Team:UCSC/Logs
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