TITLE: Integration of a cargo brick
AUTHORS:  Carolyn Bayer, Maja Rennig, Anja  Ehrmann, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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</div><br/><div class = "text-block"><span>This protocol describes the process of integrating a SEGA cargo brick. A cargo brick is integrated using </span><span style = "font-style:italic;">tetA </span><span>counterselection. This protocol also applies to integration of other bricks using tetA counterselection, e.g. splitting</span><span style = "font-style:italic;"> tetA</span><span>. </span></div></div>

[STEPS]
?. [preculture and DNA fragment- Day 1]
Prepare a PCR product of the cargo brick and purify it from an agarose gel.
?. [preculture and DNA fragment- Day 1]
Setup a preculture of the strain with pSIM19 in LB medium supplemented with Spectinomycin  and incubate at  overnight
30 33
?. [Recombineering- Day 2 ]
Prepare:
?. [Recombineering- Day 2 ]
Cold sterile water
?. [Recombineering- Day 2 ]
Cold Glycerol
?. [Recombineering- Day 2 ]
Pre-chilled centrifuge and tabletop centrifuge at
4 °C
?. [Recombineering- Day 2 ]
M9 agar plates supplemented with  NiCl2
Additionally, specific inducers to enable green-white screening can be added
?. [Recombineering- Day 2 ]
Inoculate  LB-Medium supplemented with Spectinomycin () with  of the preculture from step 3
50 mL
500 µl
?. [Recombineering- Day 2 ]
Incubate at  until cultures reached an OD600 of 0.5
30 33
?. [Recombineering- Day 2 ]
Induce expression by transferring the culture to a shaking water bath at
42 33
?. [Recombineering- Day 2 ]
Transfer culture to prechilled  falcon tubes and put on ice for
50 mL
?. [Recombineering- Day 2 ]
Spin the culture down at  and discard the supernatant
Centrifuge: 4000 34, 5 min, 4 10
?. [Recombineering- Day 2 ]
Add  of ice cold water, resuspend and transfer to a 1.5 ml tube
1 mL
?. [Recombineering- Day 2 ]
Spin at   in a tabletop centrifuge
Centrifuge: 11000 34, 30 s, 4 10
?. [Recombineering- Day 2 ]
Wash pellet twice with  ice cold water
1 mL
?. [Recombineering- Day 2 ]
Resuspend the pellet in  cold glycerol ()
600 µl
?. [Recombineering- Day 2 ]
Unused cells can be stored at
-80 °C
This is not possibe for E. coli Nissle
?. [Recombineering- Day 2 ]
Electroporate  of cells with  of purified PCR product from step 2 or  of a  single stranded oligonucleotide
50 µl
200 ng
2 µl
?. [Recombineering- Day 2 ]
Recover cells  in a tabletop shaker using SOC medium.
30 33
?. [Recombineering- Day 2 ]
Transfer the cells into  LB medium supplemented with Spectinomycin
5 mL
?. [Recombineering- Day 2 ]
Incubate at  overnight
30 33
?. [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.
Streaking out of the colonies at least twice is necessary to ensure a clean culture
?. [Plating- Day 2 ]
Wash  of the recovered cells twice with sterile water. Centrifuge at
1 mL
Centrifuge: 11000 33, 30 s, 20 10
?. [Plating- Day 2 ]
Make a dilution series and plate  of the 1:10 - 1:1000 dilution on M9 agar supplemented with  NiCl2
100 µl
the appropriate inducer can be added to the agar plates to enable green-white screening
?. [Plating- Day 2 ]
incubate the plates at  for  to
30 °C
Cells can be incubated at   if it is the last step of recombination and pSIM19 is no longer needed in the cells