Notebook
May Entries
June Entries
July Entries
August Entries
September Entries
October Entries
May 18
Julian and Sophia
Mason jars purchased for growing plants. Filled with soil and autoclaved.
May 19
Julian
Second autoclaving of jars with soil
May 21
All
45 Brachypodium seeds were sterilized, then 5 seeds planted in each jar. Jars were placed into the cold room.
May 22
Julian, Sophia, Ashley, Sarah, and Casper
Design and ordering of acdS genes
NEBuilder assembly design
May 24
Julian and Erin
Isolation of endophytes from plant 1
 |
Sophia
Obtained cultures of E. coli with pRL814
Overnight culture of above prepped
May 25
Sarah and Ashley
Miniprep of pRL814
Nanodropped miniprep
May 27
Erin and Julian
Endophyte plating and isolation
 |
May 28
Julian
Brachypodium jars were removed from the cold room, foil was removed from the top and sterilized miracloth was placed on the jar
May 30
Sophia and Sarah
Nanodropped pRL814 miniprep
Julian, Sarah, and Ashley
DNA extraction of Fusarium oxysporum and Trichoderma isolates
PCR 1 acdS of fungal DNA extractions with ITS1F - LR3 to verify quality of extraction
May 31
Julian
First Brachypodium sprouts observed
 |
Julian, Sarah, and Ashley
Gel of PCR 1 acdS
PCR 2 acdS with Fo and Ta acdSF2-R2 primers
 |
 |
Sophia
pRL814 cultures were prepared with DAP + Spec
June 1
Sarah and Sophia
Miniprep of pRL814
Julian, Sarah, and Ashley
Gel of PCR 2 acdS
June 4
Julian
PCR 3 acds of Burkholderia thailandensis isolate
Sophia
Prep of pRL814 cultures
June 5
Sophia and Sarah
Miniprep of pRL814 cultures
Nanodropped minipreps
Julian
ACCD assay try, with B. thailandensis
June 6
Julian
PCR 5 acdS of Fusarium oxysporum with assembly primers Fo_acdSF1-R1
PCR 6 acdS of Ralstonia picketti with assembly primers Rp_acdSF1-R1
Julian and Ashley
ACCD assay spectrophotometry, standard curve and samples
Sophia and Sarah
Prepped pRL814 cultures
June 7
Julian
PCR 7 acdS with F. oxysporum with primer sets Fo_acdSF1-R1 and F2-R2
PCR 8 acdS with F. oxysporum with primer sets ITS1F-LR3, Fo_acdSF1-R1, and F2-R2
 |
Julian and Erin
PCR troubleshooting
Sarah and Sophia
Miniprep of pRL814
Restriction digest of minipreps
June 8
Julian
PCR 9 acdS of F. oxysporum with a range of annealing temperatures
 |
PCR 10 acdS of R. picketti with a range of annealing temps
Sophia
Ran RDs of pRL814 on gel
Cloning of pRL814 in E. coli
June 9
Julian
PCR 11 acdS reamplify tube 6 from PCR 9 acdS
 |
Julian and Erin
PCR 3 endo of soil isolates w/ 27F-1492R
|
Brainstorming of plans of which vectors to use, possible conjugation, and fungal co-inoculation
Sophia
Miniprep and digestion of pRL814
June 10
Sophia
Ran digestion on gel and cut out band
June 11
Julian
PCR 12 acdS, redo of PCR 5 acdS with primer concentration of 500 nM
PCR 13 acdS, F. oxysporum IG37 with Fo_acdSF2-R2
 |
PCR 14 acdS, template from PCR 13 acdS with Fo_acdSF1-R1
 |
Julian and Erin
PCR troubleshooting
Made ExoAP Master Mix
Sophia
Gel extraction of pRL814 fragment
June 12
Julian
PCR 15 acdS, with templates from PCRs 13 and 14, primers are Fo_acdSF1-R1
Gel band excised, ready for extraction. IG37 acdS with overhangs for expression in pRL814
June 13
Sophia
Gel band purification of IG37 acdS
Julian
Solid DF Agar prepared, nitrogen free
Assembly of pRL814 + Fo_acdS with NEBuilder HiFi Assembly Master Mix
PCR 16 acdS with two Trichoderma isolates and primers Ta_acdSF2-R2
 |
June 14
Julian
Planning of ACC deaminase activity assay
ExoAP of PCR 3 endo
Submission of sequencing reactions from PCR 3 endo
June 15
Ashley, Jessica, Jordan, Julian, and Sarah
Tour of Monsanto farm, interview Troy
Sophia
PCR 17A acdS of Trichoderma asperellum 6C-1
Gel extraction of PCR 17A acdS product
June 18
Julian and Erin
Streaking of identified isolates on DFA + ACC or + NH4 to test for ACC deaminase activity
Julian
Prepared more DFA, accidentally added 2X FeSO4
Streaked FP0-02 on the 2X Fe agar + NH4 to test toxicity
June 19
Erin
PCR 4 endo of soil isolates w/ 27F-1492R
PCR 17B acdS of R. picketii w/ Rp_acdSF2-R2
 |
June 20
Julian and Ashley
PCR 5 endo w/ 27F-1492R
Imaged plates for ACCD assay results
 |
June 21
Julian
PCR 18 acdS with P_UW4_acdSF2-R2 of ACC deaminase + isolates
 |
Erin
Prep of overnight cultures of Pseudomonas strains for transformation attempt
June 22
Julian
PCR 5B endo, repeat of PCR 5
PCR 19 acdS of F. oxysporum IG37 w/ Fo_acdSF2-R2
PCR 20 acdS of Pseudomonas isolates w/ P_UW4_acdSF2-R2
 |
PCR 21 acdS of PCR 19 acdS product w/ Fo_acdSF1-Fo_acdS-spl1R, Fo_acdS-spl1F-Fo_acdSR1
 |
Julian and Sophia
Electroporation of Pseudomonas using pRL814, plating on LB + Strep
June 25
Julian
ExoAP cleanup of soil isolates PCRs for sequencing
PCR 6 endo of two as of yet unamplified isolates
Confocal microscopy of putatively transformed GFP endophytes
June 26
Julian
PCR cleanup of Ta_syn_acdS and spliced Fo_gene
Prep of SOC media
Julian, Ashley and Sarah
Assembly w/ NEBuilder HiFi Assembly using Ta_syn_acdS and Fo_acdS spliced
Transformation of Pseudomonas UAP1-01 and UCP2-01 w/ electroporation and plasmid pRL814 + Fo_acdS or Ta_acdS
June 27
Julian
ExoAP of PCRs 3 (one sample), 4, and 5B endo, and 20 acdS
Jordan, Casper, and Sarah
Planted Brachypodium seeds
Sarah
Made competent cells of UAP1-01
Erin and Casper
Plated acdS transformants
June 28
Julian
Submission of previous day’s samples for sequencing
Brachypodium seed sterilization
PCR 22 acdS of PCR 20 product with P_UW4_acdSF1-R1
 |
Gel band excision
Ashley
Plating of seeds on MS/2 plates
Sarah
Assay for chloramphenicol resistance by plating on chlor plates
ACC deaminase assay on DFA
All
Public forum
June 29
Julian
Electroporation of Pseudomonas isolates with RFP
Julian and Sarah
Measured OD of all isolates from overnight cultures
Sophia
Miniprep of MT001
June 30
Julian
Antibiotic resistance plates photographed
 |
All
MSU iGEM meetup
July 2
Julian
ACC deaminase assay modeling
Filled culture tubes with soil and autoclaved
Sophia
Miniprep of BBr1
July 3
Erin
Dilutions of all isolates made at same starting OD for measuring growth
2nd autoclaving of culture tubes with soil inside
Planting Brachypodium seeds in culture tubes
Sophia
Transformation of NEB 5 alpha with High Expression cassette BioBrick
July 5
Jordan, Jessica, and Casper
Sequencing prep of endophytic isolates
July 6
Sophia
Miniprep of high expression cassette Biobrick plasmid (pBB)
July 9
Julian
Analysis of sequence data on endophytes
PCR 23 acdS on Pseudomonas isolates positive for ACC deaminase activity
 |
PCR 24 acdS to splice IG37 exons, template is PCR 21 acdS both lanes
 |
Creation of ABA regulation mechanism figure
Email UM about regulation with dCas9
PCR 25 acdS of Pseudomonas isolates w/ P_UW4_acdSF2-R2
 |
July 10
Julian
PCR 7 endo, to ID soil isolates
 |
PCR 26 acdS of 1C2P-04 w/ P_UW4_acdSF3-R3 and IG37 spliced w/ Fo_acdSF3-R3
 |
July 11
Sophia
Gel extraction of acdS from 1C2P-04 with P_UW4_acdSF3-R3
Julian and Sophia
Assembly of acdS into pBB
Erin
Kirby-Bauer results for isolates
July 12
Julian
PCR 27 acdS w/ Burkholderia stabilis and range of annealing temps, primers Bs_acdSF2-R2
July 13
Julian
PCR 28 acdS for amplifying P_UW4_acdS from putatively transformed colonies
PCR28B, repeat of above using a Fast Extraction of colonies and test w/ 27F-1492R
July 23
Sophia
Miniprep of pBB w/ P_UW4_acdS from 1C2P-04
Julian
ExoAP cleanup of PCR endo 4
Sequencing prep of endo PCR 4 isolates
July 24
Julian
Made Phosphate-buffered Sucrose + MgCl2 for electroporation
Submitted sequencing reactions
July 25
Sophia
Miniprep of pBB w/ P_UW4_acdS from 1C2P-04
His-tag purification of acdS
July 26
Julian
Saw that colonies of FCP2-01 electroporated at 1.8 kV and recovered w/ SOC had many more colonies than other conditions
Screened for presence of plasmid in electroporated and conjugated cells (PCR 32 acdS with P_UW4_acdSF2-R2 and with 27F-1492R)
 |
Sophia
Transformed E. coli with pGFP
July 27
Julian
Prepared overnight cultures of FCP2-01 tentatively transformed with GFP
PCR 33 acdS w/ proB_F-R, from pJK_proB_eGFP fast extraction
 |
PCR 34 acdS w/ Linker_GFP_F-R, from pJK_proB_eGFP fast extraction
PCR 35 acdS w/ P_UW4_acdS_GFP_F-R, from acdS PCR product from 1C2P-04
PCR 36 acdS w/ pSB1C3_F-R, from pBB+acdS miniprep
PCR 37 acdS with spliced IG37 acdS for sequencing (Fo_acdSF2-R2)
Erin and Julian
Inoculation of plant roots with putatively transformed FCP2-01+acdS
July 28
Julian
Repeated PCRs 34 and 35
Submitted additional isolates for sequencing, and the spliced IG37
July 30
Julian
PCR38 acdS of conjugation colonies expression GFP, used 27F-1492R
 |
 |
Restriction digest w/ HindIII or MfeI to determine if the colonies expressing GFP were E. coli or Enterobacter
 |
July 31
Julian
Repeat PCR 34 w/ decreased amount of template (1 ul to 0.1 ul)
Cell washing and resuspension for FCP2-01 transformation, using Peiqi Zhang’s protocol
Electroporation with pBB+acdS, pGFP, and control
August 1
Julian
pGFP miniprep
Gel band purification for acdS eGFP fusion plasmid
In-fusion cloning to assemble plasmid
August 2
Sarah
Interlab dilution plating
Julian
Transformation of FCP2-01
 |
August 3
Julian
First FCP2-01 transformants observed
 |
August 24
Julian
FCP2-01 GFP added to overnight culture
Transformation of FCP2-01 with pGFP-acdS
August 26
Julian
4 colonies of GFP-acdS transformants observed, overnight cultures made
Glycerol stocks of FCP2-01 with GFP(BBa_K608011) were prepared
Brachypodium seeds sterilized and placed in MS/2 plates
August 27
Julian
Poured DFA plates
Streaked wild-type and GFP-acdS FCP2-01 on DF-ACC plates
Transformed FCP2-01 with 60 or 120 minutes post-electroporation incubation
August 28
Julian
PCR 39 acdS to confirm presence of acdS in FCP2-01 in GFP-acdS
Collected Enterobacter transformation results
August 29
Julian
Normalized ODs of GFP and GFP_acds cultures for seed inoculation
Made FCP2-01 competent cells
August 30
Erin and Julian
Brachypodium seed sterilization and vernalization
August 31
Erin and Julian
Soil preparation for endophyte persistence assay
Restriction digest of isolated from inoculated roots, negative for FCP2-01
September 1
Erin and Sophia
Autoclave soil for endophyte persistence assay
FCP2-01 + GFP culture made
FCP2-01 + GFP_acds culture made
September 2
Sophia
Autoclave soil for endophyte persistence assay
September 3
Erin and Sophia
Brachypodium seed inoculation and planting
5 negative control
5 FCP2-01 + GFP
5 FCP2-01 + GFP_acds
September 8
Julian
Added soil to jars for persistence assay
September 10
Erin and Julian
Persistence assay time point 1 using confocal microscopy
Endo PCR 8 with Hsp60F-R of FCP2-01
September 18
Erin and Julian
Persistence assay time point 2 using confocal microscopy
September 21
Julian
Gibson of pDB1C3-proB-acdS-link-GFP-term (BBa_K2633000)
Transformation into E. coli
September 24
Julian
PCR acdS 41 of transformants to confirm transformation
September 25
Erin and Julian
Persistence assay time point 3 using confocal microscopy
September 26
Julian
Miniprep of BBa_K2633000
September 27
Julian
Nanodrop of BBa_K2633000 minipreps
October 2
Erin and Julian
Persistence assay time point 4 using confocal microscopy
October 9
Erin and Julian
Persistence assay time point 5 using confocal microscopy
October 11
Julian
PCR 42 acdS of BioBrick BBa_K2633000
October 14
Julian
PCR 43 acdS of BioBrick BBa_K2633000 for sequencing
