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

PCR of EBL1 and EBL2 Gibson fragments for pSB1C3

Redoing failed PCR, modified protocol by having elongation time be extended to be 30s/kb; annealing temperature set to 72C; ran 25uL and 50uL rxn

Fragments:

• EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 210s)
• EBL2 in pSB1C3 frag 1 (Tm = 65C, ext time = 180s)
• EBL2 in pSB1C3 frag 4 (Tm = 64C, ext time = 120s)

Cycling conditions:

• 98C -- 30s
• 98C -- 10s
• Tm -- 30s
• 72C -- ext time
• 72C -- 2min
• 14C -- infinite

28 cycles



ladder; EBL2 frag 1; EBL2 frag 4; EBL1 frag 2 (25uL); EBL1 frag 2 (50uL)

• only primer dimers observed

Troubleshooting PCR of EBL1 frag 2, EBL2 frag 1, EBL2 frag 4

Redoing failed PCR, modified protocol by doubling and quadrupling template dna amount; changed from 28 to 32 cycles

• cycling conditions remained constant


ladder; 2x EBL1 frag 2; 4x EBL1 frag 2; 2x EBL1 frag 1; 4 EBL1 frag 1; 2x EBL1 frag 4; 4x EBL1 frag 4

• doubling template DNA resolved EBL2 frag 2 and 4 problem; most likely faulty primers for EBL1 frag 2

Transformation of DH5-alpha with EC93 cosmid and pSK33

transform with 1ng of DNA

EC93: AmpR; pSK33: KanR

Troubleshooting PCR of EBL1 frag 2 in pSB1C3 Cont.

redid protocol in 7/6/16 with newly diluted primers

inoculated starter cultures of DH5-alpha w/ EC93, DH5-alpha w/ pSK33, and EPI100 w/ EC93

Verification of EC93 cosmid and miniprepping for more pSK33

pSK33 had poor yield since it is a pSC101 derivative and is very low copy --> next time miniprep 10mL of culture

ran test digest to confirm EC93 cosmid:

• 500 ng DNA
• 5uL NEB 2.1
• 1uL EcoRI
• H2O to 50uL

expecting bands at 10kb and 8kb

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.

Innoculation of transformed colonies

Recap from previous day: Colonies on all transformations were successful. Interestingly, the 1:50 dilution of DNA produced the largest number of colonies. All plates were very full, with the 1x plating dilutions being entirely unusable due to how covered they were. This may also be due to the long saving time from yesterday.

I decided to pick colonies from the 10x plating dilution of the 1:50 transformation.

Goals: The only thing I’ll really be able to do is pick colonies and grow them overnight for tomorrow.

Inoculation:

• Autoclaved culture tubes

• 2 isolated colonies picked

• 5mL LB per culture tube

**Tubes in 37C incubator from 3:30pm

Conclusions: The commercial cells were far more successful than our old cells. I used two colonies, which may not end up perfect, so I’ve stored the plate in a 4C cooler. Tomorrow, I’ll store the cells and/or miniprep and possibly make glycerol stocks of them, though I don’t have primers to sequence or PCR them beyond that.