Inoculation of strains obtained from Dr. Christopher Hayes

Inoculated overnight cultures of:

• EPI100 w/ EC93 cosmid
• X90 w/ EC869 cosmid
• ATCC 13048

Conditions:

• 10mL LB
• 10uL ampicillin
• 37 C for 16h shaking

Isolation of template DNA

Miniprep of EC93 and EC869

• EC93 #1: 145.42 ng/μL
• EC93 #2: 198.85 ng/μL
• EC869 #1: 90.81 ng/μL
• EC869 #2: 45.95 ng/μL

gDNA isolation of ATCC 13048

• 34.87 ng/μL

Gibson primers designed for EBL1 and EBL2 toxin switched constructs in pSB1C3 and pSC101

PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3

50uL rxn:

• 2.5 uL F primer
• 2.5 uL R primer
• 25 uL Q5 2X MM
• 19 uL dH2O

Fragments:

• EBL1 in pSB1C3 frag 2
• EBL2 in pSB1C3 frag 2
• EBL2 in pSB1C3 frag 3

Cycling conditions:

• 98C -- 30s

• 98C -- 10s
• TmC -- 30s
• 72C -- ext time

• 72C -- 2min

28 cycles

Troubleshooting PCR of Gibson fragments

• try using KAPA Hifi instead of Q5
• add more template gDNA (1ng, 10ng, 35ng, 70ng)

25uL rxn:

• 0.75 uL F primer
• 0.75 uL R primer
• 12.5 uL KAPA Hifi 2X MM
• dH2O up to 25 uL

Fragments:

• EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 140s)
• EBL2 in pSB1C3 frag 2 (Tm = 65C, ext time = 88s)
• EBL2 in pSB1C3 frag 3 (Tm = 65C, ext time = 180s)

Cycling conditions:

• 98C -- 30s
• 98C -- 10s
• TmC -- 30s
• 72C -- ext time
• 72C -- 2min

28 cycles



ladder; EBL2 frag 2; EBL1 frag 2; EBL2 frag 3

Plate Making and Transformation of O3-33 WT

Goals for today: We have a vector of O333 from Neil King contained in a pET vector, the goal for today is to transform this vector, grow it tomorrow and PCR it for a large number of copies to work with and creation of a biobrick. Primers will need to be designed including the T7 promoter to terminator, with iGEM prefix and suffix.

Made plates

• 12.5g LB

• 7.5 Bacto Agar

• 500mL dH2O

• 500uL Kanomycin (50mg/mL)

• 20 minutes autoclave

• 4 hours total time to solidify

=> 32 plates total

Transformation

• 30uL iGEM electrocompetent cells

• 1uL O333 vector 1:100 dilution

• 970uL SOC save

• 45 minutes incubation @ 800rpm/37C

• 1 and 1:10 dilution plating

=> 3 transformations total

=> arc time: 5.5s, 5.6s, 5.6s

**Plates in 37C inoculation from 6:40pm

Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.

Transformed O3-33 WT

Recap from previous day: No colonies were shown from any of the transformations. Competent cells may have been of low quality (relatively long arc times).

Goals: Today, commercial electro-competent cells are going to be transformed, and different concentrations of DNA will be tested for transformation as well.

Transformation:

• 3x 25uL commercial electrocompetent cells

• 1uL of 1:5, 1:50, 1:100 serial dilutions of plasmid from Neil King

• 970uL SOC save

• 60 minutes save (800rpm @ 37C), then additional 50 minutes at 37C incubator (no shaking) as I was waiting for plating beads to be autoclaved and cooled

• 1 and 1:10 dilution platings

=> 1:5 DNA: 5.1s arc time

=> 1:50 DNA: 5.0s arc time

=> 1:100 DNA: 4.8s arc time

**Plates in 37C incubation from 4:10pm

Conclusions: Same as yesterday, though today arc times were slightly better. Unfortunately, my timing was off for plating because I was waiting to autoclave new beads (thanks for sharing, grad students!), so when I plated the transformants smelled pretty awful. Hopefully that doesn’t make too much of a difference.