></li> |
− | Wednesday 7/15 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Filter paper that’s been bleached</span></li> |
− | pick a strain of E.coli | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Avicel microcrystalline cellulose </span></li> |
− | chemically competent | + | </ol> |
− | Thursday 7/16 | + | <p><strong>Tuesday, June 30</strong></p> |
− | Monday 7/20 Mini-Prep/ Sequencing Prep | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Continued the narrowing down of the cellulase candidates with respect to the presence of signal peptide</span></li> | |
− | Thursday, July 9 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Found the sequence of the cellulase (Hu-CBH1) from </span><em><span style="font-weight: 400;">H. utahensis</span></em><span style="font-weight: 400;"> that has been proven to work in the paper “Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance” Zhang, Tao et al </span></li> |
− | Need to prepare gene blocks for: | + | </ol> |
− | + | <p><span style="font-weight: 400;"> </span></p> | |
− | C-Terminus | + | <p><strong>July 2015</strong></p> |
− | -Shewanella | + | <p><strong><strong> </strong></strong></p> |
− | -tiamaten | + | <p><strong>Wednesday, July 1</strong></p> |
− | -Hu-CBH1 | + | <ol> |
− | -b-glucosidase (hispanica) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Have enzymes categories as beta glucanase and alpha amylase, but also looking for endo glucanase and exo glucanase</span></li> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Discuss whether or not we can utilize the cellobiohydrolase Hu-CBH1 since it is patented </span></li> | |
− | Friday, July 10 | + | </ol> |
− | While choosing cellulase candidates | + | <p><strong><strong> </strong></strong></p> |
− | Checked for signal peptides | + | <p><strong>Thursday, July 2 </strong></p> |
− | Searched through the archaeal genome browser | + | <ol> |
− | As of now we are deciding between: H.utahensis, H.hispanica, H.tiamaten | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Weekly team meeting (Moved to Thursday due to 4th of July Weekend) </span></li> |
− | Found that we already have stock of H.hispanica in the freezer | + | </ol> |
− | + | <ul> | |
− | Saturday, July 11 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Discussed a more narrow focus on Exoglucanases, Endoglucanases and B-glucosidases </span></li> |
− | Meeting to design gene blocks for: | + | <li style="font-weight: 400;"><span style="font-weight: 400;">We did not want to focus on Alpha amylases since they are used for breaking down starch </span></li> |
− | Shewenella fusion gene | + | </ul> |
− | Hu-CBH1 cellobiohydrolase | + | <p><strong>Monday, July 6</strong></p> |
− | Cellobiose phosphorylase | + | <ol> |
− | Pyruvate decarboxylase | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Design protocols for inverse PCR and Gibson Assembly</span></li> |
− | Note: Used IDT Gene Block Editor/ oligoanalyzer 3.1 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Performed inverse PCR to linearize and amplify our expression plasmid (pta963) </span></li> |
− | Picture | + | </ol> |
− | + | <ul> | |
− | Monday, July 13 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Protocols labelled as “ Polymerase Chain Reaction (PCR) for Amplification of pTA963 Expression Plasmids” and “Gibson Assembly Protocol”</span></li> |
− | TBE buffer was autoclaved this morning | + | </ul> |
− | Ran inverse PCR to amplify N-term pTA 963 and ran on a gel | + | <p><strong><strong> </strong></strong></p> |
− | Professor Bernick teaching PCR Fragment Assembly, Gibson Assembly and Flagged primers using sticks | + | <p><strong>Tuesday, July 7</strong></p> |
− | Picture | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Acquired Q5 2x Master mix for PCR reactions</span></li> | |
− | Tuesday, July 14 | + | <li style="font-weight: 400;"><em><span style="font-weight: 400;">H.tiamaten</span></em><span style="font-weight: 400;"> cellulase → homologs present, but still need to find the extension number</span></li> |
− | Reran inverse PCR, changing annealing temperature and extension time | + | </ol> |
− | Grant Team Meeting: | + | <ul> |
− | UCSC Crowdfunding page | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Gene is about 2,700 nucleotides long x 3 ~ 8,100 </span></li> |
− | Reaching out to department chairs | + | </ul> |
− | Created program for codon optimizing proteins using the codon frequency table and amino acid sequence (Called optimizer.py) | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">We have about 4 gene candidates + </span><em><span style="font-weight: 400;">H.tiamaten</span></em><span style="font-weight: 400;"> cellulase that has already been tested</span></li> | |
− | Wednesday, July 15 | + | </ol> |
− | Found protocol for making growth media for H. Hispanica in the Halohandbook pg. 14 | + | <p><span style="font-weight: 400;"> </span></p> |
− | 23% Modified Growth Medium (MGM) | + | <p><strong>Wednesday, July 8</strong></p> |
− | + | <p><span style="font-weight: 400;">GOALS</span></p> | |
− | Thursday, July 16 | + | <ul> |
− | Protocol for DNA isolation for H.hispanica (page 69 of Halohandbook) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Thursday 7/9 → Inverse PCR (2 experiments)</span></li> |
− | Found that DNAWorks algorithm for codon optimization removes accuracy | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Friday 7/10 → Analysis - Gel/ plate prep</span></li> |
− | It does not take into account | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Wednesday 7/15 → Gibson/ PCR Analysis/ Gel, Transformation of E.coli</span></li> |
− | + | <ul> | |
− | Friday, July 17 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">pick a strain of E.coli</span></li> |
− | Weekly team meeting | + | <li style="font-weight: 400;"><span style="font-weight: 400;">chemically competent</span></li> |
− | Use of DNA works for codon optimization | + | </ul> |
− | Inverse PCR for amplification of C-term pta963 plasmid | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Thursday 7/16 → Plate</span></li> |
− | Paused during extension phase because we forgot touchdown conditions, which may greatly affect results | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Monday 7/20 Mini-Prep/ Sequencing Prep</span></li> |
− | + | </ul> | |
− | Monday, July 20 | + | <p><strong><strong> </strong></strong></p> |
− | N-term and 2 C-term PCRs completed | + | <p><strong>Thursday, July 9</strong></p> |
− | EMERGENCY LAB MEETUP: | + | <ol> |
− | Breakthrough with codon optimization code (FOCUS) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to prepare gene blocks for: </span></li> |
− | Want to pay attention to conserved rare codons | + | </ol> |
− | Need to look for this pattern across organism of different domains | + | <p><span style="font-weight: 400;">C-Terminus</span><span style="font-weight: 400;"> </span><span style="font-weight: 400;">N-Terminus</span><span style="font-weight: 400;"> </span><span style="font-weight: 400;">?</span></p> |
− | 3 genes to test (All different forms of B. Glucosidase) | + | <p><span style="font-weight: 400;">-</span><em><span style="font-weight: 400;">Shewanella</span></em><span style="font-weight: 400;"> -</span><em><span style="font-weight: 400;">Shewanella </span></em><span style="font-weight: 400;"> -cellobiose phosphorylase </span></p> |
− | Wild type (H.hispanica) - control | + | <p><span style="font-weight: 400;">-</span><em><span style="font-weight: 400;">tiamaten</span></em></p> |
− | optimized (DNA Works) + control | + | <p><span style="font-weight: 400;">-Hu-CBH1 </span></p> |
− | optimized ( | + | <p><span style="font-weight: 400;">-b-glucosidase (</span><em><span style="font-weight: 400;">hispanica</span></em><span style="font-weight: 400;">) -</span><em><span style="font-weight: 400;">H.tiamaten </span></em><span style="font-weight: 400;"> </span> <span style="font-weight: 400;"> -B 1,4 glucanase (</span><em><span style="font-weight: 400;">utahensis</span></em><span style="font-weight: 400;">)</span></p> |
− | Assay | + | <p><strong><strong> </strong></strong></p> |
− | micro-crystalline cellulose and X-Glu plates to test for enzymatic activity | + | <p><strong>Friday, July 10</strong></p> |
− | Professor Bernick explanation of more to test for Signal Peptide: | + | <ol> |
− | psortB | + | <li style="font-weight: 400;"><span style="font-weight: 400;">While choosing cellulase candidates</span></li> |
− | TM Pred | + | </ol> |
− | Introduction to the UCSC archaeal Genome Browser | + | <ul> |
− | Intro to Kasava Research Paper | + | <ul> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Checked for signal peptides</span></li> | |
− | Tuesday, July 21 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Searched through the archaeal genome browser</span></li> |
− | EMERGENCY BREAKDOWN TEAM MEETUP: | + | <li style="font-weight: 400;"><span style="font-weight: 400;">As of now we are deciding between:</span><em><span style="font-weight: 400;"> H.utahensis, H.hispanica, H.tiamaten</span></em></li> |
− | Our hypothesis: Rare and conserved codons allows better folding of the protein structures = more efficient protein production | + | </ul> |
− | Obtain the secondary structures of the protein sequences through PSS Pred | + | </ul> |
− | Decided to codon optimize our beta glucosidase protein without optimizing the signal peptide since we know signal peptide still works based on the Hu-CBH1 paper | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Found that we already have stock of </span><em><span style="font-weight: 400;">H.hispanica</span></em><span style="font-weight: 400;"> in the freezer</span></li> | |
− | Wednesday, July 22 | + | </ol> |
− | Looking for 2,565 bp band for beta-glucosidase gene in Haloarcula hispanica | + | <p><strong><strong> </strong></strong></p> |
− | Continued runs for N-term and C-term PCR | + | <p><strong>Saturday, July 11</strong></p> |
− | + | <ol> | |
− | Thursday, July 23 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Meeting to design gene blocks for: </span></li> |
− | Streaking in C-term (7/21) due to pause while running | + | </ol> |
− | too much primer for the N-term; we used 5 uL instead of 1.25 uL | + | <ul> |
− | codon bias for H.volcanii and H. | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Shewenella fusion gene</span></li> |
− | completed another N-term and C-term reaction | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Hu-CBH1 cellobiohydrolase </span></li> |
− | working on perfecting conditions | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Cellobiose phosphorylase </span></li> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Pyruvate decarboxylase </span></li> | |
− | + | </ul> | |
− | Friday, July 24 | + | <p><span style="font-weight: 400;">Note: Used IDT Gene Block Editor/ oligoanalyzer 3.1</span></p> |
− | General Meeting | + | <p><span style="font-weight: 400;">Picture </span></p> |
− | Introducing entire team to F.O.C.U.S | + | <p><strong>Monday, July 13</strong></p> |
− | Plan to test the protein expression levels of the beta-glucosidase from Haloarcula hispanica between the wild type, DNA Works Codon Optimized and F.O.C.U.S Codon optimized | + | <ol> |
− | Need to prove that the rate limiting step is the incorporation of a particular tRNA | + | <li style="font-weight: 400;"><span style="font-weight: 400;">TBE buffer was autoclaved this morning</span></li> |
− | Working out the kinks for isolation of wild type beta glucosidase | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Ran inverse PCR to amplify N-term pTA 963 and ran on a gel </span></li> |
− | Smears may be due to a high concentration of genomic DNA during isolation | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Professor Bernick teaching PCR Fragment Assembly, Gibson Assembly and Flagged primers using sticks</span></li> |
− | Use of DPN1 could get rid of non-methylated (i.e non-genomic DNA) which could mean more access to PCR product and amplification | + | </ol> |
− | + | <p><span style="font-weight: 400;">Picture</span></p> | |
− | + | <p><strong>Tuesday, July 14</strong></p> | |
− | Monday, July 27 | + | <ol> |
− | Multiple sequence alignment of conserved proteins conserved in Archaea, Bacteria, and Eukaryotes | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Reran inverse PCR, changing annealing temperature and extension time </span></li> |
− | Alanyl-tRNA, DNA polymerase III subunit, rRNA dimethylase | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Grant Team Meeting:</span></li> |
− | Further proteins amongst model organisms can be found in the paper | + | </ol> |
− | + | <ul> | |
− | Tuesday, July 28 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">UCSC Crowdfunding page</span></li> |
− | Searched for protein sequences and DNA sequences of conserved proteins in model organisms | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Reaching out to department chairs</span></li> |
− | Note, some protein sequences in certain organisms are not of equal length to others. Therefore, they may not be orthologs | + | </ul> |
− | Inverse PCR to amplify N-terminal version of plasmid pta963 | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Created program for codon optimizing proteins using the codon frequency table and amino acid sequence (Called optimizer.py)</span></li> | |
− | Wednesday, July 29 | + | </ol> |
− | Need to regrow Hispanica to the optimal OD (0.6 - 0.8) for wild type beta glucosidase isolation | + | <p><strong><strong> </strong></strong></p> |
− | + | <p><strong>Wednesday, July 15</strong></p> | |
− | August 2015 | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Found protocol for making growth media for </span><em><span style="font-weight: 400;">H. Hispanica </span></em><span style="font-weight: 400;">in the Halohandbook pg. 14</span></li> | |
− | Monday, August 3 | + | </ol> |
− | Gel for hispanica isolation did not work on Friday | + | <ul> |
− | Tested to see if the track dye and DNA ladder show up under UV light | + | <li style="font-weight: 400;"><span style="font-weight: 400;">23% Modified Growth Medium (MGM)</span></li> |
− | Reran another gel to test if the results were due to track dye or gel | + | </ul> |
− | Redid another isolation since results were negative | + | <p><strong><strong> </strong></strong></p> |
− | + | <p><strong>Thursday, July 16</strong></p> | |
− | Tuesday, August | + | <ol> |
− | Isolation of hispanica (2nd time) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Protocol for DNA isolation for </span><em><span style="font-weight: 400;">H.hispanica</span></em><span style="font-weight: 400;"> (page 69 of Halohandbook)</span></li> |
− | 50 uL Rxn (Iso, and negative control) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Found that DNAWorks algorithm for codon optimization removes accuracy </span></li> |
− | Turned attention towards using the Dali Server for protein structure information and structural alignment comparisons | + | </ol> |
− | Prepared protocol for E. coli cell electroporation | + | <ul> |
− | + | <ul> | |
− | Wednesday, August 5 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">It does not take into account “rare” codons</span></li> |
− | Isolated Hispanica from a different aliquot (Still with an OD of 1.4) | + | </ul> |
− | Multiple PCR reactions to find optimal conditions for isolating wild type b-glucosidase (Run at 71 degrees) | + | </ul> |
− | Negative control (No DNA) | + | <p><strong><strong> </strong></strong></p> |
− | Repeat of the same PCR protocol but NO PCR ENHANCER | + | <p><strong>Friday, July 17</strong></p> |
− | 2x Primers (No PCR Enhancer) | + | <ol> |
− | 2x Template ( NO PCR Enhancer) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Weekly team meeting</span></li> |
− | Amplification of the first isolation that worked (tube labelled ++) | + | </ol> |
− | + | <ul> | |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Use of DNA works for codon optimization </span></li> | |
− | Redid PCR for gene isolation (Brought the annealing temperature to 66 degrees) | + | </ul> |
− | + | <ol> | |
− | Friday, August 7 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Inverse PCR for amplification of C-term pta963 plasmid </span></li> |
− | Performed isolation from a 3rd aliquot of Hispanica | + | </ol> |
− | Performed PCR after diluting concentration 100 fold for both aliquot 1 and 3 | + | <ul> |
− | Meeting with Professor Bernick to discuss one method of measuring importance of rare codons for F.O.C.U.S | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Paused during extension phase because we forgot touchdown conditions, which may greatly affect results </span></li> |
− | Encoding cost (Method of hyper weighting a codon that is more rare, especially if it is a rare codon in a group of more frequent codons) | + | </ul> |
− | Need to develop a training set and test set to measure consistency of rare codon idea | + | <p><strong><strong> </strong></strong></p> |
− | Prepare sequence information for engineered cellulase developed by 2014 UCSC iGEM team to perform site directed mutagenesis | + | <p><strong>Monday, July 20</strong></p> |
− | + | <ol> | |
− | Tuesday, August 11 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">N-term and 2 C-term PCRs completed </span></li> |
− | Fragment assembly of codon optimized Beta-Glucosidase | + | <li style="font-weight: 400;"><span style="font-weight: 400;">EMERGENCY LAB MEETUP:</span></li> |
− | Fragment concentrations from stock: | + | </ol> |
− | F1 | + | <ul> |
− | The Fw fixed Flag primer was diluted with 216 uL of TE buffer, vortexed, and stored | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Breakthrough with codon optimization code (FOCUS)</span></li> |
− | Primers used: Fw Flag Fixed5 and Bglu_ Rv flag | + | </ul> |
− | Nested PCR part 2 (adding flag primers to the isolated WT beta Glucosidas | + | <ul> |
− | Primers: Fw Flag fixed5 and Bglu_ WT_Rv flag | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Want to pay attention to conserved rare codons</span></li> |
− | Proteins for F.O.C.U.S | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to look for this pattern across organism of different domains</span></li> |
− | EF-G, Ef-Tu, DNA Polymerase III, tRNA synthetase | + | </ul> |
− | Dali server search stored on Kerika | + | <ul> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">3 genes to test (All different forms of B. Glucosidase)</span></li> | |
− | Wednesday, August 12 | + | </ul> |
− | Working on getting the codon bias tables and nucleotide sequences to test out FOCUS | + | <ul> |
− | Reinoculated Hispanica to isolate it from a lower OD | + | <ul> |
− | Tested Fragment assembly with 1/10 dilution of fragments and non dilution | + | <ul> |
− | The lightbulb in the UV transilluminator broke, so | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Wild type (</span><em><span style="font-weight: 400;">H.hispanica</span></em><span style="font-weight: 400;">) - control </span></li> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">optimized (DNA Works) + control</span></li> | |
− | Thursday, August 13 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">optimized (Jairo’s code) F.O.C.U.S</span></li> |
− | General Meeting | + | </ul> |
− | check 260/280 and 260/230 for the isolation | + | </ul> |
− | absorbance of nucleic acids/ protein | + | </ul> |
− | Run fragment assembly with 1/5 dilution, not 1/10 | + | <ul> |
− | Length of the fragment assembly = | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Assay</span></li> |
− | Length of the wild type = 2644 bp | + | </ul> |
− | Isolation of Hispanica from a culture with an OD of 0.4 | + | <ul> |
− | concentration: 116.4 ng/uL | + | <ul> |
− | 260/280: 1.64 | + | <ul> |
− | 260/230: 0.59 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">micro-crystalline cellulose and X-Glu plates to test for enzymatic activity</span></li> |
− | WT isolation and fragment assembly PCR | + | </ul> |
− | dilution (1:10) of new isolation | + | </ul> |
− | diluted fragments and non-diluted fragments | + | </ul> |
− | Annealing temp= 63.8 degrees | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Professor Bernick explanation of more to test for Signal Peptide: </span></li> | |
− | Friday, August 14 | + | </ol> |
− | Isolation PCR reaction using isolation from OD 0.4 and 1.4 to see which is better | + | <ul> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">psortB</span></li> | |
− | Sunday, August 16 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">TM Pred</span></li> |
− | Running the gel from | + | </ul> |
− | The isolation with an OD of 0.4 worked the best! | + | <ol> |
− | dilute fragments reaction does not work | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Introduction to the UCSC archaeal Genome Browser </span></li> |
− | lower the annealing temperature | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Intro to Kasava Research Paper </span></li> |
− | used an annealing temperature of 62.8 degrees | + | </ol> |
− | Elongation time changed to 45 seconds | + | <p><strong><strong> </strong></strong></p> |
− | Q5 reads 1000 bp every 15-45 seconds | + | <p><strong>Tuesday, July 21</strong></p> |
− | PCR Reactions | + | <ol> |
− | 0.4, 1.4, nd, d, i, F, | + | <li style="font-weight: 400;"><span style="font-weight: 400;">EMERGENCY BREAKDOWN TEAM MEETUP:</span></li> |
− | + | </ol> | |
− | + | <ul> | |
− | Monday, August 17 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Our hypothesis: Rare and conserved codons allows better folding of the protein structures = more efficient protein production</span></li> |
− | Prepared 500 mL of LB agar in preparation for growing transformed E. coli cultures | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Obtain the secondary structures of the protein sequences through PSS Pred </span></li> |
− | Protocol: https://www.addgene.org/plasmid-protocols/bacterial-plates/ | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Decided to codon optimize our beta glucosidase protein without optimizing the signal peptide since we know signal peptide still works based on the Hu-CBH1 paper </span></li> |
− | nanodrop for linearized PTA963 | + | </ul> |
− | Conc. | + | <p><strong><strong> </strong></strong></p> |
− | C-term (7/21) | + | <p><strong>Wednesday, July 22</strong></p> |
− | C-term (7/23) | + | <ol> |
− | N-term (7/23) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Looking for 2,565 bp band for beta-glucosidase gene in </span><em><span style="font-weight: 400;">Haloarcula hispanica</span></em></li> |
− | N-term (7/28) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Continued runs for N-term and C-term PCR</span></li> |
− | More PCR reactions | + | </ol> |
− | 0.4 flag, annealing temp @ 70 degrees | + | <p><strong><strong> </strong></strong></p> |
− | 1.4 re-isolation, run @ 71 degrees | + | <p><strong>Thursday, July 23</strong></p> |
− | Fragment Assembly @ 65 degrees | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Streaking in C-term (7/21) due to pause while running</span></li> | |
− | Tuesday, August 18 | + | </ol> |
− | Bright band present for fragment assembly = SUCCESS! | + | <ul> |
− | Performed purification using Bernick Kit. Results: | + | <li style="font-weight: 400;"><span style="font-weight: 400;">too much primer for the N-term; we used 5 uL instead of 1.25 uL</span></li> |
− | + | </ul> | |
− | N1 | + | <ol> |
− | N2 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">codon bias for</span><em><span style="font-weight: 400;"> H.volcanii </span></em><span style="font-weight: 400;">and</span><em><span style="font-weight: 400;"> H.hispanic</span></em><span style="font-weight: 400;">a are similar</span></li> |
− | C1 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">completed another N-term and C-term reaction </span></li> |
− | C2 | + | </ol> |
− | + | <ul> | |
− | Wednesday, August 19 | + | <ul> |
− | Re-running two isolation reactions with an annealing temp of 71 C and 42 sec extension | + | <li style="font-weight: 400;"><span style="font-weight: 400;">working on perfecting conditions</span></li> |
− | Gel extraction of fragment assembly to run Gibson assembly | + | </ul> |
− | + | </ul> | |
− | Thursday, August 20 | + | <p><strong><strong><br /><br /></strong></strong></p> |
− | PCR reactions (Annealing temp. 65 degrees) | + | <p><strong>Friday, July 24</strong></p> |
− | gel-extracted iso | + | <ul> |
− | gel-extracted frag (dil) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">General Meeting</span></li> |
− | non gel-extracted iso | + | </ul> |
− | non-dil frag | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Introducing entire team to F.O.C.U.S</span></li> | |
− | Friday, August 21 | + | </ol> |
− | To Do: | + | <ul> |
− | Confirm correct isolation band | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Plan to test the protein expression levels of the beta-glucosidase from Haloarcula hispanica between the wild type, DNA Works Codon Optimized and F.O.C.U.S Codon optimized</span></li> |
− | redo fragment assembly | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to prove that the rate limiting step is the incorporation of a particular tRNA </span></li> |
− | 1 uL each fragment (10 uM) | + | </ul> |
− | correct concentrations (0.88 uL,1.25 uL,1uL) | + | <ol> |
− | 65 degrees annealing temperature | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Working out the kinks for isolation of wild type beta glucosidase </span></li> |
− | Details for fragment assembly that was gel extracted: | + | </ol> |
− | 19 uL at concentration of 66.4 ng/uL | + | <ul> |
− | 260/280: 2.03 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Smears may be due to a high concentration of genomic DNA during isolation</span></li> |
− | 260/230: 0.61 | + | </ul> |
− | Gibson Assembly | + | <ul> |
− | 2.65 uL of C-term pta963 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Use of DPN1 could get rid of non-methylated (i.e non-genomic DNA) which could mean more access to PCR product and amplification </span></li> |
− | 1.2 uL of Fragment Assembly | + | </ul> |
− | + | <p><strong><strong> </strong></strong></p> | |
− | Saturday, August 22 | + | <p><strong>Monday, July 27</strong></p> |
− | Inverse PCR of N-Terminal pta963 for Ethanol Team | + | <ol> |
− | Transformed NEB 5-alpha competent E. coli with Gibson Assembly product | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Multiple sequence alignment of conserved proteins conserved in Archaea, Bacteria, and Eukaryotes </span></li> |
− | Attempted to isolate which band corresponded to the wild type Beta Glucosidase | + | </ol> |
− | Fw_Flg Fixed5 and Seq Rv3 primers, looking for band of 1000 | + | <ul> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Alanyl-tRNA, DNA polymerase III subunit, rRNA dimethylase</span><span style="font-weight: 400;"> </span></li> | |
− | Sunday, August 23 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Further proteins amongst model organisms can be found in the paper “ Universal trees based on large combined protein” Brown, James R. et al</span></li> |
− | Checked transformation plate, put found no growth | + | </ul> |
− | Most likely due to cells having lost competence since they were not stored at -80 C for at least a week | + | <p><strong>Tuesday, July 28</strong></p> |
− | Prepared reagents for making spheroplast | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Searched for protein sequences and DNA sequences of conserved proteins in model organisms</span></li> | |
− | Monday, August 24 | + | </ol> |
− | Made | + | <ul> |
− | Protocol found on pgs. 59-62 of the Halohandbook | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Note, some protein sequences in certain organisms are not of equal length to others. Therefore, they may not be orthologs</span></li> |
− | Redid inverse PCR of N-term pTA963 | + | </ul> |
− | Elongation lowered to 2 min and 30 sec | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Inverse PCR to amplify N-terminal version of plasmid pta963</span></li> | |
− | Wednesday, August 26 | + | </ol> |
− | Electroporation using electrocompetent E. coli cells | + | <p><strong>Wednesday, July 29</strong></p> |
− | 1800 volts | + | <ol> |
− | Used Electroporation protocol made by Fermentation team | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to regrow Hispanica to the optimal OD (0.6 - 0.8) for wild type beta glucosidase isolation</span></li> |
− | Ag Tech Meet up Presentation | + | </ol> |
− | + | <p><strong><strong> </strong></strong></p> | |
− | Thursday, August 27 | + | <p><strong>August 2015</strong></p> |
− | Nested PCR Part 2 | + | <p><strong><strong> </strong></strong></p> |
− | Used Seq/iso reaction product from 8/27 as template DNA since it has the brightest band corresponding to the wild type beta glucosidase | + | <p><strong>Monday, August 3</strong></p> |
− | Concentration: 472.2 ng/uL ; 262/280: 1.79 | + | <ol> |
− | Used Fw Flag Fixed5, Rv Flag and Rv Flag fixed as primers | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Gel for hispanica isolation did not work on Friday</span></li> |
− | + | </ol> | |
− | Monday, August 31 | + | <ul> |
− | Colony PCR from Transformation plate | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Tested to see if the track dye and DNA ladder show up under UV light</span></li> |
− | Picked 6 colonies | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Reran another gel to test if the results were due to track dye or gel</span></li> |
− | Used miliq water to lyse cells (Tubes labelled 1P-6P) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Redid another isolation since results were negative </span></li> |
− | Used Flagged primers: Fw Flag Fixed 5 and Rv Flag Fixed | + | </ul> |
− | Ran another reaction using sequencing primers: Seq Fw2 and Seq Rv2 | + | <p><strong><strong> </strong></strong></p> |
− | + | <p><strong>Tuesday, August 4</strong></p> | |
− | September 2015 | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Isolation of hispanica (2nd time)</span></li> | |
− | Tuesday, September 1 | + | </ol> |
− | Meeting with the Dean of Students | + | <ul> |
− | Rerun a gel including fermentation team colony PCR samples and Breakdown sample (D - dilute, and ND- non-Dilute) of wild type isolation test | + | <li style="font-weight: 400;"><span style="font-weight: 400;">50 uL Rxn (Iso, and negative control)</span></li> |
− | Made 236.25 uL of Q5 Master Mix | + | </ul> |
− | Enough for 27 reactions if we use 25 uL PCR reactions | + | <ol> |
− | Recipe (X10): | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Turned attention towards using the Dali Server for protein structure information and structural alignment comparisons</span></li> |
− | 20 uL Q5 Buffer | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Prepared protocol for </span><em><span style="font-weight: 400;">E. coli</span></em><span style="font-weight: 400;"> cell electroporation</span></li> |
− | 2 uL of | + | </ol> |
− | 0.5 uL of Q5 enzyme | + | <p><strong>Wednesday, August 5</strong></p> |
− | 11.25 uL of Miliq water | + | <ol> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Isolated Hispanica from a different aliquot (Still with an OD of 1.4)</span></li> | |
− | Wednesday, September 2 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Multiple PCR reactions to find optimal conditions for isolating wild type b-glucosidase (Run at 71 degrees) </span></li> |
− | Colony PCR (4 reactions): | + | </ol> |
− | P2 with iso Primers and PCR enhancer (1:10 dilution) | + | <ul> |
− | P2 with iso Primers, No PCR enhancer (1:10 dilution) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Negative control (No DNA) </span></li> |
− | P2 with Flagged Primers, PCR enhancer (1:10 dilution) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Repeat of the same PCR protocol but NO PCR ENHANCER</span></li> |
− | P2 with flagged Primers, No PCR enhancer (1:10 dilution) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">2x Primers (No PCR Enhancer) </span></li> |
− | Electroporation of Fermentation Gibson reactions (G2, G6, G7, and G8) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">2x Template ( NO PCR Enhancer) </span></li> |
− | Colony PCR of P1 - P6 using Sequencing FW1 and Rv4 primers | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Amplification of the first isolation that worked (tube labelled ++)</span></li> |
− | Annealing Temp: 59 C | + | </ul> |
− | Extension time: 3 minutes | + | <p><strong><strong> </strong></strong></p> |
− | Need to prepare HVCA plates for Spheroplast analysis after transformation | + | <p><strong> Thursday, August 6</strong></p> |
− | Protocol for HVCA agar on Pg. 21 of Halohandbook | + | <ol> |
− | Transformants should grow on plates without Uracil | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Redid PCR for gene isolation (Brought the annealing temperature to 66 degrees)</span></li> |
− | + | </ol> | |
− | Friday, September 4 | + | <p><strong><strong> </strong></strong></p> |
− | General Meeting | + | <p><strong>Friday, August 7</strong></p> |
− | FOCUS Discussion | + | <ol> |
− | Transition Probability Modeling | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Performed isolation from a 3rd aliquot of Hispanica</span></li> |
− | Only need to produce a model with 2 states: fast and stall (excluding steady state) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Performed PCR after diluting concentration 100 fold for both aliquot 1 and 3</span></li> |
− | John has a paper about | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Meeting with Professor Bernick to discuss one method of measuring importance of rare codons for F.O.C.U.S</span></li> |
− | Colony PCR of P1 - P6 | + | </ol> |
− | + | <ul> | |
− | Annealing Temp. 51 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Encoding cost (Method of hyper weighting a codon that is more rare, especially if it is a rare codon in a group of more frequent codons)</span></li> |
− | Inoculated H. volcanni for making spheroplast | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to develop a training set and test set to measure consistency of rare codon idea</span></li> |
− | Optimal absorbance reading at 600 between 0.8- 1.0 for late exponential phase | + | </ul> |
− | Nested PCR part 2 | + | <ol> |
− | Template (0.4 isolation from 8/13, 8/14 and 8/17) to test which is best | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Prepare sequence information for engineered cellulase developed by 2014 UCSC iGEM team to perform site directed mutagenesis </span></li> |
− | Primers: Bglu_Fw_flagFixed5 and Bglu_wt_rv_fixedFlag | + | </ol> |
− | Annealing temp. : 69 C as suggested by TM Calculator | + | <p><strong><strong> </strong></strong></p> |
− | + | <p><strong>Tuesday, August 11</strong></p> | |
− | Saturday, September 5 | + | <ol> |
− | Absorbance reading of inoculated H. volcanii | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Fragment assembly of codon optimized Beta-Glucosidase </span></li> |
− | 0.621 A | + | </ol> |
− | Plated Fermentation Team Electroporated Cells ( G2, G6, G7, and G8) | + | <ul> |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Fragment concentrations from stock: </span></li> | |
− | Monday, September 7 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">F1 → 0.88 uL, F2 → 1.25 uL, F3 → 1 uL</span></li> |
− | Colony PCR positive controls (using Gibson Assembly reaction as template) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">The Fw fixed Flag primer was diluted with 216 uL of TE buffer, vortexed, and stored </span></li> |
− | Sequencing Primers Fw1 and Rv4 (Titaq) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Primers used: Fw Flag Fixed5 and Bglu_ Rv flag </span></li> |
− | Sequencing Primers Fw1 and Rv4 (OneTaq) | + | </ul> |
− | + | <ol> | |
− | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Nested PCR part 2 (adding flag primers to the isolated WT beta Glucosidas</span></li> | |
− | Wednesday, September 9 | + | </ol> |
− | Chose 8 new colony picks from original plate having Transformed E. coli cells with codon optimized B Glucosidase | + | <ul> |
− | Performed colony PCR using | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Primers: Fw Flag fixed5 and Bglu_ WT_Rv flag</span></li> |
− | Thursday, September 10 | + | </ul> |
− | Started Preparing BioBricks of Codon Optimized Beta Glucosidase, and Fermentation enzymes | + | <ol> |
− | Added Ampicilin to LB Agar plates and replated electroporated E. coli cells | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Proteins for F.O.C.U.S</span></li> |
− | Added 26.3 ul of 50 ng/uL ampicillin to each plate | + | </ol> |
− | Colony PCR using Q5 Master Mix | + | <ul> |
− | 3 Reactions from fermentation and 2 from Breakdown | + | <li style="font-weight: 400;"><span style="font-weight: 400;">EF-G, Ef-Tu, DNA Polymerase III, tRNA synthetase </span></li> |
− | Friday, September 11 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Dali server search stored on Kerika</span></li> |
− | Professor Bernick Discussion | + | </ul> |
− | Dislikes homopolymer in both Breakdown and | + | <p><strong><strong> </strong></strong></p> |
− | Prefers | + | <p><strong>Wednesday, August 12</strong></p> |
− | Suggests 10 cycle touchdown from 56 - 51 using | + | <ol> |
− | Saturday, September 12 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Working on getting the codon bias tables and nucleotide sequences to test out FOCUS</span></li> |
− | Verification that primers anneal to the C-term construct of pTA963 | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Reinoculated Hispanica to isolate it from a lower OD </span></li> |
− | Aldy5Fw1 anneals from 485 - 508 (15 nucleotides from His Tag) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">Tested Fragment assembly with 1/10 dilution of fragments and non dilution </span></li> |
− | Aldy5Rv5 anneals from 569 - 587 (31 nucleotides from His Tag) | + | </ol> |
− | SeqFw1 anneals fromm 462 to 486 | + | <ul> |
− | SeqRv4 anneals from 570 - 593 (Note, first G is supposed to be a C) | + | <li style="font-weight: 400;"><span style="font-weight: 400;">The lightbulb in the UV transilluminator broke, so weren’t able to capture a picture of the gel </span></li> |
− | Might have to redo Gibson Assembly of fragment assembly based on positive control results | + | </ul> |
− | + | <p><strong><strong> </strong></strong></p> | |
− | Sunday, September 13 | + | <p><strong>Thursday, August 13 </strong></p> |
− | Touchdown of Nested PCR Part 2 | + | <ol> |
− | Annealing temp from 70 - 65 C | + | <li style="font-weight: 400;"><span style="font-weight: 400;">General Meeting</span></li> |
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">check 260/280 and 260/230 for the isolation </span></li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">absorbance of nucleic acids/ protein</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Run fragment assembly with 1/5 dilution, not 1/10</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Length of the fragment assembly = 2641 bp </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Length of the wild type = 2644 bp</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Isolation of Hispanica from a culture with an OD of 0.4</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">concentration: 116.4 ng/uL</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">260/280: 1.64</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">260/230: 0.59</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">WT isolation and fragment assembly PCR </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">dilution (1:10) of new isolation </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">diluted fragments and non-diluted fragments</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Annealing temp= 63.8 degrees</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Friday, August 14 </strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Isolation PCR reaction using isolation from OD 0.4 and 1.4 to see which is better</span></li> | ||
+ | </ol> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Sunday, August 16</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Running the gel from Friday’s PCR reaction </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">The isolation with an OD of 0.4 worked the best!</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">dilute fragments reaction does not work</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">lower the annealing temperature</span></li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">used an annealing temperature of 62.8 degrees</span></li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Elongation time changed to 45 seconds</span></li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Q5 reads 1000 bp every 15-45 seconds</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">PCR Reactions </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">0.4, 1.4, nd, d, i, F, …, _</span><span style="font-weight: 400;"><br /><br /></span></li> | ||
+ | </ul> | ||
+ | <p><strong>Monday, August 17</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Prepared 500 mL of LB agar in preparation for growing transformed </span><em><span style="font-weight: 400;">E. coli </span></em><span style="font-weight: 400;">cultures</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Protocol: https://www.addgene.org/plasmid-protocols/bacterial-plates/</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">nanodrop for linearized PTA963</span></li> | ||
+ | </ol> | ||
+ | <p><span style="font-weight: 400;">Conc.</span> <span style="font-weight: 400;">260/280</span> <span style="font-weight: 400;">260/230</span></p> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">C-term (7/21)</span> <span style="font-weight: 400;">462.7 ng/uL</span> <span style="font-weight: 400;">1.82</span> <span style="font-weight: 400;">0.87</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">C-term (7/23)</span> <span style="font-weight: 400;">561.9 ng/uL</span> <span style="font-weight: 400;">1.83</span> <span style="font-weight: 400;">0.93</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">N-term (7/23)</span> <span style="font-weight: 400;">794.6 ng/uL</span> <span style="font-weight: 400;">1.84</span> <span style="font-weight: 400;">1.24</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">N-term (7/28)</span> <span style="font-weight: 400;">335.3 ng/uL</span> <span style="font-weight: 400;">1.84</span> <span style="font-weight: 400;">0.74</span></li> | ||
+ | </ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">More PCR reactions</span></li> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">0.4 flag, annealing temp @ 70 degrees → Today</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">1.4 re-isolation, run @ 71 degrees → Tomorrow</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Fragment Assembly @ 65 degrees → Tomorrow</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Tuesday, August 18 </strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Bright band present for fragment assembly = SUCCESS!</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Performed purification using Bernick Kit. Results: </span></li> | ||
+ | </ol> | ||
+ | <p><span style="font-weight: 400;"> </span><span style="font-weight: 400;">DNA</span><span style="font-weight: 400;"> </span><span style="font-weight: 400;"> Conc.</span><span style="font-weight: 400;"> </span><span style="font-weight: 400;">260/280</span><span style="font-weight: 400;"> </span><span style="font-weight: 400;">260/230</span></p> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">N1 32.2ng/ul 1.58 0.14</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">N2 7.4ng/ul 2.07 0.03</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">C1 6.1ng/ul 1.76 0.02</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">C2 14.5 ng/ul 1.57 0.06</span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Wednesday, August 19 </strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Re-running two isolation reactions with an annealing temp of 71 C and 42 sec extension</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Gel extraction of fragment assembly to run Gibson assembly</span></li> | ||
+ | </ol> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Thursday, August 20 </strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">PCR reactions (Annealing temp. 65 degrees)</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">gel-extracted iso </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">gel-extracted frag (dil)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">non gel-extracted iso </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">non-dil frag</span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Friday, August 21</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">To Do:</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Confirm correct isolation band </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">redo fragment assembly</span></li> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">1 uL each fragment (10 uM)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">correct concentrations (0.88 uL,1.25 uL,1uL)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">65 degrees annealing temperature</span></li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Details for fragment assembly that was gel extracted: </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">19 uL at concentration of 66.4 ng/uL</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">260/280: 2.03</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">260/230: 0.61</span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Gibson Assembly </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">2.65 uL of C-term pta963</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">1.2 uL of Fragment Assembly</span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Saturday, August 22</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Inverse PCR of N-Terminal pta963 for Ethanol Team</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Transformed NEB 5-alpha competent </span><em><span style="font-weight: 400;">E. coli</span></em><span style="font-weight: 400;"> with Gibson Assembly product</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Attempted to isolate which band corresponded to the wild type Beta Glucosidase</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Fw_Flg Fixed5 and Seq Rv3 primers, looking for band of 1000</span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Sunday, August 23</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Checked transformation plate, put found no growth </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Most likely due to cells having lost competence since they were not stored at -80 C for at least a week</span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Prepared reagents for making spheroplast </span></li> | ||
+ | </ol> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Monday, August 24 </strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Made “Unbuffered spheroplasting solution”, “Buffered spheroplasting solution” and “Regeneration solution” </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Protocol found on pgs. 59-62 of the Halohandbook </span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Redid inverse PCR of N-term pTA963</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Elongation lowered to 2 min and 30 sec </span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Wednesday, August 26</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Electroporation using electrocompetent </span><em><span style="font-weight: 400;">E. coli </span></em><span style="font-weight: 400;">cells </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">1800 volts </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Used Electroporation protocol made by Fermentation team </span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Ag Tech Meet up Presentation </span></li> | ||
+ | </ol> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Thursday, August 27</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Nested PCR Part 2 </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Used Seq/iso reaction product from 8/27 as template DNA since it has the brightest band corresponding to the wild type beta glucosidase</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Concentration: 472.2 ng/uL ; 262/280: 1.79 ; 260/230: 0.68</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Used Fw Flag Fixed5, Rv Flag and Rv Flag fixed as primers </span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Monday, August 31</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Colony PCR from Transformation plate</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Picked 6 colonies</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Used miliq water to lyse cells (Tubes labelled 1P-6P)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Used Flagged primers: Fw Flag Fixed 5 and Rv Flag Fixed </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Ran another reaction using sequencing primers: Seq Fw2 and Seq Rv2</span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>September 2015</strong></p> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Tuesday, September 1</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Meeting with the Dean of Students</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Rerun a gel including fermentation team colony PCR samples and Breakdown sample (D - dilute, and ND- non-Dilute) of wild type isolation test </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Made 236.25 uL of Q5 Master Mix </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Enough for 27 reactions if we use 25 uL PCR reactions</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Recipe (X10): </span></li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">20 uL Q5 Buffer</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">2 uL of dNTP’s</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">0.5 uL of Q5 enzyme</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">11.25 uL of Miliq water </span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Wednesday, September 2</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Colony PCR (4 reactions): </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">P2 with iso Primers and PCR enhancer (1:10 dilution) </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">P2 with iso Primers, No PCR enhancer (1:10 dilution) </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">P2 with Flagged Primers, PCR enhancer (1:10 dilution)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">P2 with flagged Primers, No PCR enhancer (1:10 dilution) </span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Electroporation of Fermentation Gibson reactions (G2, G6, G7, and G8)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Colony PCR of P1 - P6 using Sequencing FW1 and Rv4 primers </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Annealing Temp: 59 C</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Extension time: 3 minutes </span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Need to prepare HVCA plates for Spheroplast analysis after transformation </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Protocol for HVCA agar on Pg. 21 of Halohandbook </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Transformants should grow on plates without Uracil </span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Friday, September 4</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">General Meeting </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">FOCUS Discussion </span></li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Transition Probability Modeling </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Only need to produce a model with 2 states: fast and stall (excluding steady state) </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">John has a paper about “modeling ribosomal speed”</span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Colony PCR of P1 - P6</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Dominic’s plasmid specific primers</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Annealing Temp. 51 </span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Inoculated </span><em><span style="font-weight: 400;">H. volcanni </span></em><span style="font-weight: 400;">for making spheroplast </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Optimal absorbance reading at 600 between 0.8- 1.0 for late exponential phase </span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Nested PCR part 2 </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Template (0.4 isolation from 8/13, 8/14 and 8/17) to test which is best </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Primers: Bglu_Fw_flagFixed5 and Bglu_wt_rv_fixedFlag</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Annealing temp. : 69 C as suggested by TM Calculator </span></li> | ||
+ | </ul> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Saturday, September 5</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Absorbance reading of inoculated </span><em><span style="font-weight: 400;">H. volcanii </span></em></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">0.621 A</span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Plated Fermentation Team Electroporated Cells ( G2, G6, G7, and G8)</span></li> | ||
+ | </ol> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Monday, September 7</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Colony PCR positive controls (using Gibson Assembly reaction as template)</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Sequencing Primers Fw1 and Rv4 (Titaq) </span><span style="font-weight: 400;">Touchdown: 66 - 59 C</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Sequencing Primers Fw1 and Rv4 (OneTaq) </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Dominic’s Primers Aldy5Seq1F and Aldy5Seq5R (Titaq) </span><span style="font-weight: 400;">Touchdown 66 - 59 C</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Dominic’s Primers Aldy5Seq1F and Aldy5Seq5R (OneTaq)</span></li> | ||
+ | </ul> | ||
+ | <p><strong>Wednesday, September 9</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Chose 8 new colony picks from original plate having Transformed E. coli cells with codon optimized B Glucosidase </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Performed colony PCR using Dominic’s primers, Titaq and No PCR Enhancer </span></li> | ||
+ | </ul> | ||
+ | <p><strong>Thursday, September 10</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Started Preparing BioBricks of Codon Optimized Beta Glucosidase, and Fermentation enzymes </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Added Ampicilin to LB Agar plates and replated electroporated E. coli cells </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Added 26.3 ul of 50 ng/uL ampicillin to each plate</span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Colony PCR using Q5 Master Mix </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">3 Reactions from fermentation and 2 from Breakdown </span></li> | ||
+ | </ul> | ||
+ | <p><strong>Friday, September 11</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Professor Bernick Discussion </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Dislikes homopolymer in both Breakdown and Dominic’s Primers </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Prefers Dominic’s primers because they have a base change after the homopolymer </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Suggests 10 cycle touchdown from 56 - 51 using Dominic’s Primers, using Titaq and PCR enhancer </span></li> | ||
+ | </ul> | ||
+ | <p><strong>Saturday, September 12</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Verification that primers anneal to the C-term construct of pTA963</span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Aldy5Fw1 anneals from 485 - 508 (15 nucleotides from His Tag)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Aldy5Rv5 anneals from 569 - 587 (31 nucleotides from His Tag)</span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">SeqFw1 anneals fromm 462 to 486 </span></li> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">SeqRv4 anneals from 570 - 593 </span><span style="font-weight: 400;">(Note, first G is supposed to be a C)</span></li> | ||
+ | </ul> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Might have to redo Gibson Assembly of fragment assembly based on positive control results </span></li> | ||
+ | </ol> | ||
+ | <p><strong><strong> </strong></strong></p> | ||
+ | <p><strong>Sunday, September 13</strong></p> | ||
+ | <ol> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Touchdown of Nested PCR Part 2 </span></li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li style="font-weight: 400;"><span style="font-weight: 400;">Annealing temp from 70 - 65 C</span></li> | ||
+ | </ul> | ||
</div> | </div> |
Revision as of 21:40, 17 September 2015
Daily Logs
June 2015
Wednesday, June 24
- Discussed overall project goals for the research period
- Need to test the efficiency of multiple cellulases
- preferably those that are already present in halophiles and is compatible with Haloferax volcanii
- identified the positions of the four different cellulases associated with Halorhabdus utahensis
- Cellulosome construction possibility
Thursday, June 25
- Looked through the halophile phylogenetic tree and found that Haloquadratum walsbyi (Hwa) is closer to Haloferax volcanii, and has cellulase that occurs in the natural metabolic pathway as well
- Did a pairwise alignment between Halorhabdus utahensis (Hut) and Haloquadratum walsbyi (Hwa)
- Concluded that it was probably not a good idea to risk using Haloquadratum walsbyi since it only contains a “probable” cellulase, and it does not contain any other beta-glucanase enzymes
Friday, June 26
- Learned how to design primers for Gibson Assembly
- Have two potential cellulase candidates
- one from H.tiamaten
- one from H.utahensis
- Using pTA963 as an expression plasmid
- Most of the cellulases seen need C-terminal His Tag since the N-terminus is found in the transmembrane
- Need to use inverse PCR to linearize and amplify plasmid. This will also remove 6x His Tag and stop codon
- His Tag and stop codon will be added using flagged primers
- Introduced to idea of codon optimization
- Worked on the pI Finder program, which takes in a FASTA amino acid sequence and calculates the theoretical isoelectric point
- Narrowed criteria for usable cellulases:
- Signal Peptide for excretion or transmembrane position
- pI range between 4 and 5
Monday, June 29
- Worked with different plasmid editors and decided on Geneious
- Narrowed down the cellulase candidates from CAZy with respect to the signal peptide sequences
- Introduction to PredSignal. Software that uses hidden markov models to predict the presence and purpose of archaeal signal peptides
- Selection options for testing with cellulose:
- Rayon
- Filter paper that’s been bleached
- Avicel microcrystalline cellulose
Tuesday, June 30
- Continued the narrowing down of the cellulase candidates with respect to the presence of signal peptide
- Found the sequence of the cellulase (Hu-CBH1) from H. utahensis that has been proven to work in the paper “Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance” Zhang, Tao et al
July 2015
Wednesday, July 1
- Have enzymes categories as beta glucanase and alpha amylase, but also looking for endo glucanase and exo glucanase
- Discuss whether or not we can utilize the cellobiohydrolase Hu-CBH1 since it is patented
Thursday, July 2
- Weekly team meeting (Moved to Thursday due to 4th of July Weekend)
- Discussed a more narrow focus on Exoglucanases, Endoglucanases and B-glucosidases
- We did not want to focus on Alpha amylases since they are used for breaking down starch
Monday, July 6
- Design protocols for inverse PCR and Gibson Assembly
- Performed inverse PCR to linearize and amplify our expression plasmid (pta963)
- Protocols labelled as “ Polymerase Chain Reaction (PCR) for Amplification of pTA963 Expression Plasmids” and “Gibson Assembly Protocol”
Tuesday, July 7
- Acquired Q5 2x Master mix for PCR reactions
- H.tiamaten cellulase → homologs present, but still need to find the extension number
- Gene is about 2,700 nucleotides long x 3 ~ 8,100
- We have about 4 gene candidates + H.tiamaten cellulase that has already been tested
Wednesday, July 8
GOALS
- Thursday 7/9 → Inverse PCR (2 experiments)
- Friday 7/10 → Analysis - Gel/ plate prep
- Wednesday 7/15 → Gibson/ PCR Analysis/ Gel, Transformation of E.coli
- pick a strain of E.coli
- chemically competent
- Thursday 7/16 → Plate
- Monday 7/20 Mini-Prep/ Sequencing Prep
Thursday, July 9
- Need to prepare gene blocks for:
C-Terminus N-Terminus ?
-Shewanella -Shewanella -cellobiose phosphorylase
-tiamaten
-Hu-CBH1
-b-glucosidase (hispanica) -H.tiamaten -B 1,4 glucanase (utahensis)
Friday, July 10
- While choosing cellulase candidates
- Checked for signal peptides
- Searched through the archaeal genome browser
- As of now we are deciding between: H.utahensis, H.hispanica, H.tiamaten
- Found that we already have stock of H.hispanica in the freezer
Saturday, July 11
- Meeting to design gene blocks for:
- Shewenella fusion gene
- Hu-CBH1 cellobiohydrolase
- Cellobiose phosphorylase
- Pyruvate decarboxylase
Note: Used IDT Gene Block Editor/ oligoanalyzer 3.1
Picture
Monday, July 13
- TBE buffer was autoclaved this morning
- Ran inverse PCR to amplify N-term pTA 963 and ran on a gel
- Professor Bernick teaching PCR Fragment Assembly, Gibson Assembly and Flagged primers using sticks
Picture
Tuesday, July 14
- Reran inverse PCR, changing annealing temperature and extension time
- Grant Team Meeting:
- UCSC Crowdfunding page
- Reaching out to department chairs
- Created program for codon optimizing proteins using the codon frequency table and amino acid sequence (Called optimizer.py)
Wednesday, July 15
- Found protocol for making growth media for H. Hispanica in the Halohandbook pg. 14
- 23% Modified Growth Medium (MGM)
Thursday, July 16
- Protocol for DNA isolation for H.hispanica (page 69 of Halohandbook)
- Found that DNAWorks algorithm for codon optimization removes accuracy
- It does not take into account “rare” codons
Friday, July 17
- Weekly team meeting
- Use of DNA works for codon optimization
- Inverse PCR for amplification of C-term pta963 plasmid
- Paused during extension phase because we forgot touchdown conditions, which may greatly affect results
Monday, July 20
- N-term and 2 C-term PCRs completed
- EMERGENCY LAB MEETUP:
- Breakthrough with codon optimization code (FOCUS)
- Want to pay attention to conserved rare codons
- Need to look for this pattern across organism of different domains
- 3 genes to test (All different forms of B. Glucosidase)
- Wild type (H.hispanica) - control
- optimized (DNA Works) + control
- optimized (Jairo’s code) F.O.C.U.S
- Assay
- micro-crystalline cellulose and X-Glu plates to test for enzymatic activity
- Professor Bernick explanation of more to test for Signal Peptide:
- psortB
- TM Pred
- Introduction to the UCSC archaeal Genome Browser
- Intro to Kasava Research Paper
Tuesday, July 21
- EMERGENCY BREAKDOWN TEAM MEETUP:
- Our hypothesis: Rare and conserved codons allows better folding of the protein structures = more efficient protein production
- Obtain the secondary structures of the protein sequences through PSS Pred
- Decided to codon optimize our beta glucosidase protein without optimizing the signal peptide since we know signal peptide still works based on the Hu-CBH1 paper
Wednesday, July 22
- Looking for 2,565 bp band for beta-glucosidase gene in Haloarcula hispanica
- Continued runs for N-term and C-term PCR
Thursday, July 23
- Streaking in C-term (7/21) due to pause while running
- too much primer for the N-term; we used 5 uL instead of 1.25 uL
- codon bias for H.volcanii and H.hispanica are similar
- completed another N-term and C-term reaction
- working on perfecting conditions
Friday, July 24
- General Meeting
- Introducing entire team to F.O.C.U.S
- Plan to test the protein expression levels of the beta-glucosidase from Haloarcula hispanica between the wild type, DNA Works Codon Optimized and F.O.C.U.S Codon optimized
- Need to prove that the rate limiting step is the incorporation of a particular tRNA
- Working out the kinks for isolation of wild type beta glucosidase
- Smears may be due to a high concentration of genomic DNA during isolation
- Use of DPN1 could get rid of non-methylated (i.e non-genomic DNA) which could mean more access to PCR product and amplification
Monday, July 27
- Multiple sequence alignment of conserved proteins conserved in Archaea, Bacteria, and Eukaryotes
- Alanyl-tRNA, DNA polymerase III subunit, rRNA dimethylase
- Further proteins amongst model organisms can be found in the paper “ Universal trees based on large combined protein” Brown, James R. et al
Tuesday, July 28
- Searched for protein sequences and DNA sequences of conserved proteins in model organisms
- Note, some protein sequences in certain organisms are not of equal length to others. Therefore, they may not be orthologs
- Inverse PCR to amplify N-terminal version of plasmid pta963
Wednesday, July 29
- Need to regrow Hispanica to the optimal OD (0.6 - 0.8) for wild type beta glucosidase isolation
August 2015
Monday, August 3
- Gel for hispanica isolation did not work on Friday
- Tested to see if the track dye and DNA ladder show up under UV light
- Reran another gel to test if the results were due to track dye or gel
- Redid another isolation since results were negative
Tuesday, August 4
- Isolation of hispanica (2nd time)
- 50 uL Rxn (Iso, and negative control)
- Turned attention towards using the Dali Server for protein structure information and structural alignment comparisons
- Prepared protocol for E. coli cell electroporation
Wednesday, August 5
- Isolated Hispanica from a different aliquot (Still with an OD of 1.4)
- Multiple PCR reactions to find optimal conditions for isolating wild type b-glucosidase (Run at 71 degrees)
- Negative control (No DNA)
- Repeat of the same PCR protocol but NO PCR ENHANCER
- 2x Primers (No PCR Enhancer)
- 2x Template ( NO PCR Enhancer)
- Amplification of the first isolation that worked (tube labelled ++)
Thursday, August 6
- Redid PCR for gene isolation (Brought the annealing temperature to 66 degrees)
Friday, August 7
- Performed isolation from a 3rd aliquot of Hispanica
- Performed PCR after diluting concentration 100 fold for both aliquot 1 and 3
- Meeting with Professor Bernick to discuss one method of measuring importance of rare codons for F.O.C.U.S
- Encoding cost (Method of hyper weighting a codon that is more rare, especially if it is a rare codon in a group of more frequent codons)
- Need to develop a training set and test set to measure consistency of rare codon idea
- Prepare sequence information for engineered cellulase developed by 2014 UCSC iGEM team to perform site directed mutagenesis
Tuesday, August 11
- Fragment assembly of codon optimized Beta-Glucosidase
- Fragment concentrations from stock:
- F1 → 0.88 uL, F2 → 1.25 uL, F3 → 1 uL
- The Fw fixed Flag primer was diluted with 216 uL of TE buffer, vortexed, and stored
- Primers used: Fw Flag Fixed5 and Bglu_ Rv flag
- Nested PCR part 2 (adding flag primers to the isolated WT beta Glucosidas
- Primers: Fw Flag fixed5 and Bglu_ WT_Rv flag
- Proteins for F.O.C.U.S
- EF-G, Ef-Tu, DNA Polymerase III, tRNA synthetase
- Dali server search stored on Kerika
Wednesday, August 12
- Working on getting the codon bias tables and nucleotide sequences to test out FOCUS
- Reinoculated Hispanica to isolate it from a lower OD
- Tested Fragment assembly with 1/10 dilution of fragments and non dilution
- The lightbulb in the UV transilluminator broke, so weren’t able to capture a picture of the gel
Thursday, August 13
- General Meeting
- check 260/280 and 260/230 for the isolation
- absorbance of nucleic acids/ protein
- Run fragment assembly with 1/5 dilution, not 1/10
- Length of the fragment assembly = 2641 bp
- Length of the wild type = 2644 bp
- Isolation of Hispanica from a culture with an OD of 0.4
- concentration: 116.4 ng/uL
- 260/280: 1.64
- 260/230: 0.59
- WT isolation and fragment assembly PCR
- dilution (1:10) of new isolation
- diluted fragments and non-diluted fragments
- Annealing temp= 63.8 degrees
Friday, August 14
- Isolation PCR reaction using isolation from OD 0.4 and 1.4 to see which is better
Sunday, August 16
- Running the gel from Friday’s PCR reaction
- The isolation with an OD of 0.4 worked the best!
- dilute fragments reaction does not work
- lower the annealing temperature
- used an annealing temperature of 62.8 degrees
- Elongation time changed to 45 seconds
- Q5 reads 1000 bp every 15-45 seconds
- PCR Reactions
- 0.4, 1.4, nd, d, i, F, …, _
Monday, August 17
- Prepared 500 mL of LB agar in preparation for growing transformed E. coli cultures
- Protocol: https://www.addgene.org/plasmid-protocols/bacterial-plates/
- nanodrop for linearized PTA963
Conc. 260/280 260/230
- C-term (7/21) 462.7 ng/uL 1.82 0.87
- C-term (7/23) 561.9 ng/uL 1.83 0.93
- N-term (7/23) 794.6 ng/uL 1.84 1.24
- N-term (7/28) 335.3 ng/uL 1.84 0.74
- More PCR reactions
- 0.4 flag, annealing temp @ 70 degrees → Today
- 1.4 re-isolation, run @ 71 degrees → Tomorrow
- Fragment Assembly @ 65 degrees → Tomorrow
Tuesday, August 18
- Bright band present for fragment assembly = SUCCESS!
- Performed purification using Bernick Kit. Results:
DNA Conc. 260/280 260/230
- N1 32.2ng/ul 1.58 0.14
- N2 7.4ng/ul 2.07 0.03
- C1 6.1ng/ul 1.76 0.02
- C2 14.5 ng/ul 1.57 0.06
Wednesday, August 19
- Re-running two isolation reactions with an annealing temp of 71 C and 42 sec extension
- Gel extraction of fragment assembly to run Gibson assembly
Thursday, August 20
- PCR reactions (Annealing temp. 65 degrees)
- gel-extracted iso
- gel-extracted frag (dil)
- non gel-extracted iso
- non-dil frag
Friday, August 21
- To Do:
- Confirm correct isolation band
- redo fragment assembly
- 1 uL each fragment (10 uM)
- correct concentrations (0.88 uL,1.25 uL,1uL)
- 65 degrees annealing temperature
- Details for fragment assembly that was gel extracted:
- 19 uL at concentration of 66.4 ng/uL
- 260/280: 2.03
- 260/230: 0.61
- Gibson Assembly
- 2.65 uL of C-term pta963
- 1.2 uL of Fragment Assembly
Saturday, August 22
- Inverse PCR of N-Terminal pta963 for Ethanol Team
- Transformed NEB 5-alpha competent E. coli with Gibson Assembly product
- Attempted to isolate which band corresponded to the wild type Beta Glucosidase
- Fw_Flg Fixed5 and Seq Rv3 primers, looking for band of 1000
Sunday, August 23
- Checked transformation plate, put found no growth
- Most likely due to cells having lost competence since they were not stored at -80 C for at least a week
- Prepared reagents for making spheroplast
Monday, August 24
- Made “Unbuffered spheroplasting solution”, “Buffered spheroplasting solution” and “Regeneration solution”
- Protocol found on pgs. 59-62 of the Halohandbook
- Redid inverse PCR of N-term pTA963
- Elongation lowered to 2 min and 30 sec
Wednesday, August 26
- Electroporation using electrocompetent E. coli cells
- 1800 volts
- Used Electroporation protocol made by Fermentation team
- Ag Tech Meet up Presentation
Thursday, August 27
- Nested PCR Part 2
- Used Seq/iso reaction product from 8/27 as template DNA since it has the brightest band corresponding to the wild type beta glucosidase
- Concentration: 472.2 ng/uL ; 262/280: 1.79 ; 260/230: 0.68
- Used Fw Flag Fixed5, Rv Flag and Rv Flag fixed as primers
Monday, August 31
- Colony PCR from Transformation plate
- Picked 6 colonies
- Used miliq water to lyse cells (Tubes labelled 1P-6P)
- Used Flagged primers: Fw Flag Fixed 5 and Rv Flag Fixed
- Ran another reaction using sequencing primers: Seq Fw2 and Seq Rv2
September 2015
Tuesday, September 1
- Meeting with the Dean of Students
- Rerun a gel including fermentation team colony PCR samples and Breakdown sample (D - dilute, and ND- non-Dilute) of wild type isolation test
- Made 236.25 uL of Q5 Master Mix
- Enough for 27 reactions if we use 25 uL PCR reactions
- Recipe (X10):
- 20 uL Q5 Buffer
- 2 uL of dNTP’s
- 0.5 uL of Q5 enzyme
- 11.25 uL of Miliq water
Wednesday, September 2
- Colony PCR (4 reactions):
- P2 with iso Primers and PCR enhancer (1:10 dilution)
- P2 with iso Primers, No PCR enhancer (1:10 dilution)
- P2 with Flagged Primers, PCR enhancer (1:10 dilution)
- P2 with flagged Primers, No PCR enhancer (1:10 dilution)
- Electroporation of Fermentation Gibson reactions (G2, G6, G7, and G8)
- Colony PCR of P1 - P6 using Sequencing FW1 and Rv4 primers
- Annealing Temp: 59 C
- Extension time: 3 minutes
- Need to prepare HVCA plates for Spheroplast analysis after transformation
- Protocol for HVCA agar on Pg. 21 of Halohandbook
- Transformants should grow on plates without Uracil
Friday, September 4
- General Meeting
- FOCUS Discussion
- Transition Probability Modeling
- Only need to produce a model with 2 states: fast and stall (excluding steady state)
- John has a paper about “modeling ribosomal speed”
- Colony PCR of P1 - P6
- Dominic’s plasmid specific primers
- Annealing Temp. 51
- Inoculated H. volcanni for making spheroplast
- Optimal absorbance reading at 600 between 0.8- 1.0 for late exponential phase
- Nested PCR part 2
- Template (0.4 isolation from 8/13, 8/14 and 8/17) to test which is best
- Primers: Bglu_Fw_flagFixed5 and Bglu_wt_rv_fixedFlag
- Annealing temp. : 69 C as suggested by TM Calculator
Saturday, September 5
- Absorbance reading of inoculated H. volcanii
- 0.621 A
- Plated Fermentation Team Electroporated Cells ( G2, G6, G7, and G8)
Monday, September 7
- Colony PCR positive controls (using Gibson Assembly reaction as template)
- Sequencing Primers Fw1 and Rv4 (Titaq) Touchdown: 66 - 59 C
- Sequencing Primers Fw1 and Rv4 (OneTaq)
- Dominic’s Primers Aldy5Seq1F and Aldy5Seq5R (Titaq) Touchdown 66 - 59 C
- Dominic’s Primers Aldy5Seq1F and Aldy5Seq5R (OneTaq)
Wednesday, September 9
- Chose 8 new colony picks from original plate having Transformed E. coli cells with codon optimized B Glucosidase
- Performed colony PCR using Dominic’s primers, Titaq and No PCR Enhancer
Thursday, September 10
- Started Preparing BioBricks of Codon Optimized Beta Glucosidase, and Fermentation enzymes
- Added Ampicilin to LB Agar plates and replated electroporated E. coli cells
- Added 26.3 ul of 50 ng/uL ampicillin to each plate
- Colony PCR using Q5 Master Mix
- 3 Reactions from fermentation and 2 from Breakdown
Friday, September 11
- Professor Bernick Discussion
- Dislikes homopolymer in both Breakdown and Dominic’s Primers
- Prefers Dominic’s primers because they have a base change after the homopolymer
- Suggests 10 cycle touchdown from 56 - 51 using Dominic’s Primers, using Titaq and PCR enhancer
Saturday, September 12
- Verification that primers anneal to the C-term construct of pTA963
- Aldy5Fw1 anneals from 485 - 508 (15 nucleotides from His Tag)
- Aldy5Rv5 anneals from 569 - 587 (31 nucleotides from His Tag)
- SeqFw1 anneals fromm 462 to 486
- SeqRv4 anneals from 570 - 593 (Note, first G is supposed to be a C)
- Might have to redo Gibson Assembly of fragment assembly based on positive control results
Sunday, September 13
- Touchdown of Nested PCR Part 2
- Annealing temp from 70 - 65 C
Week 3 (7/6/15 - 7/10/15)
- Discussed the formation of the team
- Used KEGG to view the metabolic pathway of Haloferax volcanii and identify genes involved with pyruvate breakdown in order to isolate potential genes to knockout
- Lactate dehydrogenase & NADP-dependent malic enzyme
- Found Zymomonas mobilis genome (2,056,363 bp) and Pyruvate decarboxylase gene, pdc, from Zymomonas mobilis subsp. ZM4 on NCBI
Week 4 (7/13/15 - 7/17/15)
- We contacted Julie Maupin-Furlow from the University of Florida because of previous research she had done with H.vo and the Z.mo pdc gene
- We optimized the Z.mo pdc for insertion into H.vo using DNAWorks
- Designed gene blocks, an overlap region, and primers (flagged with our pTA963 plasmid) for gibson assembly of our H.vo pdc
Week 5 (7/20/15 - 7/24/15)
- Began designing our knockouts
- We used the UCSC archaea browser to retrieve genes before and after knockout gene and intergenic sequences
Week 6 (7/27/15 - 7/31/15)
- Finalized our knockout sequences
- Ran knockout genes through blastx and there are no protein matches in any organism
- Designed primers for our knockout genes
- Ordered our H.vo pdc gene blocks and primers along with our knockout gene blocks and primers
Week 7 (8/3/15 - 8/7/15)
- Our gene blocks and primers arrived
- We looked into making spheroplasts for transformation into H.vo
Week 8 (8/10/15 - 8/14/15)
- Ran PCR for fragment assembly of our H.vo pdc gene
- We ran PCR on or knockouts genes to amplify them
- Purified H.vo pdc to be used for gibson assembly
Week 9 (8/17/15 - 8/21/15)
- Did gibson assembly of our H.vo pdc into the N-term His-tagged plasmid pTA963
- Transformed our product into chemically competent E. coli by heat shocking them, then plated these onto ampicillin plates and let them grow overnight
- Upon plating there was no growth and we determined that the problem was with the E. coli cells so we switched to top ten electro-competent E. coli
Week 10 (8/24/15 - 8/28/15)
- Redid transformations
- Electroporation was used to transform our Gibson assembly product into the electro-competent E. coli cells
- Had significant growth on our ampicillin plate showing that our plasmid had been successfully transformed into the E. coli cells
Week 11 (8/31/15 - 9/4/15)
- Ran colony PCR with the primers we used for our H.vo pdc fragment assembly
- We chose 9 colonies from separate areas on the plate for a better chance of success
- Results were inconclusive but we were able to select 5 candidates that were more likely to give us positive results
- Reran colony PCR using plasmid specific primers
- Again results were inconclusive
Week 12 (8/7/15 - 8/11/15)
- Reran colony PCR using the same plasmid specific primers but doing a touchdown PCR
- No results to examine
- Reran colony PCR using a different pair of plasmid specific primer doing another touchdown PCR
- No results to examine
- Troubleshooting these errors