Team:British Columbia/Notebook

British_Columbia_Base

Autoinduction Notebook

The bacterial strains used were Agrobacterium tumefaciens GV3101 and Escherichia coli DH5-α unless otherwise stated. All E. coli cultures were grown in LB media at 37℃ unless otherwise stated. All A. Tumefaciens were grown in LB at 30℃ unless otherwise stated. All plasmid DNA extractions were performed with ABM Column-Pure Plasmid Mini-Prep Kit. DNA purification from gels with ABM Column-Pure DNA Gel Recovery Kit. All gels were run with 0.8 TAE unless otherwise stated. The same protocols for DNA gel electrophoresis, restriction enzyme digest, DNA ligation, and chemical and electro transformation were used throughout unless otherwise stated and can be found under Protocols.


September 9, 2017

  • Performed the following transformations
    • traAC + pK1357010
    • traR + pK1357010
    • virB1 + pK1357010
    • traAC + pJ04650
    • traR + pJ04650
    • virB1 + pJ04650
    • traAC + pSB1C3
    • traR + pSB1C3
    • virB1 + pSB1C3
    • Positive transformation control: pK13 miniprep
    • Negative transformation control: dH2O
    into DH5α competent cells
  • plated on chloramphenicol (Cam) plates

September 10, 2017

  • picked and inoculated two clones containing traAC, traR and virB1 in pSB1C3

September 11, 2017

  • miniprepped grown cultures containing traAC, traR and virB1 in pSB1C3
  • made glycerol stocks of grown cultures containing traAC, traR and virB1 in pSB1C3
  • picked and inoculated two clones containing
    • traAC + pK1357010
    • traR + pK1357010
    • virB1 + pK1357010
    • traAC + pJ04650
    • traR + pJ04650
    • virB1 + pJ04650

September 12, 2017

  • miniprepped grown cultures containing
    • traAC + pK1357010
    • traR + pK1357010
    • virB1 + pK1357010
    • traAC + pJ04650
    • traR + pJ04650
    • virB1 + pJ04650

September 15, 2017

  • diagnostic digest of all minipreps with EcoR1 and Pst1
  • ran gel of diagnostic digests (Figure 1)

September 16, 2017

    Redoing the digestion and ligation

  • Digestions
    • traAC, traR and virB1 fragements with EcoR1 and Spe1
    • K1357010 with EcoR1 and Xba1
  • ligated the following combinations
    • traAC in K1357010
    • traR in K1357010
    • virB1 in K1357010
  • transformed into into DH5α competent cells with the controls
    • positive control: 2μL pJ04650 plasmid
    • negative control: competent cells

September 17, 2017

  • performed a digestion identical to the one on september 15th
  • picked and inoculated two biological replicates containing
    • traAC + pK1357010
    • traR + pK1357010
    • virB1 + pK1357010
    • traAC + pJ04650
    • traR + pJ04650
    • virB1 + pJ04650

September 18, 2017

  • miniprepped grown cultures containing
    • traAC + pK1357010
    • traR + pK1357010
    • virB1 + pK1357010
    • traAC + pJ04650
    • traR + pJ04650
    • virB1 + pJ04650

September 20, 2017

  • Digestion and gel electrophoresis (Figure 2) of constructs
    • September 9th cloning

    • traAC in pK1357010 replicate A
    • traR in pK1357010 replicate A
    • virB1 in pK1357010 replicate A
    • traAC in pK1357010 replicate B
    • traR in pK1357010 replicate B
    • virB1 in pK1357010 replicate B
    • September 16th cloning

    • traAC in pK1357010 replicate A
    • traR in pK1357010 replicate A
    • virB1 in pK1357010 replicate A
    • traAC in pK1357010 replicate B
    • traR in pK1357010 replicate B
    • virB1 in pK1357010 replicate B
    • pSB1C3 constructs

    • traAC in pSB1C3 replicate A
    • traR in pSB1C3 replicate A
    • virB1 in pSB1C3 replicate A
    • traAC in pK1357010 replicate B
    • traR in pSB1C3 replicate B
    • virB1 in pSB1C3 replicate B

    succesful digestions are

    • traAC in pK1357010 replicate B OR
    • traR in pK1357010 replicate A from September 16th
    • traAC in pSB1C3 replicate A
    • virB1 in pSB1C3 replicate A
    • traR in pSB1C3 replicate B

September 24, 2017

  • Innoculate colonies of potentially succesful clones:
    • traAC in pK1357010 replicate B
    • traR in pK1357010 replicate A from September 16th

September 25, 2017

  • miniprepped samples from September 24th

September 26, 2017

  • digested the following plasmids with Eco1 and Pst1
    • traAC in pK1357010 replicate B (traR A)
    • traR in pK1357010 replicate A
    • pCambia Multi-clonal site (MCS)
  • ran gel electrophoresis of the digests (Figure 3)
  • gel purified the digested fragments of traR A and pCambia MCS

September 28, 2017

  • ligation of traR A fragment with pCambia MCS (controlled with ligation into pCambia MCS with one cut)
  • transformed ligations into E. coli S17-1 competent cells
  • plated transformants onto Kanomycin (Kan) plates

September 29, 2017

  • innoculated transformants from plates into liquid LB and Kan

September 30, 2017

  • innoculated cultures looked clear - 3 more colonies innoculated from September 28th plate
  • innoculated the following 5 cultures
    • pSB1C3-TraR construct from the 11 of September 2017
    • pSB1C3-TraAC construct from the 11 of September 2017
    • pSB1C3-virB1 construct from the 11 of September 2017
    • K135010 from the 30th of August 2017
    • JO4650 from the 30th of August 2017

October 1, 2017

  • innoculated cultures did not grow again
  • miniprepped the overnight cultures from September 30th

October 3, 2017

  • Performed the following digestions
    • pSB1C3-ptraAC with Spe1 and Pst1
    • pSB1C3-ptraR with Spe1 and Pst1
    • pSB1C3-virB1 with Spe1 and Pst1
    • pJ04650 with Xba1 and Pst1
    • pK1357010 with Spe1 and Pst1
    • CONTROL: traAC with Pst1

October 6, 2017

  • ran gel of October 3rd digests (Figure 4)
  • the following bands were excised and gel purified
    • traAC = 2.3kb
    • traR = 2.3kb
    • virB1= 2.6kb
    • traAC control = 2.3kb
    • J04650 = 869kb
    • K1357010 = 857kb

October 9, 2017

  • Ligated the following digests together
    • pTraAC fragment + J04650 fragment
    • pTraAC fragment + K1357010 fragment
    • pTraR fragment + J04650 fragment
    • pTraR fragment + K1357010 fragment
    • pvirB1 fragment + J04650 fragment
    • pvirB1 fragment + K1357010 fragment
    • pTraAC fragment only (control)
    • pTraR fragment only (control)
    • pvirB1 fragment only (control)
    • pTraAc single digest (control)

October 10, 2017

  • Transformed the ligations from October 9th into E. Coli S17-1 competent cells
  • Plated all transformations on Kan plates

October 11, 2017

  • picked and inoculated 2 transformants from each plate

October 12, 2017

  • Miniprepped October 11th colonies

October 13, 2017

  • Digested all minipreps from October 12th with Pst1 and EcoR1

October 17, 2017

  • ran all digests from October 13th on a gel (Figure 5)

October 21-27, 2017

  • re-cloned traR and virB1 from synthesized fragments into reporter-containing backbones and subsequently into pCAMBIA

October 28, 2017

  • transformated pCAMBIA-MCS and promoter fragments into Agrobacterium
    • traR with pK1357010
    • traR with pJ04650
    • virB1 with pK1357010
    • virB1 with pJ04650
  • innoculated colonies of transformants in LB + Kan

October 29, 2017

  • Performed the acetosyringone assay on transformants in technical triplicate
    • treatments =no treatment, 50μL acetosyringone, 100μL acetosyringone for each succesful transformant
    • timepoints = 0,1,2,3,4,5,6 hrs
    • no fluorescence observed

CRISPR-Cas9 Notebook

The bacterial strains used were Agrobacterium tumefaciens GV3101 and Escherichia coli DH5-α unless otherwise stated. All E. coli cultures were grown in LB media at 37℃ unless otherwise stated. All A. Tumefaciens were grown in LB at 30℃ unless otherwise stated. All plasmid DNA extractions were performed with ABM Column-Pure Plasmid Mini-Prep Kit. DNA purification from gels with ABM Column-Pure DNA Gel Recovery Kit. All gels were run with 0.8 TAE unless otherwise stated. The same protocols for DNA gel electrophoresis, restriction enzyme digest, DNA ligation, and chemical and electro transformation were used throughout unless otherwise stated and can be found under Protocols.


July 14, 2017

  • Inoculate Agrobacterium in LB+Kanamycin, Grow overnight at 30°C
  • Inoculate DH5α with following plasmids/antibiotics and Grow overnight at 37°C:

  • Cas9/Chloramphenicol
  • pCambia/Kanamycin

July 15, 2017

  • Plasmid extraction of inoculated bacteria(from July14,2017) according to miniprep protocol

July 26, 2017

  • Diagnostic digest of pCambia and pCas9-CR4 with SacII & BamHI:
    Incubate for 1 hr at 37°C
    Incubate for 20min at 30°C
  • Run digested plasmids on a 1% agarose gell at 110V for 20 min.(Figure 1):
    20ul of each digest
    20ul of 1Kb ladder

July 27, 2017

  • Diagnostic digest #2 for pCambia and pCas9-CR4 with SacII & BamHI:
    Incubate for 2 hr at 37°C
    Digested 625ng of pCambia in 10ul reaction
    Digested 280ng of pCas9 in 20ul reaction
  • Digestion of pCambia for future cloning with SacII & Sph1:
    Digested 1400ng of pCambia in a 10ul reactions

August 9, 2017

  • Digest pCas9 with Spe1 & SacII:
    Incubate for 1 hr at 37°C
    Digested 1ug total of pCas9
  • Run digested plasmids from July 27 and Aug 9 on a 1% agarose gell at 100V (Figure 2)
  • Gel extraction was unsuccesful

August 11, 2017

  • Digest pCambia & pCas9:
    pCambia with Sph1 & SacII
    pCas9-Cr4 with Spe1 and SacII
    50ul reactions for both
    Incubate at 37°C for 1 hours
  • Run digested plasmids a 1% agarose gell at 101V (Figure 3)
  • Gel extraction of both top and bottom band of pCambia

August 17, 2017

  • Made agrobacterium competent cells as specified in protocols

August 22, 2017

  • Blunting of pCas9 from SpeI and SacII digestion
  • Blunting of pCambia from SphI and SacII digestion
  • Ligated pCas9+pCambia fragments, left reaction overnight

August 23, 2017

  • Transform E coli competent cells with 3ul of ligation
  • Grow cells in Kanamycin+Chloramphenicol plates

August 31, 2017

    Competent Cell test:
  • Transformation efficiency of our competent E. coli cells tested as in Competent Cell test kit protocol*

  • *This procedure was done due to some unsatisfying transformations in our previous experiments

September 1, 2017

  • Results from competent test showed no growth of our competent cells in selective plates

September 8, 2017

  • Inoculate E.coli with pCas9 in 5ml of LB with Chloramphenicol

September 9, 2017

  • Miniprep pCas9:
    Concentration = 62ng/ul

  • Cloning of pCas9 to pCambia+MCS(Multiple Cloning Site):
  • Digest pCambia+MCS with SpeI & SacII
  • Digest pCambia+MCS with SacII(Positive Control)
  • Digest Cas9 with SpeI & SacII
  • All digestions done for 2 hrs at 37°C

September 10, 2017

  • Run gel for digested samples from Sepetmber 9th
  • Gel extraction of 6Kb band from pCambia+MCS(SpeI+SacII) and pCambia+MCS(SacII Only).
  • Gel extraction of 6Kb band from pCas9(SpeI+SacII) sample
  • Set up ligation for pCambia+Cas9

September 12, 2017

    Transfrom DH5α:
  • pCambia+Cas9 on Kanmycin+Chloramphenicol
  • pCambia on Kanamycin
  • pCambia+Cas9 on plain LB
  • no Plasmid on Kanamycin+Chloramphenicol

September 13, 2017

  • Plates from Sepember 12th transformation showed pCambia+Cas9 on Kanmycin+Chloramphenicol plate was only one with colonies
  • Grow a 5ml culture from two colonies of the pCambia+Cas9 plate

September 14, 2017

  • Validate ligation of pCambia+Cas9
  • Miniprep cultures inoculated on September 13th
  • Digest pCambia+Cas9 with EcoRI and SphI for validation

September 15, 2017

    Transformation
  • E.coli S17, pCambia+Cas9 ligation from September 10
  • E.coli S17, no plasmid
  • Plate on Kanamycin+Chloramphenicol

September 16, 2017

    Run gel with pCambia+Cas9 digested samples from September 14th(Figure 4)
  • Ran both undigested and digested samples
  • Saw expected bands and will further validate ligated plasmid by sequencing



  • S17 pCambia+Cas9 grew on plate, and the following cultures were grown:
  • S17 LB + Kan + CM
  • S17 LB + Kan
  • S17 LB + CM
  • S17 LB + Gen
  • Agrobacterium LB + Gent

  • S17 were grown at 37°C, Agro was grown at 30°C

September 17, 2017

    Mating of S17(pCambia+Cas9) with Agrobacterium
  • Mix 300 ul of each S17 and Agrobacterium
  • Spin them down at 13,000 rpm for 2 min
  • Remove 300ul
  • Repeat procedure with E.coli and Agrobacterium, as negative controls
  • Incubate 4-5 hours at 30°C
  • Grow mated cells in plates with Rif+Kan and No antibiotic

September 21, 2017

  • 3 colonies grew in the selective plates with S17+Agro mating
  • Inoculated 8mL LB+Rif+Kan and grow overnight at 30°C

September 23, 2017

  • Miniprep Agrobacterium inoculated on LB+Rif+Kan and digest with XbaI & SpeI
  • Inoculated Agro in CM + Rif

September 29, 2017

  • Inoculated 3 X 5 mL cultures of Agro with pCambiaCas9 in LB+Rif+Kan

October 1, 2017

  • Run CRISPR toxicity assay as in protocol found here

October 4, 2017

  • CRISPR toxicity assay re run as in modified protocol found here
    Included no pCambia+Cas9 agro control this time

October 5, 2017

  • Digestion of pCambia and guide RNAs
  • Digest 1ug of pCambia with PstI and SpeI
  • Digest 100ng of guides A,B,C and N with PstI and SpeI
  • Digest both for 2hrs
  • Heat inactivate 80°C
  • Run digested samples on agarose gel and perform Gel extraction

October 6, 2017

  • Ligation of pCambia and guide RNAs:

  • 10ul of sgRNA
    2ul of pCambia+Cas9
    2ul of Buffer
    1ul of T4 ligase
    5ul of water

October 10, 2017

    Tranfromation of ligated gRNAs into E.coli S17:
  • sgRNA "A" + pCambia
  • sgRNA "B" + pCambia
  • sgRNA "C" + pCambia
  • sgRNA "N" + pCambia
  • H2O, no plasmid
  • Grow in LB+Kan+CM plates
    Transform with 1ul of ligated DNA

October 12, 2017

  • No growth on tranfromations from October 10
  • Repeat tranformations using DH5α competent cells
  • Used 2ul of DNA for transformation instead of 1ul

October 14, 2017

    Digest guide RNAs with respective enzymes:
  • Digest guide A with SpeI and HindIII
  • Digest guide B with SpeI and HindIII
  • Digest guide B with PstI and HindIII
  • Digest guide C with PstI and HindIII
  • Ligate guide RNAs A+B, B+C and A+C

October 15, 2017

  • Digest pCambiaCas9 with SpeI and PstI
  • Digest guideA+B ligation with SpeI and PstI
  • Digest guideA+C ligation with SpeI and PstI
  • Digest guideB+C ligation with SpeI and PstI
  • Gel purify pCambiaCas9 digestion
  • Ligate each combination of guides with pCambiaCas9, incubate at room termperature overnight

October 15, 2017

    Transformations, sgRNA in E.coli Bl121:
  • pCambia+Cas9 + sgRNA A
  • pCambia+Cas9 + sgRNA B
  • pCambia+Cas9 + sgRNA C
  • pCambia+Cas9 + sgRNA N
  • pCambia+Cas9 + sgRNA A+B
  • pCambia+Cas9 + sgRNA A+C
  • pCambia+Cas9 + sgRNA B+C
  • pCambiaCr4 no guides
  • no plasmid
  • Grown on Lb+Kan plates

October 19, 2017

  • Inoculate 10 ml LB+Kan with 2 colonies from each October 15th transformation

October 20, 2017

  • Miniprep 2 colonies from each gRNA transformation

October 27, 2017

  • Digest pCambia+Cas9 plasmids with ligated gRNAs
  • Used SakII and PstI for digestion
  • Run digest on agarose gel (Figure 5)
  • Grew overnight culture for pCambia+Cas9 ligations with: B1, BC-1, Neg1, and Neg2

October 29, 2017

  • Screen of potential guide RNA's on agrobacterium. Procotol found here

Conjugation Notebook

The bacterial strains used were Agrobacterium tumefaciens GV3101 and Escherichia coli DH5-α unless otherwise stated. All E. coli cultures were grown in LB media at 37℃ unless otherwise stated. All A. Tumefaciens were grown in LB at 30℃ unless otherwise stated. All plasmid DNA extractions were performed with ABM Column-Pure Plasmid Mini-Prep Kit. DNA purification from gels with ABM Column-Pure DNA Gel Recovery Kit. All gels were run with 0.8 TAE unless otherwise stated. The same protocols for DNA gel electrophoresis, restriction enzyme digest, DNA ligation, and chemical and electro transformation were used throughout unless otherwise stated and can be found under Protocols.


July 25, 2017

  • Making Primer Freezer Stock
  • TraB_F added 263 ul to make 100 uM
  • TraB_R added 291 ul to make 100 uM
  • TrbB_F added 309 ul to make 100 uM
  • TrbB_R added 240 ul to make 100 uM
  • TraG_F added 361 ul to make 100 uM
  • TraG_R added 309 ul to make 100 uM
  • TraL_F added 232 ul to make 100 uM
  • TraL_R added 216 ul to make 100 uM
  • Add 90 ul of nuclease free water to make stocks of 10 uM for each primer

July 27, 2017

  • Run PCR reaction with primers on RP1 from stock made on July 25, 2017
  • PIC

August 16, 2017

  • Inoculating RP1 in E. coli with kanamycin, tetracycline, and ampicillin
  • Inoculated 5 ml tubes:
  • E. coli on LB
  • E. coli + RP1 on LB
  • E. coli on LB w/ tetra
  • E. coli on LB w/ amp
  • E. coli on LB w/ kan
  • E. coli on LB w/ tetra + amp + kan
  • E. coli + RP1 w/ on LB tetra
  • E. coli + RP1 w/ on LB amp
  • E. coli + RP1 w/ on LB kan
  • E. coli + RP1 w/ on LB tetra + amp + kan

August 17, 2017

  • All 5 ml tubes from August 16, 2017 miniprepped
  • 71 ng/ml of plasmid DNA RP1

August 30, 2017

  • Start day 1 of conjugation protocol of E. coli w/ Agrobacterium
  • Grow donor and recipient cultures
  • Inoculate 3ml of LB + Kanamycin with RP1 in E. coli
  • Inoculate 3ml of LB + Rifampicin with Agrobacterium
  • Incubate E. coli overnight at 37C
  • Incubate A. tumefaciens overnight at 30C

August 31, 2017

  • Day 2 of conjugation protocol
  • Set up mating reaction mixtures (one for each time point):
  • Mating reactions:
  • 500ul donor : 100ul recipient
  • 300ul donor : 300ul recipient
  • 100ul donor : 500ul recipient
  • Control reactions:
  • 200ul donor
  • 200ul recipient

September 1, 2017

  • Day 3 of conjugation protocol
  • Prepare plates for conjugation assay
  • Incubate at 30C for the following lengths of time:
  • 6 Hours
  • Overnight (~16 hours)
  • Remove mixtures from 6 hour time point and plate on:
  • Mating LB + Kan + Rif plates
  • Donor on LB + Kan + Rif plates
  • Recipient on LB + Kan + Rif plates
  • Donor on LB
  • Recipient on LB

September 2, 2017

  • Day 4 of conjuation protocol
  • Remove mixtures from overnight hour time point and plate on:
  • Mating LB + Kan + Rif plates
  • Donor on LB + Kan + Rif plates
  • Recipient on LB + Kan + Rif plates
  • Donor on LB
  • Recipient on LB
  • Results from 6 hour time point showed growth where expected and no growth on all negative control
  • Plated equal volumes of E. coli, E. coli + RP1, Agrobacterium, Agrobacterium + RP1 on CM-LB

September 3, 2017

  • Day 5 of conjugation protocol
  • Results from overnight time point showed growth where expected and no growth on all negative control
  • Results from plating on CM-LB showed no growth on all CM-LB tubes and growth without

September 19, 2017

  • Antibiotic phenotypes of Agrobacterium strains GV3101, GV3101 + RP1, LBA4404 inoculated in tubes of:
  • LB
  • LB + KAN (75 ug/ml)
  • LB + GENT (100 ug/ml)
  • LB + STREP (200 ug/ml)

September 20, 2017

  • Results show that antibiotics worked in all the right places except for streptomycin on GV3101 and GV3101 + RP1
  • Made plates:
  • 20 strep (200 ug/ml) plates
  • 20 kan (75 ug/ml) plates
  • 20 gent (100 ug/ml) plates

September 21, 2017

  • Strep gradient test on GV3101 and LBA4404:
  • 2 ul streptomycin (200 ug/ml) in 2ml
  • 4 ul streptomycin (200 ug/ml) in 2ml
  • 8 ul streptomycin (200 ug/ml) in 2ml
  • 12 ul streptomycin (200 ug/ml) in 2ml
  • 20 ul streptomycin (200 ug/ml) in 2ml
  • Streaked GV3101 + RP1 on Kan and LB
  • Made 5 tubes of 2ml LB and strep

September 22, 2017

  • No growth above 400 ug/ml of strep concentration for GV September 24, 2017
  • Day 1 of conjugation assay for agrobacterium and agrobacterium
  • Inoculation of GV3101 + RP1 and LBA

September 25, 2017

  • OD’s taken from cultures from

September 24

  • Mating incubated at 11:30
  • Plated matings onto plates

October 14, 2017

  • Conjugation Assay 2 started (same protocol as conjugation assay 1)
  • Strep gradient test on GV3101 + RP1
  • Inoculated LBA4404 and GV3101 + RP1
  • Made plates for assay

October 22, 2017

  • Conjugation Assay 3 started

October 26, 2017

  • Conjugation assay 3 results: Growth on negative donor plates Counted colonies on those plates

October 26, 2017

  • Conjugation assay 4 started October 27, 2017
  • Replicate plating of GV3101 from “dead” plate

October 30, 2017

  • Conjugation assay 4 results: Growth on negative donor plates
  • Replicate plating results: GV3101 grew in kan, but not in strep

October 31, 2017

  • Conjugation assay 5 started November 1
  • OD’s taken from replicate plating tubes

Plasmid Maintenance Notebook

The bacterial strains used were Agrobacterium tumefaciens GV3101 and Escherichia coli DH5-α unless otherwise stated. All E. coli cultures were grown in LB media at 37℃ unless otherwise stated. All A. Tumefaciens were grown in LB at 30℃ unless otherwise stated. In the notes below, "Kan" refers to Kanamycin (50 mg/ml) and "Cm" refers to Cloramphenicol (25 mg/ml). All plasmid DNA extractions were performed with ABM Column-Pure Plasmid Mini-Prep Kit. DNA purification from gels with ABM Column-Pure DNA Gel Recovery Kit. All gels were run with 0.8 TAE unless otherwise stated. The same protocols for DNA gel electrophoresis, restriction enzyme digest, DNA ligation, and chemical and electro transformation were used throughout unless otherwise stated and can be found under Protocols.


August 22, 2017

  • Digestion and ligation of ietA and pSB1C3 with EcoRI and SpeI.
  • Transformation of construct into E. coli.
  • Cm selective plating of transformed E. coli.

August 28, 2017

  • Miniprep of E. coli + pSB1C3.
  • Ran digest of pSB1C3 on gel and purified. (Figure 1)
  • Transformed pCAMBIA into Agrobacterium tumefaciens competent cells.
  • Repeat of ligation of ietA and pSB1C3.

Figure 1 (left to right): 1 kb ladder, duplicate pSB1C3 ietA.


August 29, 2017

  • Repeat of transformation of pSB1C3 + ietA into E. coli.

August 30, 2017

  • Two ietA colonies observed from August 22 transformation.
  • Agrobacterium tumefaciens competent cells appeared contaminated.
  • Digestion of pCambia with EcoRI and XbaI.
  • Digestion of ietA with EcoRI and SpeI.
  • Digestion of MCS with SphI and SacII.
  • Ran pCAMBIA digest on gel. (Figure 2).
  • Inoculated ietA + pSB1C3 in LB-Cm.

Figure 2 (left to right): 1 kb ladder, pSB1C3 ietA, duplicate pCAMBIA MCS.


August 31, 2017

  • Minprep of pSB1C3 + ietA.
  • Made glycerol stock of pSB1C3 + ietA.
  • Plated pSB1C3 + ietA.
  • Sequenced pSB1C3 + ietA.
  • Digested pSB1C3 + ietA with EcoRI and SpeI.
  • Ligation of pCAMBIA + MCS.
  • Ligation of ietS + pSB1C3.

August 31, 2017

  • Transformation of pCAMBIA + MCS & pSB1C3 + ietS into E. coli.
  • Ran gel of pSB1C3 (Figure 3).

Figure 3 (left to right): 1 kb ladder, duplicate pSB1C3 ietA.


September 1, 2017

  • One colony observed from ietS + pSB1C3 tranformation, inoculated in LB-Cm.
  • No colonies from MCS + pCAMBIA transformation.

September 4, 2017

  • Digestion and gel purification of pSB1C3 (Figure 4).
  • Ligation of pSB1C3 and ietS.
  • Sequenced ietA + pSB1C3.
  • Transformation of pSB1C3 + ietS, and pCAMBIA + MCS.

Figure 4 (left to right): 1 kb ladder, duplicate pSB1C3 ietS.


September 7, 2017

  • Inoculated two colonies from pCAMBIA + MCS & pSB1C3 + ietS in LB + antibiotics for miniprep.

September 7, 2017

  • Miniprep of colonies from September 7.
  • Sequence confirmation of ietA.

September 8, 2017

  • MCS ran on 2% TBE gel.
  • ietS ran on gel.
  • Gel purification of ietA and ietS completed.

September 11, 2017

  • Ligation of pSB1C3 + ietA with ietS.
  • Sequenced ietS.

September 12, 2017

  • Transformation of ietAS + pSB1C3 into E. coli.

September 13, 2017

  • Miniprep of ietAS.
  • Glycerol stock made for pSB1C3 + ietAS.
  • Digested ietAS + pSB1C3 minprep and pCAMBIA + MCS with EcoRI and PstI.
  • Ran digests on gel (Figure 5).

Figure 5 (left to right): 1 kb ladder, pCAMBIA MCS, duplicate pSB1C3 ietAS.


September 20, 2017

  • Repeated ietAS + pSB1C3 miniprep and digestion.
  • Gel purification of digest. (Figure 6).
  • Ligation of ietAS into pCAMBIA.

Figure 6 (left to right): 1 kb ladder, duplicate pSB1C3 ietAS.


September 26, 2017

  • Repeat of digestion of ietAS and pCAMBIA.
  • Gel purification of ietAS + pCAMBIA. (Figure 7).
  • Ligated purified ietAS + pCAMBIA.

Figure 7 (left to right): 1 kb ladder, duplicate pSB1C3 ietAS, duplicate pCAMBIA MCS.


September 27, 2017

  • Transformation of ietAS + pCAMBIA into S17 E. coli (no colonies seen).

October 4, 2017

  • Repeat of transformation of ietAS + pCAMBIA into S17 E. coli.

October 10, 2017

  • Miniprep of ietAS + pSB1C3.
  • Ran gel of ietAS.
  • Ligated ietAS into pCAMBIA.
  • Transformed ietAS + pCAMBIA into E. coli.

October 11, 2017

  • Miniprep of October 10 transformation and digested with EcoRI and PstI.
  • Ran digest on gel (did not get correct band) (Figure 8)

Figure 8 (left to right): 5 replicates pCAMBIA ietAS, 2 log ladder, duplicate pCAMBIA MCS.


October 19, 2017

  • Digest of ietAS + pSB1C3 with EcoRI and SpeI.
  • Ligation of digest into pCAMBIA.
  • Transformation into electrocompetent Agrobacterium tumefaciens.

October 24, 2017

  • Miniprep and digest of pCAMBIA + ietAS with HindIII.
  • Ran gel of digest with expected results. (Figure 9)

Figure 9 (left to right): 2 log ladder, pCAMBIA ietAS.


October 28, 2017

  • Day 1 of plating assay.

October 29, 2017

  • Day 2 of plating assay.

October 30, 2017

  • Day 3 of plating assay.

October 31, 2017

  • Day 4 of plating assay.

British_Columbia_Base

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