Rapid fine tuning of Eschericia coli gene
expression
PROTOCOL FOR:
Escherichia coli strains with promoter libraries constructed by Red/ET
recombination pave the way for transcriptional fine-tuning
Stephan Braatsch1, Søren Helmark2, Harald Kranz1, Brian Koebmann2, and Peter
Ruhdal Jensen2,3
1
Gene Bridges GmbH, Commercial Centre, Heidelberg, Germany, 2Fermenco ApS c/o
Technical University of Denmark, and 3Center for Systems Microbiology, Department of
Systems Biology, Technical University of Denmark, Lyngby, Denmark
BioTechniques 45:335-337 (September 2008)
LEGEND
ATTENTION
* HINT
REST

REAGENTS
Deoxynucleotide Mix (5 PRIME, Hamburg, Germany)
MinElute DNA Cleanup System (Qiagen, Hilden, Germany)
Glycerol (AppliChem, Darmstadt, Germany)
Kanamycin sulfate (AppliChem, Darmstadt, Germany)
L-arabinose (Sigma-Aldrich, Seelze, Germany)
Quick & Easy E. coli Gene Deletion Kit (Gene Bridges, Heidelberg, Germany)
Tetracycline hydrochloride (AppliChem, Darmstadt, Germany)
TripleMaster PCR System (Eppendorf, Hamburg, Germany)
pRed/ET (Gene Bridges, Heidelberg, Germany)
BioTechniques Protocol
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PROCEDURE
CONSTRUCTION OF THE SPL-LIBRARY BY PCR
It is necessary to PCR-attach 50 bp of flanking homology arms (primer 1, 2)
and the degenerated SPL sequence (N5TTGACAN17 TATAATN5; primer 2) to the
kmR cassette supplied with the Quick & Easy E. coli Gene Deletion Kit.
*
Consult the Quick & Easy E. coli Gene Deletion Kit manual for primer design.
1. Use primers 1 and 2 to PCR-amplify an SPL library from the FRT-PGK-gb2-neoFRT template supplied with the Quick & Easy E. coli Gene Deletion Kit.
2. Purify the PCR fragment using the MinElute DNA Cleanup system.
3. Analyse the PCR product on an agarose gel.
4. Quantify the DNA concentration by UV spectroscopy and adjust it to 200 ng/µl.
*
The FRT-PGK-gb2-neo-FRT template supplied with the Quick & Easy E. coli
Gene Deletion Kit is encoded by a R6K-based plasmid. It can not be replicated in
most E. coli strains. Thus, the PCR product usually does not need be gel-purified
or DpnI-digested.

The resulting km -SPL constructs can be stored at -20°C for several weeks.
R
TRANSFORMATION OF THE E. coli TARGET STRAIN WITH PLASMID pRed/ET
*
Before starting, chill 2.5 ml of 10% glycerol (v/v) and an electroporation cuvette;
cool benchtop centrifuge to 2°C.
5. Set up an overnight (o.n.) culture of your E. coli strain: pick a single colony and
inoculate a 1 ml LB culture in a 2 ml microfuge tube. Puncture the lid for air
supply. Incubate o.n. at 37°C, 700 rpm.
6. Set up a new microfuge tube with 1.4 ml of fresh LB medium and inoculate with
30 μl of the o.n. culture from step 5; incubate for 2 h at 37°C, 700 rpm.
7. Prepare electrocompetent cells: wash 2× with 1 ml ice cold 10% glycerol (v/v).
Tip out the supernatant and resuspend cells in the remaining liquid (∼50 µl).
8. Add 1 μl (20 ng) of pRed/ET (Gene Bridges) and electroporate at 1350 V, 10 µF,
600 Ω.
*
This setting applies to an Eppendorf Electroporator 2510 using a 1-mm electroporation cuvette. Other devices can be used, but 1350 V and a 5-ms pulse are
recommended. For recommended settings of other electroporators, consult
http://www.genebridges.com/gb/tech_info.php?id=142.
BioTechniques Protocol
Page 2 of 4
9. Resuspend cells in 1 ml LB and incubate the culture in a microfuge tube for 60
min at 30°C, 700 rpm.
10. Streak out 100 µl culture on LB plates + tetracycline (Tc; 3 µg/ml). Incubate o.n.
at 30°C in order to get single colonies.
Make sure the Red/ET-competent cells are incubated at 30°C since pRed/ET can
not be replicated at 37°C.
Red/ET-competent E. coli colonies can be stored at 4°C in the dark for several
days.
CONSTRUCTION OF THE E. coli SPL LIBRARY
*
Before starting, chill 5 ml of 10% glycerol (v/v) and 2 electroporation cuvettes
on ice; cool benchtop centrifuge to 2°C.
11. Set up an o.n. culture of your Red/ET-competent E. coli strain from step 10: pick
a single colony and inoculate 1 ml of LB + Tc (3 μg/ml) in a 2 ml microfuge tube.
Puncture the lid for air supply. Incubate o.n. at 30°C, 700 rpm.
12. Set up two microfuge tubes with 1.4 ml of fresh LB + Tc (3 μg/ml) medium and
inoculate each tube with 50 μl of the o.n. culture from step 11; incubate for
2 h at 30°C, 700 rpm.
13. Add 50 μl of 10% L-arabinose (w/v) to one of the cultures. Leave the other
tube without induction as negative control. Incubate for 30 min at 37°C, 700 rpm.
14. Prepare electrocompetent cells as described in step 7.
15. Add 1 μl (200 ng) of the SPL-kmR construct from step 4 to the competent cells
from step 14 and electroporate at 1350 V, 10 μF, 600 Ω.
16. Resuspend cells in 1 ml LB and incubate in microfuge tubes for 1 h at 37°C,
700 rpm.
17. Streak out 100 and 900 µl of the cultures on LB plates + kanamycin (Km;
15 μg/ml). Incubate o.n. at 37°C in order to get single colonies.
The plates should not contain Tc.
* You should obtain >100 colonies; the ratio of L-arabinose induced to uninduced
colonies should clearly exceed 10:1.

Plates with SPL-transformants can be stored at 4°C for several days.
18. Verification by colony PCR: resuspend single colonies in 30 μl of sterile H2O.
Boil the samples at 98°C for 5 min and briefly spin the debris down at 13,000
rpm. Take 2 μl of supernatant as template for your control PCR reaction.
BioTechniques Protocol
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*The km
R
selection cassette supplied with the Quick & Easy E. coli Gene Deletion
Kit is flanked with FRT-sites. Thus, the marker can subsequently be removed by
Flp recombinase where appropriate.
TROUBLESHOOTING
NO COLONIES ON YOUR PLATE AFTER Red/ET RECOMBINATION
The Red/ET reaction did not take place⎯pRed/ET was lost because the cells were
grown at 37°C instead of 30°C.
Electroporation problems⎯cells are not competent enough to take up the linear
DNA. Keep electrocompetent cells cool at all stages. For electroporation settings
consult http://www.genebridges.com/gb/tech_info.php?id=142.
SIMILAR NUMBER OF INDUCED AND UNINDUCED COLONIES
Probably caused by traces of the template plasmid used to PCR-amplify the SPLconstruct. Set up a DpnI digest prior to electroporation.
EQUIPMENT
Centrifuge 5415R (Eppendorf, Hamburg, Germany)
Electroporator 2510 (Eppendorf, Hamburg, Germany)
Flex Cycler (Analytik Jena, Jena, Germany)
Thermomixer compact (Eppendorf, Hamburg, Germany)
BioTechniques Protocol
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