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

[DESCRIPTION]
<div class = "text-blocks"><br/><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 to process of integrating a SEGA control brick. A control brick is integrated using </span><span style = "font-style:italic;">galK</span><span> counterselection. </span></div></div>

[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.
?. [preculture and DNA fragment- Day 1]
Setup a preculture of the strain harbouring pSIM19 in LB medium supplemented with Spectinomycin . Incubate overnight at
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 ]
M63 agar plates supplemented with   2-deoxy-galactose,  glycerol and L-rhamnose
?. [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 ]
Transfer cells into  LB medium in a 250 ml baffled conical shake flask and recover overnight at
50 mL
30 33
Alternatively, cells can be recovered in   medium for
?. [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
?. [Plating- Day 3]
Wash  of the recovered cells twice with 1X M9 salts. Centrifuge at
1 mL
Centrifuge: 11000 33, 30 s, 20 10
?. [Plating- Day 3]
Make a dilution series and plate  of the 1:100 - 1:1000 dilution on M63 agar supplemented with   2-deoxy-galactose,  glycerol and  L-rhamnose.
100 µl
In case the cells were recovered for    dilution 1:10 and 1:100 need to be plated
?. [Plating- Day 3]
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