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From 2014.igem.org

Contents 1 Week 1 2 Week 2 3 Week 3

Week 1

Notes: Unless stated otherwise, all gels contain 2-log ladder

5/9

• •Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene
  • -Will also be called BW23115 or BW
  • -Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)
• •Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)
  • -Will also be called ER. Assume that all ER samples contain the F’ episome
  • -Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome

5/10

• •Did receive colonies from 5/9 selection

Week 2

5/12

• •Need to conjugate BW23115 KanR cells with the F’ episome
  • -Set up overnight cultures of ER2738 and BW23115 KanR
  • -When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance

5/13

• •Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage
  • -Let precipitated in NaCl/PEG solution overnight
  • -Possible sources of error

Did not sterilize 2.5M NaCl/20% PEG-8000 solution

Added 4-fold PEG solution

• • • • •Compensated by adding more LB

During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze

• •Conjugated BW23115 with F’ episome
  • -Added 1mL BW23115 to 1mL ER2738 overnight culture
  • -Incubated at 37C for 30 minutes, shaking
  • -Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)

To select for BW cells that took the F’ episome (containing Tet resistance)

5/14

• •Finished the M13 Amplification
  • -Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.
• •Results of BW23115 Conjugation
  • -Many colonies indicating successful conjugation of F’ episome into BW23115
  • -Set up overnight to make freeze down tomorrow
• •Set up overnight of ER2738 to make chemically competent tomorrow

5/15

• •Made freeze down of BW23115 KanR F’
  • -BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome
• •Made chemically competent ER2738 cells
• •Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells
  • -Added 1ul Litmus28i plasmid to 40ul competent cells
  • -Plated on LB + Amp(100ug/mL)
  • -Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage.

5/16

• •Results of 5/15 transformation
  • -No growth for No DNA control
  • -Many colonies for sample

Week 3

5/19

• •M13 Amplification to isolate M13-Litmus28i phage
  • -Cells: ER2738 cells containing Litmus28i phagemid
  • -Helper Phage: M13K07
  • -Not much phage was precipitated
• •Set up overnight culture of ER2738 to infect tomorrow

5/20

• •Infected ER2738 cells with M13-Litmus28i phage
  • -Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)
  • -Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect

5/21

• •Results from M13-Litmus28i infection of ER2738
  • -Solid lawn of growth for diluted and non-diluted
  • -Also sickly looking growth
• •Set up overnights
  • -ER2738 cells containing Litmus28i for freeze down
  • -BW23115 with F’ episome to make chemically competent cells
  • -ER2738 to redo infection

5/22

• •Tested absorbance of phage produced through M13 amplification on 5/19
  • -Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm
  • -Decided to redo M13 amplification
• •Made chemically competent BW23115 with f-episome
• •Made freeze down of ER2738 containing Litmus28i
• •Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow

5/23

• •Protocol switch to make phage using phagemid
  • -“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid
  • -Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)
  • -Making phage….

• •Made fresh antibiotics
  • -Chloramphenicol (34 ng/mL)

1.44g chloramphenicol into 42mL EtOH

• • -Ampicillin (50 ng/mL)

4g ampicillin into 80mL mili-Q H2O