June 2015

 

Wednesday, June 24

1. Discussed overall project goals for the research period
2. Need to test the efficiency of multiple cellulases

• preferably those that are already present in halophiles and is compatible with Haloferax volcanii

1. identified the positions of the four different cellulases associated with Halorhabdus utahensis
2. Cellulosome construction possibility

 

Thursday, June 25

1. 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

1. Learned how to design primers for Gibson Assembly
2. Have two potential cellulase candidates

• one from H.tiamaten
• one from H.utahensis

1. 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

1. Introduced to idea of codon optimization
2. Worked on the pI Finder program, which takes in a FASTA amino acid sequence and calculates the theoretical isoelectric point
3. Narrowed criteria for usable cellulases:

• Signal Peptide for excretion or transmembrane position
• pI range between 4 and 5

Monday, June 29

1. Worked with different plasmid editors and decided on Geneious

1. 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

1. Selection options for testing with cellulose:

1. Rayon
2. Filter paper that’s been bleached
3. Avicel microcrystalline cellulose

Tuesday, June 30

1. Continued the narrowing down of the cellulase candidates with respect to the presence of signal peptide
2. 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

1. Have enzymes categories as beta glucanase and alpha amylase, but also looking for endo glucanase and exo glucanase
2. Discuss whether or not we can utilize the cellobiohydrolase Hu-CBH1 since it is patented

 

Thursday, July 2

1. 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

1. Design protocols for inverse PCR and Gibson Assembly
2. 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

1. Acquired Q5 2x Master mix for PCR reactions
2. H.tiamaten cellulase → homologs present, but still need to find the extension number

• Gene is about 2,700 nucleotides long x 3 ~ 8,100

1. 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

1. 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

1. 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

1. Found that we already have stock of H.hispanica in the freezer

 

Saturday, July 11

1. 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

1. TBE buffer was autoclaved this morning
2. Ran inverse PCR to amplify N-term pTA 963 and ran on a gel
3. Professor Bernick teaching PCR Fragment Assembly, Gibson Assembly and Flagged primers using sticks

Picture

Tuesday, July 14

1. Reran inverse PCR, changing annealing temperature and extension time
2. Grant Team Meeting:

• UCSC Crowdfunding page
• Reaching out to department chairs

1. Created program for codon optimizing proteins using the codon frequency table and amino acid sequence (Called optimizer.py)

 

Wednesday, July 15

1. Found protocol for making growth media for H. Hispanica in the Halohandbook pg. 14

• 23% Modified Growth Medium (MGM)

 

Thursday, July 16

1. Protocol for DNA isolation for H.hispanica (page 69 of Halohandbook)
2. Found that DNAWorks algorithm for codon optimization removes accuracy

• It does not take into account “rare” codons

 

Friday, July 17

1. Weekly team meeting

• Use of DNA works for codon optimization

1. 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

1. N-term and 2 C-term PCRs completed  
2. 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

1. Professor Bernick explanation of more to test for Signal Peptide:

• psortB
• TM Pred

1. Introduction to the UCSC archaeal Genome Browser
2. Intro to Kasava Research Paper

 

Tuesday, July 21

1. 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

1. Looking for 2,565 bp band for beta-glucosidase gene in Haloarcula hispanica
2. Continued runs for N-term and C-term PCR

 

Thursday, July 23

1. 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

1. codon bias for H.volcanii and H.hispanica are similar
2. completed another N-term and C-term reaction

• working on perfecting conditions

Friday, July 24

• General Meeting

1. 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

1. 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

1. 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

1. 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

1. Inverse PCR to amplify N-terminal version of plasmid pta963

Wednesday, July 29

1. Need to regrow Hispanica to the optimal OD (0.6 - 0.8) for wild type beta glucosidase isolation

 

August 2015

 

Monday, August 3

1. 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

1. Isolation of hispanica (2nd time)

• 50 uL Rxn (Iso, and negative control)

1. Turned attention towards using the Dali Server for protein structure information and structural alignment comparisons
2. Prepared protocol for E. coli cell electroporation

Wednesday, August 5

1. Isolated Hispanica from a different aliquot (Still with an OD of 1.4)
2. 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

1. Redid PCR for gene isolation (Brought the annealing temperature to 66 degrees)

 

Friday, August 7

1. Performed isolation from a 3rd aliquot of Hispanica
2. Performed PCR after diluting concentration 100 fold for both aliquot 1 and 3
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

1. Prepare sequence information for engineered cellulase developed by 2014 UCSC iGEM team to perform site directed mutagenesis

 

Tuesday, August 11

1. 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

1. Nested PCR part 2 (adding flag primers to the isolated WT beta Glucosidas

• Primers: Fw Flag fixed5 and Bglu_ WT_Rv flag

1. 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

1. Working on getting the codon bias tables and nucleotide sequences to test out FOCUS
2. Reinoculated Hispanica to isolate it from a lower OD
3. 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

1. 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

1. 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

1. 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

1. Isolation PCR reaction using isolation from OD 0.4 and 1.4 to see which is better

 

Sunday, August 16

1. 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

1. PCR Reactions

• 0.4, 1.4, nd, d, i, F, …, _
  
  

Monday, August 17

1. Prepared 500 mL of LB agar in preparation for growing transformed E. coli cultures

• Protocol: https://www.addgene.org/plasmid-protocols/bacterial-plates/

1. 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

1. Bright band present for fragment assembly = SUCCESS!
2. 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

1. Re-running two isolation reactions with an annealing temp of 71 C and 42 sec extension
2. Gel extraction of fragment assembly to run Gibson assembly

 

Thursday, August 20

1. PCR reactions (Annealing temp. 65 degrees)

• gel-extracted iso
• gel-extracted frag (dil)
• non gel-extracted iso
• non-dil frag

 

Friday, August 21

1. 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

1. 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

1. Gibson Assembly

• 2.65 uL of C-term pta963
• 1.2 uL of Fragment Assembly

 

Saturday, August 22

1. Inverse PCR of N-Terminal pta963 for Ethanol Team
2. Transformed NEB 5-alpha competent E. coli with Gibson Assembly product
3. 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

1. 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

1. Prepared reagents for making spheroplast

 

Monday, August 24

1. Made “Unbuffered spheroplasting solution”, “Buffered spheroplasting solution” and “Regeneration solution”

• Protocol found on pgs. 59-62 of the Halohandbook

1. Redid inverse PCR of N-term pTA963

• Elongation lowered to 2 min and 30 sec

 

Wednesday, August 26

1. Electroporation using electrocompetent E. coli cells

• 1800 volts
• Used Electroporation protocol made by Fermentation team

1. Ag Tech Meet up Presentation

 

Thursday, August 27

1. 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

1. 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

1. Meeting with the Dean of Students
2. Rerun a gel including fermentation team colony PCR samples and Breakdown sample (D - dilute, and ND- non-Dilute) of wild type isolation test
3. 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

1. 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)

1. Electroporation of Fermentation Gibson reactions (G2, G6, G7, and G8)
2. Colony PCR of P1 - P6 using Sequencing FW1 and Rv4 primers

• Annealing Temp: 59 C
• Extension time: 3 minutes

1. 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

1. 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”

1. Colony PCR of P1 - P6

• Dominic’s plasmid specific primers
• Annealing Temp. 51

1. Inoculated H. volcanni for making spheroplast

• Optimal absorbance reading at 600 between 0.8- 1.0 for late exponential phase

1. 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

1. Absorbance reading of inoculated H. volcanii

• 0.621 A

1. Plated Fermentation Team Electroporated Cells ( G2, G6, G7, and G8)

 

Monday, September 7

1. 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

1. 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

1. Started Preparing BioBricks of Codon Optimized Beta Glucosidase, and Fermentation enzymes
2. Added Ampicilin to LB Agar plates and replated electroporated E. coli cells

• Added 26.3 ul of 50 ng/uL ampicillin to each plate

1. Colony PCR using Q5 Master Mix

• 3 Reactions from fermentation and 2 from Breakdown

Friday, September 11

1. 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

1. 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)

1. Might have to redo Gibson Assembly of fragment assembly based on positive control results

 

Sunday, September 13

1. Touchdown of Nested PCR Part 2

• Annealing temp from 70 - 65 C

June 2015 Wednesday, June 24 ● Outlined overall project goals ● Used NCBI’s nucleotide best BLAST to find gene orthologs from the Garcia pathway in H. volcanii. ○ Found all genes in H. volcanii except for butyraldehyde dehydrogenase and butanol dehydrogenase. ● Used BLAST to find other butyraldehyde and butanol dehydrogenases that could be used in H. volcanii. ○ Considered using an aldehyde/alcohol dehydrogenase fusion gene. ● Found a promising fusion gene in Halomonas sp. S2151 called adhE2. Also had a pI near 5, which is ideal for H. volcanii. ○ Little was known about Halomonas and the gene was only recently discovered. ● Considered using gas chromatography to confirm the presence of butyryl-CoA. Thursday, June 25 ● Continued to pursue the idea of using gas chromatography to test for butyryl-CoA. ○ Rob Franks agreed to allow us to use the GC. ● Searched for places to purchase butyryl-CoA to create a standard for GC, but it’s very expensive. ● Started searching for protocols for gas chromatography of butyryl-CoA and more generally, fatty acids. Friday, June 26 ● Created 10 L of 30% salt water stock to be used in producing media for H. volcanii. ○ Used protocol in pg 20 of Halohandbook. ● Prepared Tris buffer (500 mL of pH 8.83 and 40 mL of pH 7.54). Monday, June 29 ● Continued searching for gas chromatography protocols specific to butyryl-CoA. ● Considered using qPCR, since finding a protocol is a challenge. ● Sources suggest using HPLC to detect butyryl-CoA. ○ Rob Franks believes HPLC will be better suited for butyryl-CoA. ● Found a site with potentially helpful primer design rules (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html) Tuesday, June 30 ● Decided to stop pursuing gas chromatography of butyryl-CoA. We are assuming that H. volcanii produces it at this point. ● MGM plates have been prepared according to pg 14 of Halohandbook. July 2015 Wednesday, July 1 ● Proposed the idea of using beads that bind to CoA groups to detect the presence of butyryl-CoA. ● Trying to find a good promoter to increase the gene expression of the genes we decide to use. ○ Several searches yielded the promsing ptb promoter. ● Considered using rational design to lower the pI of the fusion protein to near 4.5. Requires generating a model structure using SWISS-MODELLER. ○ Unable to find a 3D model of adhE2. ● Jairo used his program to codon optimize the adhE2 gene for H. volcanii. Thursday, July 2 ● Decided on the pTA963 plasmid for eventual expression of our fusion gene. ● Professor Bernick urged us to focus on pI rather than percent identity of potential fusion genes. ● Found yet another alternative fusion gene in Shewanella waksmanii (http://www.ncbi.nlm.nih.gov/nuccore/655366186). ○ Found the nucleotide sequence for this fusion gene. ○ Discontinuing work on the Halomonas fusion gene adhE2. Monday, July 6 ● Attempting to find a protocol for inserting the S. waksmanii fusion gene into the expression plasmid. ○ Dominic suggested using Gibson assembly. ● Tried to codon optimize the S. waksmanii gene using Jairo’s program, but it ran into a syntax error which couldn’t be fixed. ● Asked Professor Bernick to contact an expert in S. waksmanii (Chad Saltikov). ● Reconsidered the ptb promoter as a backup. ● Found the bgaH gene, which is used to assay the promoters of haloarchaeal genes. ● Found a similar fusion gene in Shewanella sediminis. ○ Has a lower pI. ○ Unable to find the entire genome of S. waksmanii for codon optimization. ○ The full genome for S. sediminis is available, allowing codon optimization. Tuesday, July 7 ● Professor Bernick approved the idea of using the S. sediminis fusion gene. ● The fusion gene was codon optimized for H. volcanii. ● Checked nucleotide sequence for the formation of structures that would affect Gibson assembly. ● Split the S. sediminis fusion gene into smaller 500 - 700 bp fragments for ordering. ○ Fragments >1000 bp take longer and are more expensive. Wednesday, July 8 ● Finished splitting the S. sediminis fusion gene into 3 gene fragments. ● Began designing primers for the pTA963 plasmid and the His-tags. Thursday, July 9 ● Plasmid primers were ordered. ● Added overlaps on the S. sediminis fusion gene for both N- and C-terminal His tags. Friday, July 10 ● Continuing to pursue the bgaH gene as a backup. ● Started working on primers for fusion gene. Saturday, July 11 ● Finished fusion gene primers. Monday, July 13 ● Waited for approval of S. sediminis primers for ordering. ● Ran inverse PCR on the pTA963 plasmid. Tuesday, July 14 ● Checked optimized gene sequence to ensure that resulting amino acid sequence matches. ○ Did not match, so the program needs refinement. Wednesday, July 15 ● Jairo attempted to fix codon optimization program. Thursday, July 16 ● Jairo fixed his program, however Professor Bernick urged us to use DNAWorks for codon optimization. ● Discontinued work on the ptb promoter, in the interest of time. Friday, July 17 ● Plasmid N- and C-terminal protocols were accidentally swapped and were redone. ● Continued to refine the S. sediminis gene fragments and primers. ○ Need to manually reduce the GC content for ordering. Monday, July 20 ● Continued to work on the S. sediminis fragments and primers. Tuesday, July 21 ● Still working on the S. sediminis fragments and primers. ● According to Timpson et al., adh2 is an alcohol dehydrogenase in H. volcanii that shows high rates of butanol production. ○ Butanol production via reduction only occurs in acidic conditions. ○ The reverse oxidation reaction occurs in alkaline conditions. ○ Since H. volcanii is grown in alkaline conditions, oxidation is favored. ○ Considered growing H. volcanii and acidifying the environment. ● H. volcanii also has two aldehyde dehydrogenases (aldY3 and aldY5). ○ It’s unclear which results in more butanol production. ● Shifted the project to focus on developing two fusion genes consisting of adh2 and each of the aldehyde dehydrogenases. ● Started researching linker sequences to connect the two dehydrogenase genes. Wednesday, July 22 ● Finalized the S. sediminis fragments and primers. ● Made minimal media for growing H. volcanii anaerobically. ○ Will give several days for colonies to grow, before acidifying the environment. ● Reduced the number of rare codons in the S. sediminis gene. ● Contacted Rob Franks about gas chromatography usage. ○ He said it should be able to handle slightly acidic conditions. ○ Need to prepare standards before running samples. ● Looked up sequences of aldY3, aldY5, and adh2 on KEGG. Thursday, July 23 ● Prepared the S. sediminis fragments and primers for ordering. ● Started researching linker sequences to connect the two dehydrogenase genes. ○ Found a paper that describes characteristics of good linkers (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/) ● Professor Bernick urged us to find natural linkers rather than designing our own. Friday, July 24 ● ?????? Monday, July 27 ● Designed primers with Dominic’s help for amplifying aldY3 and adh2. ● Tried using Pfam to view the domains of fusion proteins to obtain natural linker sequences. Tuesday, July 28 ● Used InterPro to visualize the linker region in natural fusion genes. ● Met with Professor Bernick and established a long-term game plan: ○ Design primers for dehydrogenase genes such that they incorporate the linker region. ○ Use Gibson assembly to incorporate the dehydrogenase genes and linker into the pTA963 plasmid. ○ Transform the Gibson product into E. coli, and determine which colonies were successful in taking up the fusion gene by colony PCR. ○ Design sequencing primers and send product off for Sanger sequencing. ○ Use nested PCR on the colony PCR product. ● Dominic confirmed that he also has adh2 primers, so we only designed the aldY3 primers. ● Continued searching for linker sequences. Wednesday, July 29 ● Several linker regions were designed from scratch. Started to incorporate overlap with dehydrogenase genes. ● Other linkers were found to be conserved across several species. ● Finalized aldY3 primers for ordering. ● Settled on 4 linkers: 2 designed and 2 conserved. Thursday, July 30 ● Received the S. sediminis primers and diluted them to 100 uM. August 2015 Monday, August 3 ● Waiting for S. sediminis gene blocks. ● The anaerobic cultures haven’t grown much - pH still at 7.5. Tuesday, August 4 ● Received native gene primers. These primers were also diluted to 100 uM. ● Genomic DNA isolated from H. volcanii ○ Followed protocol in pg 70 of Halohandbook. Wednesday, August 5 ● Ran PCR on genomic DNA to isolate aldY5 (annealing temp 71℃). ○ Extension time should’ve been 1 min, but was only 30 sec. ● Ran a gel on the PCR product - mostly primers showed up with little to no PCR product. Thursday, August 6 ● Redid PCR of aldY5 and aldY3 using these conditions: ● aldY3 and aldY5 were successful, adh2 was not. ● Received the S. sediminis gene blocks. Friday, August 7 ● Ran PCR again on aldY5 and aldY3 using these conditions: ● Used a touchdown PCR on adh2 for more specific primer-binding (decrease annealing temperature by 0.5 ℃ each cycle). ● The conditions were successful Saturday, August 8 ● Re-ran PCR on adh2. ● Ran PCR on the 8 gene/linker fragments (71℃ annealing, 1 min extension, 2 min final hold) ○ aldy3BFw & aldy3Blink1Rev = A ○ aldy5BFw & aldy5Blink1Rev = B ○ aldy3BFw & aldy3Blink2Rev = C ○ aldy5BFw & aldy5Blink2Rev = D ○ aldy3BFw & aldy3Blink3Rev = E ○ aldy5BFw & aldy5Blink3Rev = F ○ aldy3BFw & aldy3Blink4Rev = G ○ aldy5BFw & aldy5Blink4Rev = H Monday, August 10 ● Unable to proceed due to a lack of supplies. Tuesday, August 11 ● Still unable to proceed further due to a lack of supplies. ● Anaerobic cultures appear to be growing. ● Started to prepare minimal HEPES media rather than Tris (pH 6.55). Wednesday, August 12 ● Continuing to make minimal media. ○ Considering eliminating potassium phosphate to avoid precipitate formation. ● Professor Bernick suggested micro-aerobic cultures, in which a small amount of oxygen is injected into the culture vessel. ○ May help solve the redox imbalance from the lack of oxygen as a terminal electron acceptor. ● Assembled S. sediminis gene fragments. ● Ran a gel with aldY5/adh2 and S. sediminis fragments. Thursday, August 13 ● The S. sediminis fragments were re-assembled (increased annealing temperature to 67℃). ● Gene/linker sequences also ran again. ● Key: ○ Lane1: aldy5BFw & aldy5Blink4Rev = H ○ Lane2: adh2Dlink1Fw & adh2DRev = I ○ Lane3: adh2Dlink2Fw & adh2DRev = J ○ Lane4: adh2Dlink3Fw & adh2DRev = K ○ Lane5: adh2Dlink4Fw & adh2DRev = L ● PCR conditions: Friday, August 14 ● Assembled the aldY and adh2 linker fragments. ○ aldy3BFw & aldy3Blink1Rev & adh2Dlink1Fw & adh2DRev = F1 ○ aldy5BFw & aldy5Blink1Rev & adh2Dlink1Fw & adh2DRev = F2 ○ aldy3BFw & aldy3Blink2Rev & adh2Dlink2Fw & adh2DRev = F3 ○ aldy5BFw & aldy5Blink2Rev & adh2Dlink2Fw & adh2DRev = F4 ○ aldy3BFw & aldy3Blink3Rev & adh2Dlink3Fw & adh2DRev = F5 ○ aldy5BFw & aldy5Blink3Rev & adh2Dlink3Fw & adh2DRev = F6 ○ aldy3BFw & aldy3Blink4Rev & adh2Dlink4Fw & adh2DRev = F7 ○ aldy5BFw & aldy5Blink4Rev & adh2Dlink4Fw & adh2DRev = F8 ● PCR conditions for fragment assembly: ● Samples were purified using a DNA cleanup kit, but purification was unsuccessful (see last lane). Sunday, August 16 ● Attempted to determine why the cleanup kit didn’t work. ○ Tested the reagents in the kit. ○ Still unsuccessful. ● Decided to not assemble fragments and then use Gibson assembly, but to go straight into Gibson assembly and see if the fragments assemble properly before annealing to the plasmid. Monday, August 17 ● Continued to work on cleanup kit. ○ Sample G1 was successfully cleaned up. ● Samples B, E, F, L need to be re-cleaned, from the kit mishap. ○ Despite cleanup, these samples had a low 260/230 value. ● Unsure whether to use N-terminal or C-terminal plasmid for our Gibson reaction. Tuesday, August 18 ● Confirmed that the N-terminal plasmid is required for Gibson of native fusion genes. ● Considered the idea of testing cultures for fermentation products such as lactic acid. ○ Rob Franks suggested using HPLC, since lactic acid won’t run in the column. ○ HPLC also somewhat non-specific. ● Ran Gibson assembly on A + I (F1) and B + I (F2) Wednesday, August 19 ● Gibson assembly failed due to a bad plasmid. Need to redo plasmid before attempting Gibson assembly again. ● Conditions used in generating the C-terminal and N-terminal plasmid: ● Started to figure out the details surrounding micro-aerobic cultures and how much oxygen needs to be added. Thursday, August 20 ● Recreated samples B, E, L, F. ● Recreated both N- and C-terminal plasmid. ● Results of plasmid (left) and samples B, E, L, F (right): ● Ran Gibson assembly on both S. sediminis samples and A + I again. ● Inoculated 3 micro-anaerobic cultures containing the following types of media: ○ HEPES buffer ○ DMSO ○ Unbuffered ● Transformed the A + I Gibson product into E. coli using heat shock and plated on ampicillin plates. Friday, August 21 ● Plates did not show growth, so transformation was unsuccessful. ● Planned to re-plate and/or possibly rerun Gibson assembly using a different ratio of plasmid to fragment. Monday, August 24 ● Created both buffered and unbuffered spheroplast solutions. ● Considered switching to transformation by electroporation rather than heat shock. Tuesday, August 25 ● Transformed E. coli with F3/G3 and C-terminal S. sediminis Gibson products using electroporation. Wednesday, August 26 ● Ran Gibson assembly on all remaining gene/linker fragments. Thursday, August 27 ● Ran colony PCR on G1 sample. ● Prepared samples and standards for gas chromatography of micro-aerobic cultures (using ethyl acetate as solvent). ○ 1% 1 mL butanol standard ○ 1% 1 mL lactic acid standard ○ 1 mL ethyl acetate blank ○ 10% 1 mL samples of each culture (Unbuffered, HEPES, DMSO, and aerobic) ● Generate more I fragment to rerun Gibson assembly on G2 (B + I). ● Ran colony PCR on G3 (C + J). Monday, August 31 ● Transformed and inoculated G4 and G5 into E. coli. ● Prepared 18% MGM to be used in establishing a new aerobic culture. September 2015 Tuesday, September 1 ● Ran colony PCR on G1, G4, and G5. ● Ran Gibson assembly on G2. ● Inoculated new aerobic cultures. Once grown, these cultures will be switched to anaerobic/micro-aerobic environments.