TITLE: Integration of a landing pad 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/><br/><div class = "text-block"><span>This protocol describes to process of integrating a SEGA landing pad brick. A landing pad brick is integrated using </span><span style = "font-style:italic;">tetA </span><span>positive selection. This protocol also applies to integration of other bricks using tetA positive selection, e.g. complementation of split</span><span style = "font-style:italic;"> tetA</span><span>. </span></div></div>

[STEPS]
?. transform your desired SEGA strain with pSIM19 (Spectinomycin resistance). From now on cultures have to be kept at  to retain the plasmid (temperature-sensitive ori)
30 °C
?. [Recombineering- Day 2 ]
Prepare:
?. [Recombineering- Day 2 ]
Cold sterile water
?. [Recombineering- Day 2 ]
Cold Glycerol
?. [Recombineering- Day 2 ]
Pre-chilled centrifuge and tabletop centrifuge to
4 °C
?. [Recombineering- Day 2 ]
LB agar plates supplemented with or   Tetracycline
Using   tetracycline will increase chances to get positive colonies in case of difficult to integrate constructs. However, less background is to be expected at higher tetracycline concentrations
?. [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  single stranded oligonucleotide
50 µl
200 ng
2 µl
?. [Recombineering- Day 2 ]
Recover cells  in a tabletop shaker using SOC medium
30 33
Cells can be recovered at   if it is the last step of recombination and pSIM19 is no longer needed in the cells
?. [Recombineering- Day 2 ]
Plate cells on LB agar plates supplemented with  or  Tetracycline
Using   tetracycline will increase chances to get positive colonies in case of difficult to integrate constructs. However, less background is to be expected at higher tetracycline concentrations
?. [Recombineering- Day 2 ]
Incubate at  for   up 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
?. [Screening- Day 3-4]
Screen for positive colonies by "green-white screening"on a blue-light table and perform colony PCR on the fluorescent colonies to identify the correct recombinants
Streaking out of the colonies at least twice is necessary to ensure a clean culture
?. [preculture and DNA fragment- Day 1]
Setup a preculture of the strain with pSIM19 in LB medium supplemented with Spectinomycin
30 33
?. [preculture and DNA fragment- Day 1]
Prepare a PCR product of the landing pad brick and purify it from an agarose gel.