Daily Logs

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Breakdown Team

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

Fermentation: Butanol

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.

Fermentation: Ethanol

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

Field Team

Tuesday June 30

The field team went over the protocol and made sure all the equipment we needed was put together

Wednesday July 1

We collected 10 samples from varying locations in the Fremont salt flats
pH and salinity will have to be measured in lab due to the battery being knocked out of the portable meter
Initial plating of field samples was pushed to tomorrow due to time constraints

Thursday July 2

All of our samples were plated on 18% MGM agar plates (protocol found in the HaloHandbook)

Tuesday July 7

Substantial growth can be seen on our 18%MGM agar plates
Single cell isolations were started on colonies that showed the most robust growth from each field sample.
We decided on using a minimal media with cellulose as the only carbon source to screen for our cellulase producing halophile.
We decided on measuring turbidity on the UV spectrometer to determine sufficient growth on MCC.

Wednesday July 8

Some single cell isolations will need to be redone due to poor spreading technique
We characterized each of the single cell isolation colonies.
To prepare for 80% glycerol stocks we need to grow the single cell isolations ing 18%MGM to raise the cell count.

Thursday July 9

More single cell isolations are ready to incubate in MGM media for our cellulose screen and glycerol stocks.

The first minimal media we made was way to cloudy, we’re going to reduce it from 5g of MCC to 3g.

Friday July 10

Discussed what happens after we identify the haloarchaea after the cellulose screen. We could sequence the 16S region of the ribosomal DNA to identify the species and if there is a new species we could get the whole genome sequenced.

We would need to order a primer to do the proper PCR.
We also found a paper about solubilizing cellulose with 14%wt NaOH. It may break the chains of glucose but not substantially. (Kuo, Yen-Ning and Hong, Juan. "Investigation of Solubility of Microcrystalline Cellulose in Aqueous NaOH." (2004).)

Sunday July 12

We are going to make a soluble MCC minimal media by treating the MCC (2g) with NaOH (14.9%wt)

Monday July 13

We inoculated 5 more samples in MGM 18% from our previous single cell isolation.

We are re-isolated some of our previous single cell isolation due to smearing.

The protocol for the Micro-crystalline cellulose minimal media is finally complete for for solubilized and unsolubilized MCC.

We had to freeze the NaOH treated MCC in order to solubilize it.

Tuesday July 14

We made our solubilized MCC minimal media today.
We need to titrate the 5M TrisHCl slowly and we needed around 50mL to reach a pH of 7.5 for a 1L of solubilized MCC.

Thursday July 16

We made the insoluble MCC minal media and brought the pH down to 7.5.
We discussed what will happen after the 16S region PCR. We would use the NCBI BLASTN program to see.
The pH was raised overnight to a pH of 8.0 but it is acceptable because H. volcanii prefer a slightly alkaline environment

Tuesday July 21

We need to do more glycerol stocks for new single cell isolates.

Friday July 24

Any organism that can breakdown insoluble cellulose can theoretically breakdown insoluble MCC, so the insoluble MCC would tell us the most, but we are going to run both soluble and insoluble MCC screen just in case.

We are also considering using a glucose test to measure the concentration of glucose in the media as time goes on. We’re thinking of using a diabetes glucometer.
We are starting to prepare for designing the primers needed for the 16S ribosomal DNA PCR.

Monday July 27

We have 10 soluble and 10 insoluble MCC screens in both incubators.

The single cell isolate P5A is showing slight signs of pink in the insoluble test. Pink indicates a high cell count (if the cells are pink in color) or a high concentration of cellular metabolites, which both indicate cells can thrive of purified cellulose.

Tuesday July 28

There is growth in MCC minimal media cultures! We just need to isolate them and we’re thinking of making agar plates with the solubilized MCC minimal media.

We’re considering using the microscope to prove growth as opposed to optical density and turbidity.

Wednesday July 29

We looked at the cells that have been growing in our media under the microscope and we saw a heterogenous mixture of cell types including bacilli, cocci, and even a fast swimming species. Somewhere in the mix is our species producing cellulase and the other species may be feeding off the glucose being released from the MCC.

Friday July 31

We decided on which samples would move onto the soluble MCC agar plates to isolate our cellulase producing species.
We made MCC minimal media agar plates

Monday August 3

We’ve started plating on the soluble MCC agar plates.
More samples are showing growth in the cellulose screen

Friday August 7

We decided to go with universal archaeal primers for the 16S region of ribosomal DNA. The primers won’t work for bacteria which is ideal because we’re trying to find an archaea.
We decided to do a touchdown PCR reaction where the annealing temperature is lowered after every cycle to improve specificity of the amplification.
The annealing temperature will be set at 68℃ and will drop to 65℃ over 15 cycles

Friday August 21

The single cell isolation on the MCC plates are grown enough for identification. We need to grow them in MGM again to increase cell count for DNA extraction.
After the PCR we are going to send our PCR product to UC Berkeley to be sequenced. We’ll need controls for the PCR so we are also going to use E. coli and H. volcanii to prove our primers work only for archaea.

Tuesday August 25

We ran our sequences on the NCBI BLASTN program and we were getting hits all under the Haloferax genus

We going to show our findings at the Sierra System Synthetic Biology Symposium this weekend