Team:UCLA/Notebook

JOURNAL

Projects

CDI

Protein Cages

Week

1

Construction of ATCC 13048 Loci 2 in pSB1C3

Isolation of template DNA from EC93, EC869, and ATCC 13048; PCR of Gibson fragments for EBL2 in pSB1C3 construct

Jun

27

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
Jun

28

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

Jun

29

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

Jun

30

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

Week

1

Title For The Week

Description blah blah blah

June

14

Clone and transform

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.