"

From 2014.igem.org

Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4

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….

DATA TABLE HERE******************************************

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

1.44g chloramphenicol into 42mL EtOH

• • -Ampicillin (50 ng/mL)

4g ampicillin into 80mL mili-Q H2O

5/24

• •Isolated phage using new protocol
  • -Resuspended pellet in 200ul TBS and 200ul 30% glycerol
  • -Measured absorbance with UV-vis

concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)

DATA TABLE HERE************************************************

• •Infect ER2738 cells with M13-Litmus28i
  • -Wanted 1:10 phage:cell ratio. Math….

At 1 OD (e.coli), cell/mL = 5x10^8

5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage

• •Set up overnights
  • -ER2738 for infection with M13-Litmus28i
  • -BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain

Week 4

5/25

• •Infect ER and BWF’ cells with M13-Litmus28i
  • -Made 5mL culture of ER and BW that was at 1 OD

DATA TABLE HERE********************************************************

• • -Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage
  • -Grew the cells for 20 minutes at 37C
  • -Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample

Incubated overnight at 37C

• •Note: During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.

5/26

• •Results from 5/25 infection with M13-Litmus28i

DATA TABLE HERE***********************************