Integration of a control brick
Carolyn N Bayer, Maja Rennig, Anja Ehrmann, Morten Norholm
Abstract
SEGA, the Standardized Genome Engineering Architecture, is a comprehensive strain collection that enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome
This protocol describes to process of integrating a SEGA control brick. A control brick is integrated using galK counterselection.
Before start
Transform a SEGA strain, harbouring the upstream gadget galK , with pSIM19 (Spectinomycin resistance). From now on cultures have to be kept at 30°C to retain the plasmid (temperature-sensitive ori ).
Steps
preculture and DNA fragment- Day 1
Prepare a PCR product of the control elements that need to be integrated and purify it from an agarose gel.
Setup a preculture of the strain harbouring pSIM19 in LB medium supplemented with Spectinomycin 0.05mg/mL. Incubate overnight at 250rpm
Recombineering- Day 2
Prepare:
Cold sterile water
Cold Glycerol 15% volume
Pre-chilled centrifuge and tabletop centrifuge at 4°C
M63 agar plates supplemented with 0.2Mass / % volume 2-deoxy-galactose, 0.2% volume glycerol and5millimolar (mM) L-rhamnose
Inoculate 50mL LB-Medium supplemented with Spectinomycin (0.05mg/mL) with 500µL of the preculture from step 3
Incubate at 250rpm until cultures reached an OD600 of 0.5
Induce expression by transferring the culture to a shaking water bath at 150rpm
Transfer culture to prechilled 50mL falcon tubes and put on ice for 0h 15m 0s
Spin the culture down at 4000x g,4°C and discard the supernatant
Add 1mL of ice cold water, resuspend and transfer to a 1.5 ml tube
Spin at 11000x g,4°C in a tabletop centrifuge
Wash pellet twice with 1mL ice cold water
Resuspend the pellet in 600µL cold glycerol (15% volume)
Unused cells can be stored at -80°C
Electroporate 50µL of cells with 200ng of purified PCR product from step 2 or 2µL of a 100micromolar (µM) single-stranded oligonucleotide
Transfer cells into 50mL LB medium in a 250 ml baffled conical shake flask and recover overnight at 250rpm
Plating- Day 3
Wash 1mL of the recovered cells twice with 1X M9 salts. Centrifuge at 11000rpm,20°C
Make a dilution series and plate 100µL of the 1:100 - 1:1000 dilution on M63 agar supplemented with 0.2Mass / % volume 2-deoxy-galactose, 0.2% volume glycerol and 5millimolar (mM) L-rhamnose.
incubate the plates at 30°C for 48h 0m 0s to 72h 0m 0s
Screening- Day 4-5
Screen for positive colonies by "green-white screening"on a blue-light table and perform colony PCR on the colorless colonies to identify the correct recombinants
