iGEM 2018 — Team MichiganState

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# Team:MichiganState/Notebook

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* [Project](https://2018.igem.org/Team%3AMichiganState)
  + [Project Home](https://2018.igem.org/Team%3AMichiganState)
  + [Overview](https://2018.igem.org/Team%3AMichiganState/Description)
  + [Design](https://2018.igem.org/Team%3AMichiganState/Design)
  + [Experiments](https://2018.igem.org/Team%3AMichiganState/Experiments)
  + [Results](https://2018.igem.org/Team%3AMichiganState/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AMichiganState/Demonstrate)
  + [Safety](https://2018.igem.org/Team%3AMichiganState/Safety)+ [Improve](https://2018.igem.org/Team%3AMichiganState/Improve)
    + [References](https://2018.igem.org/Team%3AMichiganState/References)
* [Modeling](https://2018.igem.org/Team%3AMichiganState/Model)
* [Parts](https://2018.igem.org/Team%3AMichiganState/Parts)
  + [Overview](https://2018.igem.org/Team%3AMichiganState/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3AMichiganState/Basic_Part)
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* [Human Practices](https://2018.igem.org/Team%3AMichiganState/Human_Practices)
  + [Overview](https://2018.igem.org/Team%3AMichiganState/Human_Practices)
  + [Site Visit](https://2018.igem.org/Team%3AMichiganState/HP_Site_Visit)
  + [Expert Interviews](https://2018.igem.org/Team%3AMichiganState/HP_Expert_Interviews)
  + [Survey](https://2018.igem.org/Team%3AMichiganState/HP_Survey)
  + [Public Engagement](https://2018.igem.org/Team%3AMichiganState/Public_Engagement)
  + [Education](https://2018.igem.org/Team%3AMichiganState/Education)
* [Team](https://2018.igem.org/Team%3AMichiganState/Team)
  + [Team](https://2018.igem.org/Team%3AMichiganState/Team)
  + [Collaborations](https://2018.igem.org/Team%3AMichiganState/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3AMichiganState/Attributions)
* [Notebook](https://2018.igem.org/Team%3AMichiganState/Notebook)
  + [Notebook](https://2018.igem.org/Team%3AMichiganState/Notebook)
  + [InterLab Study](https://2018.igem.org/Team%3AMichiganState/InterLab)
* [Judging Form](https://igem.org/2018_Judging_Form?id=2633)

# Notebook

[May Entries](#May)

[June Entries](#June)

[July Entries](#July)

[August Entries](#August)

[September Entries](#September)

[October Entries](#October)

#### 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
 ![Cut roots in saline](https://static.igem.org/mediawiki/2018/4/47/T--MichiganState--Roots_in_saline.jpg) |

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
 ![Endophyte colonies](https://static.igem.org/mediawiki/2018/2/2b/T--MichiganState--Endophyte_colonies_notebook.jpg) |

#### 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
 ![First sprouts!](https://static.igem.org/mediawiki/2018/4/42/T--MichiganState--First_sprout_notebook.jpg) |

Julian, Sarah, and Ashley

- Gel of PCR 1 acdS
- PCR 2 acdS with Fo and Ta acdSF2-R2 primers
 ![acds PCR 1](https://static.igem.org/mediawiki/2018/8/86/T--MichiganState--acdS_PCR1_notebook.png) | ![acds PCR 2](https://static.igem.org/mediawiki/2018/3/32/T--MichiganState--acdS_PCR2_notebook.png) |

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
 ![acds PCR 8](https://static.igem.org/mediawiki/2018/2/22/T--MichiganState--acdS_PCR8_notebook.png) |

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
 ![acds PCR 9](https://static.igem.org/mediawiki/2018/0/05/T--MichiganState--acdS_PCR9_notebook.png) |- 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
 ![acds PCR 11](https://static.igem.org/mediawiki/2018/f/f0/T--MichiganState--endo_PCR3_acdS_PCR11_notebook.png) |

Julian and Erin

- PCR 3 endo of soil isolates w/ 27F-1492R
 ![endo PCR 3](https://static.igem.org/mediawiki/2018/f/f0/T--MichiganState--endo_PCR3_acdS_PCR11_notebook.png) |- 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
 ![acds PCR 13](https://static.igem.org/mediawiki/2018/c/cd/T--MichiganState--acdS_PCR13_notebook.png) |- PCR 14 acdS, template from PCR 13 acdS with Fo\_acdSF1-R1
 ![acds PCR 14](https://static.igem.org/mediawiki/2018/4/4a/T--MichiganState--acdS_PCR14_notebook.png) |

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
 ![acds PCR 16](https://static.igem.org/mediawiki/2018/3/3b/T--MichiganState--acdS_PCR16_notebook.png) |

#### 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
 ![endo PCR 4 and acds PCR 17](https://static.igem.org/mediawiki/2018/2/2f/T--MichiganState--endo_PCR4_acdS_PCR17_notebook.png) |

#### June 20

Julian and Ashley

- PCR 5 endo w/ 27F-1492R
- Imaged plates for ACCD assay results
 ![ACCD assay plates](https://static.igem.org/mediawiki/2018/7/74/T--MichiganState--ACCD_asssay_plates_notebook.jpg.jpg) |

#### June 21

Julian

- PCR 18 acdS with P\_UW4\_acdSF2-R2 of ACC deaminase + isolates
 ![acds PCR 18](https://static.igem.org/mediawiki/2018/5/53/T--MichiganState--acdS_PCR18_notebook.png) |

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
 ![acds PCR 20](https://static.igem.org/mediawiki/2018/5/5f/T--MichiganState--acdS_PCR20_notebook.png) |- PCR 21 acdS of PCR 19 acdS product w/ Fo\_acdSF1-Fo\_acdS-spl1R, Fo\_acdS-spl1F-Fo\_acdSR1
 ![acds PCR 21 and 22](https://static.igem.org/mediawiki/2018/2/29/T--MichiganState--acdS_PCR21_and_22_notebook.png) |

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
 ![acdS PCR 22](https://static.igem.org/mediawiki/2018/2/29/T--MichiganState--acdS_PCR22_notebook.png) |- 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
 ![Antibiotic resistance test plates](https://static.igem.org/mediawiki/2018/4/43/T--MichiganState--AB_resistance_notebook.jpg) |

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
 ![acds PCR 23](https://static.igem.org/mediawiki/2018/a/a6/T--MichiganState--acdS_PCR23_notebook.png) |- PCR 24 acdS to splice IG37 exons, template is PCR 21 acdS both lanes
 ![acds PCR 24](https://static.igem.org/mediawiki/2018/b/b2/T--MichiganState--acdS_PCR24_notebook.png) |- Creation of ABA regulation mechanism figure
- Email UM about regulation with dCas9
- PCR 25 acdS of Pseudomonas isolates w/ P\_UW4\_acdSF2-R2
 ![acds PCR 25](https://static.igem.org/mediawiki/2018/d/d8/T--MichiganState--acdS_PCR25_notebook.png) |

#### July 10

Julian

- PCR 7 endo, to ID soil isolates
 ![endo PCR 7](https://static.igem.org/mediawiki/2018/4/43/T--MichiganState--endo_PCR7_notebook.png) |- PCR 26 acdS of 1C2P-04 w/ P\_UW4\_acdSF3-R3 and IG37 spliced w/ Fo\_acdSF3-R3
 ![acds PCR 26](https://static.igem.org/mediawiki/2018/d/d3/T--MichiganState--acdS_PCR26_notebook.png) |

#### 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)
 ![acds PCR 32](https://static.igem.org/mediawiki/2018/d/df/T--MichiganState--acdS_PCR32_notebook.png) |

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
 ![acds PCR 33](https://static.igem.org/mediawiki/2018/3/35/T--MichiganState--acdS_PCR33_notebook.png) |- 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
 ![Conjugation streak plate](https://static.igem.org/mediawiki/2018/thumb/e/e6/T--MichiganState--Conjuation_colonies_notebook.jpg/900px-T--MichiganState--Conjuation_colonies_notebook.jpg) | ![acds PCR 38](https://static.igem.org/mediawiki/2018/e/e5/T--MichiganState--acdS_PCR38_notebook.png) |- Restriction digest w/ HindIII or MfeI to determine if the colonies expressing GFP were E. coli or Enterobacter
 ![Digest of acds PCR 38](https://static.igem.org/mediawiki/2018/3/3a/T--MichiganState--acdS_DigestPCR38_notebook.png) |

#### 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
 ![Interlab dilution plates](https://static.igem.org/mediawiki/2018/2/27/T--MichiganState--Interlab_notebook.jpg) |

#### August 3

Julian

- First FCP2-01 transformants observed
 ![First transformants!](https://static.igem.org/mediawiki/2018/thumb/1/1a/T--MichiganState--First_transformants_notebook.jpg/861px-T--MichiganState--First_transformants_notebook.jpg) |

#### 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

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