Generation of E. coli strains

Constructing NM1100

• Grow 5 ml MG1655 in LB to an OD600 ~ 0.4-0.6.
• Pellet the cells
• Resuspend the cells in 5 ml ice cold water
• Pellet in a refrigerated centrifuge
• Resuspend in 1 ml of ice cold water
• Pellet
• Repeat washes for a total of 4 times always on ice and cold water and refrigerated centrifuge
• Resuspend pellet in 50 ml ice cold water
• Transfer to cold 0.2 cm gap electroporation cuvette
• Add 100 ng of mini-l DNA Tet (https://redrecombineering.ncifcrf.gov/references-2/general-recombineering/court-2003-gene.pdf)
• Electroporate DNA on bacterial settings for electroporator
• Add 950 ml SOC media for recovery
• Incubate 1hr at 32 C
• Plate 10 ml and 200 ml on LB-Tet₂₅
• Incubate overnight at 32 C

Constructing NM50000

• Transform NM1100 with pBeloBac-gyrA_his and plate on LB-Cm₁₀ at 32 C overnight
• Take one colony and grow in LB-Cm₁₀ to an OD600 ~ 0.4-0.6
• Induce the mini-l by shifting the culture to a 42 C water bath for 15 minutes
• Take the flask from 42 C directly into an ice water slurry and swirl continuously for 2 min
• Continue swirling on and off for up to 10 min.
• Pellet the cells in a refrigerated centrifuge at 4 C and wash as mentioned above (https://redrecombineering.ncifcrf.gov/references-2/general-recombineering/cpmb-2014.pdf)
• Cells are electrocompetent
• Electroporate with 100 ng of gyrA::zeo DNA.

gyrA::zeo

Two 60-mers were designed with one forward having 40 nt of homology upstream to gyrA and 20 to amplify a zeo antibiotic marker; while the reverse has 40 nt homology downstream and 20 nt to amplify the zeo marker. PCR product is cleaned and verified on gel to make sure it is a single band ~ 600 bp.

For additional questions, please feel free to contact me directly: majdalan@mail.nih.gov

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